Plant Breeding Reviews (Volume 31)

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PLANT BREEDING REVIEWS Volume 31

edited by

Jules Janick Purdue University


Plant Breeding Reviews is sponsored by: American Society of Horticultural Science International Society for Horticultural Science

Editorial Board, Volume 31 I. L. Goldman C. H. Michler Rodomiro Ortiz


edited by

Jules Janick Purdue University

Copyright # 2009 by John Wiley & Sons, Inc. All rights reserved. Wiley-Blackwell is an imprint of John Wiley & Sons, Inc., formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing. Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, 201-748-6011, fax 201-748-6008, or online at http://www.wiley.com/ go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical upport, please contact our Customer Care Department within the United States at 877-762-2974, outside the United States at 317-572-3993 or fax 317- 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: ISBN 978-0-470-38762-7 (cloth) ISSN: 0730-2207 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

Contents

Contributors 1. Dedication: Anthony H. D. Brown Conservation Geneticist

ix 1

Reid G. Palmer and Jeff J. Doyle I. Biographical Sketch II. Research Accomplishments III. The Man IV. Honors and Awards Selected Publications of Anthony H. D. Brown

2 6 10 12 12

2. Brassica and Its Close Allies: Cytogenetics and Evolution

21

Shyam Prakash, S. R. Bhat, C. F. Quiros, P. B. Kirti, and V. L. Chopra I. II. III. IV. V. VI.

Introduction Cytogenetics Genome Manipulation Wide Hybridization Cytoplasmic Substitution and Male Sterility Genome Dissection and Development of Chromosome Addition Lines VII. Mitochondrial Genome VIII. Plastid Genome IX. Potential Role of Arabidopsis thaliana in Brassica Improvement X. Chloroplast Genomes and their Phylogenetic Implications XI. Evolution of Morphological Characters XII. Concluding Remarks Literature Cited

24 26 56 71 95 104 110 113 114 123 137 142 146 v

vi

CONTENTS

3. Genetic Enhancement for Drought Tolerance in Sorghum

189

Belum V. S. Reddy, S. Ramesh, P. Sanjana Reddy, and A. Ashok Kumar I. Introduction II. Breeding for Drought Tolerance III. Selection among Cultivars and Landraces IV. Breeding for Drought Escape V. Growth Stage–Specific Screening Techniques VI. Physiological Response Traits for Drought Tolerance VII. Marker-Assisted Breeding for Drought Tolerance VIII. Outlook Literature Cited

4. Breeding for Resistance to Stenocarpella Ear Rot in Maize

189 190 194 197 199 207 210 212 214

223

Johannes D. Rossouw, Z. A. Pretorius, H. D. Silva, and K. R. Lamkey I. Introduction II. Distribution and Importance III. Pathogen IV. Epidemiology V. Disease Management VI. Summary and Conclusion Literature Cited

5. Cassava Genetic Resources: Manipulation for Crop Improvement

224 225 229 232 233 240 241

247

Nagib M. A. Nassar and Rodomiro Ortiz I. II. III. IV.

Introduction Wild Manihot Species: A Botanical Review Interspecific Hybrids Cassava Diversity as Revealed by DNA Markers and Genetics V. Trait Transfer VI. Outlook Literature Cited

248 252 253 257 262 267 268

CONTENTS

6. Breeding Roses for Disease Resistance

vii

277

Vance M. Whitaker and Stan C. Hokanson I. Introduction II. Causal Pathogens III. Resistance Screening IV. Breeding V. Molecular Tools VI. Future Prospects Literature Cited

7. Plant Breeding for Human Nutritional Quality

277 279 288 298 305 313 316

325

Philipp W. Simon, Linda M. Pollak, Beverly A. Clevidence, Joannne M. Holden, and David B. Haytowitz I. Introduction II. Sources of Nutrients III. Progress in Breeding for Nutrient Content and Composition IV. Plant Breeding Strategies for Increasing Intake of Shortfall Nutrients Literature Cited

327 328 350 374 377

Subject Index

393

Cumulative Subject Index

395

Cumulative Contributor Index

415

Contributors

S. R. Bhat National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012 India V. L. Chopra National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012 India Beverly A. Clevidence Food Components and Health Laboratory, United States Department of Agriculture—Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 USA Jeff J. Doyle L. H. Bailey Hortorium, Department of Plant Biology, Cornell University, Ithaca, New York 14853 USA David B. Haytowitz Nutrient Data Laboratory, United States Department of Agriculture—Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 USA Stan C. Hokanson University of Minnesota, Department of Horticultural Science, 1970 Folwell Avenue, St. Paul, MN 55108 USA Joannne M. Holden Nutrient Data Laboratory, United States Department of Agriculture—Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 USA P. B. Kirti Plant Science Department, University of Hyderabad, Hyderabad, 500046 India A. Ashok Kumar International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India K. R. Lamkey 2101 Agronomy Hall, Iowa State University, Ames, Iowa, USA Nagib M. A. Nassar Departamento de Genetica e Morfologia, Universidade de Brasilia, 70919 Brasilia, Brazil Rodomiro Ortiz Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT), El Batan, Texcoco, Apdo. Postal 6-641, 06600 Mexico, D.F. Mexico Reid G. Palmer United States Department of Agriculture, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, Department of Agronomy, Iowa State University, Ames, Iowa 50011 USA Linda M. Pollak Corn Insects and Crop Genetics Research Unit, United States Department of Agriculture, Agricultural Research Service, Department of Agronomy, Iowa State University, Ames, Iowa 50011 USA Z. A. Pretorius Department of Plant Sciences, University of the Free State, Bloemfontein, 9300 South Africa ix

x

CONTRIBUTERS

Shyam Prakash National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012 India C. F. Quiros Department of Vegetable Crops, University of California, Davis, California 95616 USA S. Ramesh International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India Belum V. S. Reddy International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India P. Sanjana Reddy International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India Johannes D. Rossouw Monsanto Singapore Co (PTE) Ltd., 151 Lorong Chuan 06-08 New Tech Park, Singapore H. D. Silva Monsanto Brazil, Rodovia Uberlaˆndia-Araxa´, Uberlandia, MG, Brazil Philipp W. Simon Vegetable Crops Research Unit, United States Department of Agriculture, Agricultural Research Service, Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706 USA Vance M. Whitaker University of Minnesota, Department of Horticultural Science, 1970 Folwell Avenue, St. Paul, MN 55108 USA

Anthony H. D. Brown

1 Dedication: Anthony H. D. Brown Conservation Geneticist Reid G. Palmer United States Department of Agriculture Agricultural Research Service Corn Insects and Crop Genetics Research Unit Department of Agronomy Iowa State University Ames, Iowa 50011 USA Jeff J. Doyle L. H. Bailey Hortorium Department of Plant Biology Cornell University Ithaca, New York 14853 USA

I. BIOGRAPHICAL SKETCH II. RESEARCH ACCOMPLISHMENTS A. Conservation Genetics B. Plant Mating Systems and Population Structure III. THE MAN IV. HONORS AND AWARDS ACKNOWLEDGMENT SELECTED PUBLICATIONS OF ANTHONY H. D. BROWN

This volume of Plant Breeding Reviews is dedicated to Anthony (Tony) H. D. Brown, known internationally for his research in conservation and population genetics and plant breeding. Dr. Brown’s primary contributions in the area of conservation genetics followed two major themes: optimum sampling strategies and core collections. His life’s

Plant Breeding Reviews, Volume 31 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 1

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R. G. PALMER AND J. J. DOYLE

activities in this area were inspired primarily by his long friendship and close working relationship with Sir Otto Frankel. His research in population genetics focused on the estimation of mating systems and their impact on plant population structure while his research in breeding was on the use of wild relatives in crop improvement. Dr. Brown started with the Commonwealth Scientific and Industrial Research Organization (CSIRO) in 1972 as a research scientist and retired as a chief research scientist in 2006. He is now an Honorary Research Fellow in the Centre for Plant Biodiversity Research, CSIRO Plant Industry, Canberra, Australia.

I. BIOGRAPHICAL SKETCH Tony Brown was born on November 25, 1941, in Waverley, Sydney NSW, Australia. It was wartime and his father, Arthur Brown, was in Darwin, Australia, serving as Squadron Leader in the Royal Australian Air Force. Arthur was from three or more generations of Australian stock. Tony’s maternal grandfather, Hugh Milligan, son of Scottish immigrants, was an eminent primary school headmaster. Hugh’s task was to register Tony’s birth, the agreed name being Anthony Hugh Dean Brown. Hugh urged that the last two names be hyphenated because plain ‘‘Brown’’ was insufficiently distinguished for a future Macquarie Street specialist doctor. However, Tony’s mother Joyce intervened and said, ‘‘Plain Brown is good enough for me, it should be for my son.’’ This ensured that name hyphenation could await future needs. Yet Hugh had other major influences on Tony, inspiring a love of plants, of arithmetic shortcuts, and of parsing sentences. That three initials were an encumbrance emerged later in the United States, where names and official forms were triplet coded, allowing only one middle initial. And the inevitable inversion happened after publications on alcohol dehydrogenase, when the AHD became ADH, which spawned a growing list of mutant miscitations in the Science Citation Index. In high school Tony was inspired to seek a research career as an agricultural chemist after watching a film made by CSIRO on how the discovery of remedying trace element deficiencies in some depleted soils of South Australia converted them to cropping. Consequently, Tony enrolled in the Faculty of Agriculture, University of Sydney, as a salaried employee of the Colonial Sugar Refining Company. This was an era when companies were competing to recruit future graduates. At Sydney, inspirational lectures on Mendelian genetics by Professor ‘‘Spinny’’ Smith-White led to a final fourth year in genetics. Jim Peacock, later to become Chief CSIRO Plant Industry, was in his final year as Spinny’s PhD

1. DEDICATION: ANTHONY H. D. BROWN CONSERVATION GENETICIST

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student. He shaped the honors course, and infused enthusiasm as the DNA era was unfolding. Jim had spent some time in Adelaide University, and brought back insights from meeting Sir R. A. Fisher and the team of population genetics students there. Jim devised an unforgettable experiment for the honors genetics class with 16 blue and 16 yellow plastic beads in a jar to simulate genetic drift theory. The jar was shaken and inverted 16 times and the color of the first two beads noted. After 16 repeats, the jar was opened and its contents adjusted to the new observed gene frequency. Over 50 population replications were run over tens of generations, or until fixation blissfully occurred. Why was fixation happening faster than predicted? Sampling with or without replacement? Late into the night the rattle of balls in the jar resounded down the college corridors, until crash. . .extinction: The neck of the jar wore through and broke. However, the experiment had sown the seed of a lifelong interest in sampling issues. On graduating (in 1963), Tony was assigned by the Colonial Sugar Refining Company to its sugarcane experiment station in Lautoka, Fiji. This was a major transition, from collegiate to colonial life, and he was fully briefed at the head office in Sydney on how to behave toward the local population. The company itself was in transition, hiring local staff as officers, and the country was preparing for independence. Tony’s immediate boss was Joe Daniels, a sugarcane breeder respected around the world and a scholarly and imaginative leader. All communications were directed through the mill manager, including scientific reports. Tony had an early lesson in communication when management enrolled him in an in-house training course on report writing. The fact that management chose Tony’s report on fiber content to be one anonymous example of bad writing firmly made a point. It was an object lesson in the ‘‘Gunning fog index,’’ which is a function of the average length of sentences and the number of words with more than two syllables. The index is intended to equate to the number of years of education that a reader requires to understand the writing. Clearly no one in the head office had sufficient schooling to read Tony’s report. Joe Daniels introduced Tony to the magnet of wild crop relatives. It may seem hard to imagine in today’s bottom-line corporate world, but Joe was able to get the company’s approval for a visionary, ambitious project entitled ‘‘Re-creating the Pathway of Evolution of Sugarcane’’ by deliberately selecting Saccharum spontaneum for sucrose content. To achieve the screening of large populations for sucrose, Joe and Tony installed an autoanalyzer to run around the clock. For two weeks, Tony was on an unforgettable night shift feeding thousands of ethanol extracts into the analyzer and reading the output. That and the project to tag leafhoppers, vectors of Fiji disease, with radiolabel P-32

4


to track their movement in cane fields in retrospect seem incongruous in a small island colony. After nearly four years in Fiji, Tony returned in 1966 to academia and graduate school. He chose to work with Dr. Robert W. Allard at the University of California, Davis, primarily because of his classic plant breeding book and his research on quantitative genetics. On arrival, however, Tony found that Professor Allard was convinced that the new isozyme technique would open the door to empirical population genetics in plants. Professor Allard recommended that the PhD project should not be on quantitative genetics but on isozyme variation in Zea mays. This would fit better with his assigning Tony as half-time research assistant to implement an isozyme lab. In so many ways, this was a opportune moment to arrive in Davis and share the excitement and friendship of the Allard lab (particularly Drs. M. T. Clegg, S. K. Jain, D. R. Marshall, and B. S. Weir). The scientific collaborations begun at Davis continued in projects for several decades and led later to sabbatical visits at Stanford University and the University of California, Riverside. UC Davis was thus a watershed in Tony’s science and life including marriage. With the completion of his PhD in 1969, Tony was appointed as a lecturer in Biology at the University of York, England. A seminar by Professor Warren Ewens in Leeds on the sampling theory of neutral genes had a lasting influence on Tony’s research. After three years, Tony returned to Australia to CSIRO Plant Industry as a research scientist in Canberra in 1972. There, two sons, Laurence and Christopher, were born. At CSIRO, Tony collaborated with Dr. Don Marshall, who had preceded him from UC Davis, and with Dr. Bruce Weir then at Massey, New Zealand. One early project was on the charge-state model of electrophoretic variation, from which a number of experiences flowed. One experience was to have their joint work scooped by Drs. T. Ohta and M. Kimura. On another occasion, a manuscript by Tony and Don was being subjected to the internal CSIRO editorial process and was sent by the panel for review to Professor P. A. P. Moran at Australian National University. Professor Moran was intrigued by the problem and concerned about some aspects of the existence and convergence properties of the distribution. He not only submitted his review, but more important also phoned Tony after hours to discuss this paper. This led Professor Moran to write a series of theoretical papers, and this thinking was referred to by Dr. J. F. C. Kingman in the history of coalescent that he wrote for Genetics in 2000. More influential than the existence of wandering distributions and the electrophoretic profile was Tony’s work on sampling strategies for plant genetic resources. Sir Otto Frankel had challenged Tony and Don


5

to think critically on this subject. Later he promoted their strategy in international meetings and used it to challenge conventional collecting practice, particularly when he thought sampling was excessive. In the field, such theoretical strategies are but a guide, requiring adjustment to reality. This is particularly so for sampling the diversity of wild relatives, where one is deliberately seeking populations in diverse habitats and of greatly varying size. Along with the excitement of discovering variation new to science in its native setting came the experience of diverse human situations. Tony’s first real germplasm collecting mission was an object lesson in adjusting theory to field reality. This was a frenetic mission to Iran with Israeli professors Dani Zohary and Eibi Nevo. The trip went from Mehran near the border with Iraq, across the Zagros Mountains and the southern Caspian shores to Gonbad-e-Qabus, just two years before the 1979 revolution. With portraits of the shah’s family in every hotel room, the future course of events was not evident. At the hotel in Andimesk, the grim faces of the hotel staff were unforgettable as they examined the scientists’ passports. Although the target of the trip was wild cereals, particularly wild barley, the diversity being grown by farmers in the many barley fields was inspiring. This led to a sampling deliberately aimed at testing the allozyme diversity and genetic structure of these landraces, particularly to see whether the richness of diversity so apparent to the eye was just a mixture of a few genotypes. That research ultimately led to Tony’s principal commitment as Honorary Research Fellow with the International Plant Genetic Resources Institute in their project on the significance of crop genetic diversity still present on farms in traditional agroecosystems (with Drs. Toby Hodgkin and Devra Jarvis). The research focus was to develop a scientific basis of the use and conservation in situ of this diversity. When are germplasm collections large enough? This question came to Sir Otto with force after a visit to the excellent large world rice collection developed by Dr. T. T. Chang at the International Rice Research Institute. Few collections would have the resources to attain the size and organization of that for rice. Furthermore the human impulse to collect beyond need would lessen the utility of many collections. Sir Otto therefore proposed that core collections should be set up and called on Tony to read the draft. It was stunning to consider that Otto Frankel, renowned internationally as a champion of crisis germplasm collecting, should now advocate the slashing of collections by an order of magnitude. After much debate, a compromise concept of the ‘‘reserve’’ collection emerged. But for years thereafter Sir Otto often said, ‘‘Tony, I have you to blame for the reserve collection,’’ feeling

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that the reserve concept had dulled his original challenge to gene-bank managers to prune their holdings.

II. RESEARCH ACCOMPLISHMENTS A. Conservation Genetics Sir Otto Frankel was a member of the FAO Expert Panel on Plant Exploration and Introduction, and was preparing for the 1973 FAO/IBP Technical Conference on Crop Plant Genetic Resources in Rome. He felt that previous papers on plant collecting were strongly biased toward the practical details of collecting expeditions and that little emphasis had been given to the science of plant exploration. The original paper presented at the technical conference by Drs. Don Marshall and Tony Brown entitled ‘‘Optimum Sampling Strategies in Genetic Conservation’’ (subsequently published in the book Crop Genetic Resources for Today and Tomorrow edited by O. H. Frankel and J. G. Hawkes) was controversial but has since been widely accepted and expanded to cover other issues, such as sampling in biological control programs. Tony followed his early work on sampling strategies by extensive work on developing the concept of core collections. This concept, first introduced in 1984, was to facilitate the use of genetic resources in the major crops. By the mid-1980s it was felt that many collections, especially in the major crops, had grown so large that their mere size was likely to deter their extensive use by individual scientists, breeders, or students, except for a few characters that could be readily and rapidly discerned on single plants. It was proposed that giving priority in evaluation to a smaller number of accessions would facilitate greater use of germplasm collections, particularly for a range of characters. In a series of papers over the last 20 years, Tony has provided much of the underlying scientific rationale for the establishment and use of core collections. When first introduced, the core collection concept, because it challenged accepted dogma, was controversial, but it now has become widely applied in practice.

B. Plant Mating Systems and Population Structure A key theme spanning Tony’s research career has been the measurement of plant mating systems and their impact on population variation and structure, and the implications of these differences in terms of evolution and the collection and conservation of genetic diversity.


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One of his earliest papers with Professor Allard, which was based on his PhD research, reported the use of isozyme polymorphisms to estimate mating system parameters in open-pollinated maize populations. Over his career Tony developed procedures not only for the estimation of mating system parameters in both predominantly inbreeding and outbreeding populations but for also for apomictic species. Tony also worked with a wide range of colleagues in applying these techniques in species as diverse as Eucalyptus, Lupinus, wild Hordeum, and a number of colonizing weed species (with Drs. Jeremy Burdon and Spencer Barrett). Tony’s work on population structure was focused on genetic polymorphism, heterozygosity, multilocus associations, and population differentiation. A theoretical project with Dr. Marc Feldman, which is enjoying renewed attention with the burgeoning DNA sequence data, dealt with the measuring and testing of multilocus associations. Another example is the analysis of published isozyme data undertaken with Dr. Dan Schoen, which showed that not only do inbreeding and outbreeding species differ in overall levels of genetic diversity, but they also differ in the amount of among population variance of gene diversity. Inbreeding species exhibited much greater variation in how their populations are structured than do the populations of individual outbreeding species. We have to be clear that the comparison is the variability between the populations of one species; that is, populations 1 and 2 of species A, not population 1 of species A with population 1 of species B. Hordeum Research. Another major research theme of Tony’s was the genetic structure of natural populations of the wild progenitor (Hordeum spontaneum) of cultivated barley and the utilization of the variation in the wild species in barley improvement. His interest in wild barley was sparked in part by his experience at Davis and because of an invitation by Professor Nevo in 1976 to spend a six-month sabbatical in the Institute of Evolution, University of Haifa, in Israel. This opened an opportunity for Tony to collect wild barleys in the field and use enzyme markers to understand their genetic structure. The finding of extensive isozyme variation in wild barley raised the issue as to whether this indicated equally high levels of potentially useful variation for barley improvement. Screening of accessions collected in Israel and Iran revealed extensive resistance to barley scald and a range of other diseases and quality traits. Tony developed advanced backcross lines carrying scald resistance from the wild species, which proved to be a useful tool in investigating the genetics of seedling resistance and the tagging and pyramiding of resistance genes.

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This work led to the release of a commercial cultivar (Tantangara) carrying a known scald resistance gene from wild barley. An allied project, also conducted with Dr. Dave Garvin, was the use of molecular markers in breeding adapted proanthocyanidin-free barley. Glycine Research. The legume genus Glycine includes G. max (soybean) and its wild progenitor, G. soja. These annual species are native to northern Asia and so would seem to have little or nothing to do with Australia. Yet, surprisingly, their closest relatives, and the only other members of the genus Glycine, are native to Australia. This group of wild perennial species, Glycine subgenus Glycine, represents the tertiary gene pool for the soybean and is thus of potential economic importance. Collecting and characterizing these perennials has been a major focus of Tony’s work. The potential of this uniquely Australian resource was recognized at CSIRO by Dr. Don Marshall, working initially with Paul Broue` and Jim Grace. Subsequent staff changes led to Tony taking over the program in 1982. At that time there were fewer than 10 species recognized in subgenus Glycine, but that has changed dramatically. In 1982, the International Board of Plant Genetic Resources (IBPGR) held a workshop on soybean genetic resources at Urbana, Illinois, where Tony met those who were already, or would become, among the key figures in soybean diversity research, including Drs. Theodore Hymowitz, Reid Palmer, Randy Nelson, Christine Newell, and Duncan Vaughn. At the time of this workshop, papers on crosses between soybean and perennial Glycine species by the Hymowitz group and by the CSIRO group (Broue` and Marshall) were in draft, and there was tremendous excitement about the potential of the perennials for plant breeding, particularly as sources of drought- and disease-resistance genes. Tony came to the workshop with rough maps of the Australian Glycine distributions, based on existing herbarium records. This led to proposals for germplasm collecting trips to various poorly explored regions of Australia. As a result, IBPGR funded field trips conducted by Tony to north Queensland (1983), the Kimberley district, Western Australia, and Northern Territory (1984), south Queensland (1985), and eastern Victoria (1985). The first two of these trips were co-led by another leading figure in perennial Glycine research, Dr. Theodore Hymowitz (University of Illinois), with whom Tony led two later trips funded by the U.S. Department of Agriculture, to Western Australia (1993) and to the central arid zone of the continent (1996). As a result of these trips, the number of accessions in the CSIRO perennial soybean germplasm collection grew from a few hundred in 1982 to over 2,000 at present.


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It was through Tony’s role as curator of the perennial Glycine seed collection that he began a longtime collaboration with Dr. Jeff Doyle (Cornell University) and his wife, Jane Doyle, when Dr. Doyle contacted CSIRO requesting seed for systematic studies in 1982. Tony’s detailed knowledge of Glycine has guided their collaboration, which has produced numerous papers on the molecular phylogenetics of the subgenus. The chloroplast phylogeny of Glycine corroborated the existence of the genome groups that were based on cytological data amassed by the CSIRO and the University of Illinois groups, and offered the first hypothesis of relationships among these groups of species. The availability of a phylogeny based on defined molecular markers shared among all species also allowed the affinities of newly described species to be determined without recourse to the painstaking studies of chromosome pairing in difficult-to-produce artificial F1 hybrids conducted by Tony and colleagues in the 1980s and by the group at Illinois. Phylogenies based on nuclear markers subsequently showed some incongruence with chloroplast sequences, and some relationships in Glycine remain unresolved. Despite these limitations, molecular systematic approaches have replaced artificial hybridization as the standard method for categorizing new species in the subgenus. The field collections made these studies possible and also enabled traditional taxonomic studies of the genus by other workers, notably Drs. Mary Tindale, Lyn Craven, and Bernard Pfeil. Thanks to all of their efforts and the ongoing work of Dr. Hymowitz and colleagues, the number of species in the subgenus has grown to 25, including 3 new species formally named in 2006. Of the 25, 11 were first found in the field trips led by Tony. Additional species remain to be described in this diverse group. Diploid (2n ¼ 38, 40) members of subgenus Glycine are confined almost exclusively to the Australian continent; one species makes it across the Torresian Strait to the tip of Papua New Guinea. But the subgenus also contains tetraploids (2n ¼ 78–80), some of which are distributed throughout the Pacific, in Timor, the Philippines, New Caledonia, Vanuatu, Taiwan, and the Ryukya Islands. Tony’s work, along with parallel studies by Dr. Hymowitz and his group, has been instrumental in unraveling the origins of these species, pinpointing their diploid progenitors, and elucidating processes such as multiple origins and lineage recombination, by which these allopolyploids have become genetically diverse and geographically widespread. With this wealth of data, and the resources developed by years of collecting, subgenus Glycine has become a significant model for studying the process of allopolyploid evolution in plants. Tony’s dynamic collaborations on Glycine are a stellar example of cooperation

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to solve biological problems and are a tribute to his congenial and collegial personality. In the two and a half decades since Tony assumed responsibility for CSIRO’s perennial Glycine research, he published papers on a number of other topics, including disease resistance, seed size, floral biology, several on population genetics, and the distribution of calcium oxalate crystals in Glycine and allies. And through all of this, Tony drew on his Glycine work as a complement to his studies on Hordeum and other plants, to refine and illustrate his views on germplasm contributions, the area to which he has dedicated himself for many years.

III. THE MAN Dr. Brown has a passion for conservation genetics, from his formative years with sugarcane to the present with Glycine species. His admiration for Otto Frankel, his diligent research at CSIRO, and his affiliation with the International Plant Genetic Resources Institute (now Bioversity International), Rome, Italy (1982–present), are evident in his many contributions. CSIRO Plant Industry as his home base has been an excellent and supportive research environment, where Tony worked jointly with many outstanding colleagues, including Drs. Jeremy Burdon (who is the current chief of Plant Industry), Curt Brubaker, Andrew Young, Jake Jacobsen, and several others. Indeed, these characteristics of Plant Industry owe much to Sir Otto who, as a former chief of division, instilled a vision of excellence in plant research. The discovery of taxa new to science is the unique reward for the collector of wild species related to important crops. Each of his many trips had memorable incidents for Tony, and three are mentioned here. If you happened to be one of the few vehicles driving the remote dirt Peninsula ‘‘highway’’ in Cape York, north Queensland, in July of 1983, you may have seen three collectors (Ted Hymowitz from Illinois and Jim Grace and Tony from CSIRO) sprawled on the lawn outside the Lakeland pub below the billboard saying ‘‘Ice Cold Beer.’’ This was no early knock off; they actually were sampling rare, tiny Glycine tomentella plants. The billboard had nothing to do with site selection; a collector must check all habitats. The roadside pub, a lone building in the rural landscape, was a haven for the thirsty traveler, and it surrounds a haven for wild plants that grazing animals would otherwise decimate. Thus, sampling strategies for germplasm collection adapt to reality. Day’s end of another trip found Tony with U.S. soybean breeder Dr. Bill Kenworthy, graduate student Michael Doyle, and Jim Grace in


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the Carnarvon Gorge of central Queensland, which proved a special site, rich in diverse new taxa. Excitement died, however, when halfway along the 150-km return trip, it became clear there was insufficient fuel to reach home at Injune. There followed a long silent drive, meeting no other vehicles. Despite eking out the last drop of fuel, the vehicle slowed to a stop 15 km north of their destination. Tony and Jim remained with the vehicle; Bill and Michael chose to jog and walk to town for help, guided by moonlight and the smell of road-killed cattle and kangaroos. During the long wait at the vehicle, the silent darkness was broken by another vehicle, the first sighted since Carnarvon and, luckily, approaching the road to town, from the property right where the vehicle had stopped. When they apprised the driver of the pickup of their situation, he pointed to the rifle hung above his rear window and replied in a rural Texas accent, ‘‘Just as well you told me. If I’d been forced to stop on the road in the dark by two desperados on foot, looking for a ride to town, I’d answer with this.’’ Meeting the wildlife is a feature of any field trip in Australia. A trip to collect wild Australian Gossypium species, and to evaluate the risks to them of GM cotton, with botanist Professor Herbert Hurka from Osnabrueck, Germany, brought them to a remote Corona farm 70 km north of Broken Hill, western NSW. Herbert was intrigued by the caged talking sulphur-crested cockatoo. The farmer’s wife had warned them that the bird had lived in a hotel in the ‘‘silver city’’ but was banished because of bad language. Clearly the garrulous bird enjoyed the attention of the team of rare visitors, and Herbert lingered to converse with it while the CSIRO team sampled. When he turned to leave, the bird had a fail-safe method to retrieve attention. To the visiting professor, it screeched ‘‘A***hole’’—a fully effective way to grab Herbert’s notice. Sir Otto Frankel was one of the major influences of Tony’s science and life. His unyielding insistence on high standards and exactness led to many legendary stories. Memorable for Tony was a Christmas Eve lunch at which Tony hosted Sir Otto and Lady Margaret, along with Professor Herbert and Ute Hurka and family members. At one point, Tony introduced a wine he was particularly enjoying, and asked who would like some of this excellent Orlando chardonnay, Otto’s response was immediate and emphatic: ‘‘That wine is good, but is certainly NOT excellent!!’’ Silence fell; then he asked, ‘‘Which vintage?’’ Stunned, Tony checked the label and replied, ‘‘1988.’’ Back came the riposte: ‘‘1987 is better!!’’ Those quips have often proved useful, not only when recalling Otto’s outspokenness. Elements of Tony’s migratory career and strategic sampling interests are reflected in the lives of his two sons. Currently, Laurence is a lecturer

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in migration history in the Department of History, University of Manchester, UK; and Chris is an investment banker with the Mergers and Acquisitions Section of UBS, New York, USA. Tony has a new younger family of three stepchildren who are themselves embarking on diverse careers.

IV. HONORS AND AWARDS Dr. Brown has been extensively recognized for his contributions and achievements to conservation genetics. To further broaden his expertise, Tony has been a visiting professor at Stanford University and the University of California, Riverside, visiting research fellow at Haifa University, Israel, and a visiting scientist at the Universitaet Osnabrueck, Germany. Tony has excelled in his editorial duties for the journals Genetics, Molecular Biology and Evolution, and Conservation Genetics as well as serving as editor or coeditor of 10 books, and conference and symposia proceedings. Of the eight plant collecting missions, Tony has been leader or coleader of six in Australia, one in Israel, and one in Iran. Tony has been the International Plant Genetic Resources Institute (IPGRI) technical advisor and on the organizing committee of 18 international workshops in 10 different countries. Perhaps the most rewarding honor was the award as Honorary Research Fellow by the IPGRI, Rome, Italy. The initial award was in 1997 and Tony has been reappointed three times, most recently with Bioversity International, IPGRI’s new name.

ACKNOWLEDGMENT The authors thank Dr. Don Marshall of Plant Breeding Solutions Pty. Ltd., Hamilton, NSW, Australia, for his contributions to the text and for his critical review of this chapter.

SELECTED PUBLICATIONS OF ANTHONY H. D. BROWN Brown, A.H.D., J. Daniels, and B.D.H. Latter. 1968. Quantitative genetics of sugarcane. I. Analysis of variation in a commercial hybrid sugarcane population. Theor. Appl. Genet. 38:361–369. Brown, A.H.D. and R.W. Allard. 1969. Inheritance of isozyme differences among the inbred parents of a reciprocal recurrent selection population of maize. Crop Sci. 9:72–75.


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Brown, A.H.D. 1970. The estimation of Wright’s fixation index from genotypic frequencies. Genetica 41:399–406. Brown, A.H.D. and R.W. Allard. 1970. Estimation of the mating system in open–pollinated maize populations using isozyme polymorphisms. Genetics 66:133–145. Brown, A.H.D. 1971. Isozyme variation under selection in Zea mays. Nature 232:570. Brown, A.H.D. and R.W. Allard 1971. Effect of reciprocal recurrent selection for yield on isozyme polymorphisms in maize (Zea mays L.). Crop Sci. 11:888–893. Marshall, D.R. and A.H.D. Brown. 1973. Stability of performance of mixtures and multilines. Euphytica 22:405–412. Brown, A.H.D., D.R. Marshall, and L. Albrecht. 1974. The maintenance of alcohol dehydrogenase polymorphism in Bromus mollis L. Aust. J. Biol. Sci. 27:545–559. Marshall, D.R. and A.H.D. Brown. 1974. Estimation of the level of apomixis in plant populations. Hered. 32:321–333. Brown, A.H.D., A.C. Matheson, and K.G. Eldridge. 1975. Estimation of the mating system of Eucalyptus obliqua L. Herit using allozyme polymorphisms. Aust. J. Bot. 23:931–949. Brown, A.H.D. 1975. Efficient experimental designs for the estimation of genetic parameters in plant populations. Biometrics 31:145–160. Brown, A.H.D. 1975. Sample sizes required to detect linkage disequilibrium between two or three loci. Theor. Pop. Biol. 8:184–210. Brown, A.H.D., D.R. Marshall, and L. Albrecht. 1975. Profiles of electrophoretic alleles in natural populations. Genet. Res. Camb. 25:137–143. Brown, A.H.D., D.R. Marshall, and B.S. Weir. 1975. Population differentiation under the charge state model. Genetics 81:739–748. Marshall, D.R. and A.H.D. Brown. 1975. The charge state model of protein polymorphism in natural populations. J. Molec. Evol. 6:149–163. Marshall, D.R. and A.H.D. Brown. 1975. Optimum sampling strategies in genetic conservation. pp. 53–80. In: O.H. Frankel and J.G. Hawkes (eds.), I.B. P.2. Crop Genetic Resources for Today and Tomorrow. Cambridge Univ. Press, Cambridge. Brown, A.H.D., D.R. Marshall, and J. Munday. 1976. The adaptedness of variants at an alcohol dehydrogenase locus in Bromus mollis L. (Soft Bromegrass). Aust. J. Biol. Sci. 29:389–396. Weir, B.S., A.H.D. Brown, and D.R. Marshall. 1976. Testing for selective neutrality of electrophoretically detectable protein polymorphisms. Genetics 84:639–659. Brown, A.H.D., E. Nevo, and D. Zohary. 1977. Association of alleles at esterase loci in wild barley Hordeum spontaneum. Nature 268:430–431. Brown, A.H.D. 1978. Isozymes, plant population genetic structure and genetic conservation. Theor. Appl. Genet. 52:145–157. Brown, A.H.D., E. Nevo, D. Zohary, and O. Dagan. 1978. Genetic variation in natural populations of wild barley (Hordeum spontaneum). Genetica 49:97–108. Brown, A.H.D., D. Zohary, and E. Nevo. 1978. Outcrossing rates and heterozygosity in natural populations of Hordeum spontaneum Koch in Israel. Hered. 41:49–62. Brown, A.H.D. 1979. Enzyme polymorphism in plant population. Theor. Pop. Biol. 15:1–42. Nevo, E., D. Zohary, A.H.D. Brown, and M. Haber. 1979. Genetic diversity and environmental associations of wild barley, Hordeum spontaneum, in Israel. Evolution 33:815–833. Doll, H. and A.H.D. Brown. 1979. Hordein variation in wild (Hordeum spontaneum) and cultivated (H. vulgare) barley. Can. J. Genet. Cytol. 21:391–404. Brown, A.H.D. 1980. Genetic basis of alcohol dehydrogenase polymorphism in Hordeum spontaneum. J. Hered. 70:127–128.

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Brown, A.H.D. and L. Albrecht. 1980. Variable outcrossing and the genetic structure and predominantly self-pollinated species. J. Theor. Biol. 82:591–606. Brown, A.H.D., M.W. Feldman, and E. Nevo. 1980. Multilocus structure of natural populations of Hordeum spontaneum. Genetics 96:523–536. Corrigendum May 1981, p. 238A. Green, A.G., A.H.D. Brown, and R.N. Oram. 1980. Determination of outcrossing rate in a breeding population of Lupinus albus L. (White Lupin). Z. Pflanzenzuchtg. 84:181–191. Brown, A.H.D. and M.W. Feldman. 1981. Population structure of multilocus associations. Proc. Natl. Acad. Sci. U.S. 78:5913–5916. Brown, A.H.D. and D.R. Marshall. 1981. Evolutionary changes accompanying colonization in plants. pp. 351–363. In: G.G.E. Scudder and J.L. Reveal (eds.), Evolution Today, Proc. Second Int. Congr. Syst. and Evol. Biol. Univ. British Columbia, Vancouver. Hunt Institute for Botanical Documentation, Pittsburgh. Marshall, D.R. and A.H.D. Brown. 1981. The evolution of apomixis. Hered. 47:1–15. Marshall, D.R. and A.H.D. Brown. 1981. Wheat genetic resources. pp. 21–40. In: W. J. Peacock and L.T. Evans (eds.), Wheat Science, Today and Tomorrow. Cambridge Univ. Press, Cambridge. Brown, A.H.D. and J.V. Jacobsen. 1982. Genetic basis and natural variation of alpha– amylase isozymes in barley. Genet. Res. Camb. 40:315–324. Brown, A.H.D. and J. Munday. 1982. Population genetic structure and optimal sampling of land races of barley from Iran. Genetica 58:85–96. Erratum 60:237. Nevo, E., E. Golenberg, A. Beiles, A.H.D. Brown, and D. Zohary. 1982. Genetic diversity of environmental associations of wild wheat, Triticum dicoccoides in Israel. Theor. Appl. Genet. 62:241–254. Brown, A.H.D. 1983. Barley, pp. 57–77. In: S.D. Tanksley, and T.J. Orton (eds.), Isozymes in plant genetics and breeding, Part B. Elsevier, Amsterdam. Brown, A.H.D. and J.J. Burdon. 1983. Multilocus diversity in an outbreeding weed, Echium plantagineum L. Aust. J. Biol. Sci. 36:503–509. Brown, A.H.D. and M.T. Clegg. 1983. Analysis of variation in related DNA sequences. pp. 107– 132. In: B.S. Weir (ed.), Statistical analysis of DNA sequence data. Marcel Dekker, New York. Brown, A.H.D. and B.S. Weir. 1983. Measuring genetic variability in plant populations. pp. 219–239. In: S.D. Tanksley. and T.J. Orton (eds.), Isozymes in plant genetics and breeding, Part A. Elsevier, Amsterdam. Burdon, J.J., D.R. Marshall, and A.H.D. Brown. 1983. Demographic and genetic changes in populations of Echium plantagineum L. J. Ecology 71:667–679. Brown, A.H.D. 1984. Multilocus organization of plant populations. pp. 159–169. In: K. Wohrmann and V. Loeschcke (eds.), Population biology and evolution. Springer Verlag, Berlin. Clegg, M.T., A.H.D. Brown, and P.R. Whitfeld. 1984. Chloroplast DNA diversity in wild and cultivated barley: Implications for genetic conservation. Genet. Res. Camb. 43: 339–343. Hanson, A.D. and A.H.D. Brown. 1984. Three alcohol dehydrogenase genes in wild and cultivated barley: characterization of the products of variant alleles. Biochem. Genet. 22:495–515. Grant, J.E., A.H.D. Brown, and J.P. Grace. 1984. Cytological and isozyme diversity in Glycine tomentella Hayata (Leguminosae). Aust. J. Bot. 32:665–677. Grant, J.E., J.P. Grace, A.H.D. Brown, and E. Putievsky. 1984. Interspecific hybridization in Glycine subgenus Glycine Willd. (Leguminosae). Aust. J. Bot. 32:655–663. Schroeder, H.E. and A.H.D. Brown. 1984. Inheritance of legumin and albumin contents in a cross between round and wrinkled peas. Theor. Appl. Genet. 68:101–107. Zurawski, G., M.T. Clegg, and A.H.D. Brown. 1984. The nature of nucleotide sequence divergence between barley and maize chloroplast DNA. Genetics 106:735–749.


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Brown, A.H.D., J.E. Grant, J.J. Burdon, J.P. Grace, and R. Pullen. 1985. Collection and utilization of wild perennial Glycine. pp. 345–352. In: R. Shibles (ed.), Proc. World Soybean Research Conference III. Westview Press, Boulder, Colorado. Doyle, M.J. and A.H.D. Brown. 1985. Numerical analysis of isozyme variation in Glycine tomentella. Biochem. Syst. Ecol. 13:413–419. Brown, A.H.D., J.E. Grant, and R. Pullen. 1986. Outcrossing and paternity in Glycine argyrea by paired fruit analysis. Biol. J. Linn. Soc. 29:283–294. Burdon, J.J. and A.H.D. Brown. 1986. Population genetics of Echium plantagineum L.—a target weed for biological control. Aust. J. Biol. Sci. 39:369–378. Doyle, M.J., J.E. Grant, and A.H.D. Brown. 1986. Reproductive isolation between isozyme groups of Glycine tomentella (Leguminosae), and spontaneous doubling in their hybrids. Aust. J. Bot. 34:523–535. Grant, J.E., R. Pullen, A.H.D. Brown, J.P. Grace, and P.M. Gresshof. 1986. Cytogenetic affinity between the new species Glycine argyrea and its congeners. J. Hered. 77: 423–426. Brown, A.H.D. and J.J. Burdon. 1987. Mating systems and colonizing success in plants. pp. 115–131. In: A.J. Gray, M.J. Crawley, and P.J. Edwards (eds.), Colonization, succession and stability. 26th Symposium of British Ecol. Soc. Blackwell Scientific, Oxford. Henry, R.J. and A.H.D. Brown. 1987. Variation in the carbohydrate composition of wild barley (Hordeum spontaneum) grain. Z. Pflanzenzu¨chtung 98:97–103. Hoffman, N.E., D. Hondred, A.D. Hanson, and A.H.D. Brown. 1988. Lactate dehydrogenase isozymes in barley: Polymorphism and genetic basis. J. Hered. 79:110–114. Brown, A.H.D., J. Munday, and R.N. Oram. 1988. Use of isozyme-marked segments from wild barley (Hordeum spontaneum) in barley breeding. Plant Breed. 100:280–288. Brown, A.H.D. 1989. The case for core collections. pp. 136–156. In: A.H.D. Brown, O.H. Frankel, D.R. Marshall, and T. Williams (eds.), The use of plant genetic resources. Cambridge Univ. Press, Cambridge. Brown, A.H.D. 1989. Core collections: A practical approach to genetic resources management. Genome 31:818–824. Brown, A.H.D. 1989. Genetic characterization of plant mating systems. pp. 145–162. In: A.H.D. Brown, M.T. Clegg, A.L. Kahler, and B.S. Weir (eds.), Plant population genetics, breeding and genetic resources. Sinaeuer Associates, Sunderland, Massachusetts. Brown, A.H.D., J.J. Burdon, and A.M. Jarosz. 1989. Isozyme analysis of plant mating systems. pp. 73–86. In: D. Soltis and P. Soltis (eds.), Isozymes in plant biology. Dioscorides Press, Portland, Oregon. Brown, A.H.D., G.J. Lawrence, M. Jenkin, J. Douglass, and E. Gregory. 1989. Linkage drag in backcross breeding. J. Hered. 80:234–239. Doyle, J.J. and A.H.D. Brown. 1989. 5S nuclear ribosomal gene variation in the Glycine tomentella polyploid complex. Syst. Bot. 14:398–407. Hurka, H., S. Freunder, A.H.D. Brown, and U. Plantholt. 1989. Aspartate amino transferase isozymes in the genus Capsella (Brassicaceae): Subcellular location, gene duplication and polymorphism. Biochem. Genetics 27:77–90. Kenworthy, W.J., A.H.D. Brown, and G.A. Thibou. 1989. Variation in flowering response to photoperiod in perennial Glycine species. Crop Sci. 29:678–682. Brown, A.H.D. 1990. The role of isozyme studies in molecular systematics. Aust. Syst. Bot. 3:39–46. Brown, A.H.D., J.J. Burdon and J.P. Grace. 1990. Genetic structure of Glycine canescens, a perennial relative of soybean. Theor. Appl. Genet. 79:729–736. Doyle, J.J., J.L. Doyle, and A.H.D. Brown. 1990. Analysis of a polyploid complex in Glycine with chloroplast and nuclear DNA. Aust. Syst. Bot. 3:125–136.

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Doyle, J.J., J.L. Doyle, and A.H.D. Brown. 1990. A chloroplast DNA phylogeny of the wild perennial relatives of soybean (Glycine subgenus Glycine): Congruence with morphological and crossing groups. Evolution 44:371–389. Doyle, J.J., J.L. Doyle, and A.H.D. Brown. 1990. Chloroplast DNA phylogenetic affinities of newly described species in Glycine (Leguminosae: Phaseoleae). Syst. Bot. 15:466–471. Doyle, J.J., J.L. Doyle, and A.H.D. Brown. 1990. Chloroplast DNA polymorphism and phylogeny in the B genome of Glycine subgenus Glycine (Leguminosae). Amer. J. Botany 77:772–782. Doyle, J.J., J.L. Doyle, A.H.D. Brown, and J.P. Grace. 1990. Multiple origins of polyploids in the Glycine tabacina complex inferred from chloroplast DNA polymorphism. Proc. Natl. Acad. Sci. USA 87:714–717. Doyle, J.J., J.L. Doyle, J.P. Grace, and A.H.D. Brown. 1990. Reproductively isolated polyploid races of Glycine tabacina (Leguminosae) had different chloroplast genome donors. Syst. Bot. 15:173–181. Feuerstein, U., A.H.D. Brown, and J.J. Burdon. 1990. Linkage of rust resistance genes from wild barley (Hordeum spontaneum) with isozyme markers. Plant Breed. 104:318–324. Schoen, D.J. and A.H.D. Brown. 1991. Intraspecific variation in population gene diversity and effective population size correlates with the mating system in plants. Proc. Natl. Acad. Sci. USA 88:4494–4497. Abbott, D.C., J.J. Burdon, A.M. Jarosz, A.H.D. Brown, W.J. Muller, and B.J. Read. 1991. The relationship between seedling infection types and field reactions to leaf scald in Clipper barley backcross lines. Aust. J. Agric. Res. 42:801–809. Brown, A.H.D. and J.D. Briggs. 1991. Sampling strategies for genetic variation in ex situ collections of endangered plant species. pp. 99–119. In: D.A. Falk and K.E. Holsinger (eds.), Genetics and Conservation of Rare Plants. Oxford Univ. Press, Oxford. Lagudah, E.S., R. Appels, A.H.D. Brown, and D. McNeil. 1991. The molecular-genetic analysis of Triticum tauschii, the D-genome donor to hexaploid wheat. Genome 34: 375–386. MacLeod, L.C., R.C.M. Lance, and A.H.D. Brown. 1991. Chromosomal mapping of the Glb 1 locus encoding (1!3), (1!4)–ß–D–glucan 4–glucanohydrolase EI in barley. J. Cereal Sci. 13:291–298. Schoen, D.J. and A.H.D. Brown. 1991. Whole and part-flower self-pollination in Glycine clandestina and G. argyrea and the evolution of autogamy. Evolution 45:1651–1664. Abbott, D.C., A.H.D. Brown, and J.J. Burdon. 1992. Genes for scald resistance from wild barley (Hordeum vulgare ssp spontaneum) and their linkage to isozyme markers. Euphytica 61:225–231. Brown, A.H.D. 1992. Genetic variation and resources in cultivated barley and wild Hordeum. Barley Genetics 6:669–682. Brown, A.H.D. 1992. Human impact on plant gene pools and sampling for their conservation. Oikos. 63:109–118. Schoen, D.J., J.J. Burdon, and A.H.D. Brown. 1992. Resistance of Glycine tomentella to soybean leaf rust Phakopsora pachyrhizi in relation to ploidy level and geographic distribution. Theor. Appl. Genet. 83:827–832. Schoen, D.J. and A.H.D. Brown. 1993. Conservation of allelic richness in wild crop relatives is aided by assessment of genetic markers. Proc. Natl. Acad. Sci. USA 90:10623–10627. Brown, A.H.D. and D.J. Schoen. 1994. A revised measure of association of gene diversity values. Hereditas 120:77–79. Burdon, J.J., D.C. Abbott, A.H.D. Brown, and J.S. Brown. 1994. Genetic structure of the scald pathogen (Rhynchosporium secalis) in South East Australia: Implications for control strategies. Aust. J. Agric. Res. 45:1445–1454.


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Guerin, J.R., R.C.M. Lance, A.H.D. Brown, and D.C. Abbott. 1994. Mapping of malt endopeptidase, diaphorase and esterase loci on barley chromosome 3L. Plant Breed. 112:279–284. Prober, S.M. and A.H.D. Brown. 1994. Conservation of the grassy white box woodlands. I. Population genetics and fragmentation of Eucalyptus albens Benth. Conservation Biol. 8:1003–1013. Abbott, D.C., E.S. Lagudah, and A.H.D. Brown. 1995. Identification of RFLPs flanking a scald resistance gene on barley chromosome 6. J. Hered. 86:152–154. Brown, A.H.D. 1995. The core collection at the crossroads. pp. 3–19. In: T. Hodgkin, A.H.D. Brown, T.J.L. van Hintum, and E.A.V. Morales (eds.), Core collections of plant genetic resources. John Wiley, Chichester. Brown, A.H.D. and D.R. Marshall. 1995. A basic sampling strategy: Theory and practice. pp. 75–91. In: L. Guarino, V. Ramanatha Rao, and R. Reid (eds.), Collecting plant genetic diversity technical guidelines. CAB International, Wallingford. Frankel, O.H., A.H.D. Brown, and J.J. Burdon. 1995. The conservation of plant biodiversity. Cambridge Univ. Press, Cambridge. Brown, A.H.D., D.F. Garvin, J.J. Burdon, D.C. Abbott, and B.J. Read. 1996. The effect of combining scald resistance genes on disease levels, yield and quality traits in barley. Theor. Appl. Genet. 93:361–366. Young, A., G.T. Boyle, and A.H.D. Brown. 1996. The population genetic consequences of habitat fragmentation for plants. Trends in Ecology and Evolution 11:413–418. Young, A.G. and A.H.D. Brown. 1996. Comparative population genetic structure on the rare woodland shrub Daviesia suaveolens and its common congener D. mimosoides. Conservation Biol. 10:1220–1228. Brown, A.H.D., C.L. Brubaker, and J.P. Grace. 1997. The regeneration of germplasm samples: Wild versus cultivated species. Crop Sci. 37:7–13. Brown, A.H.D., C.L. Brubaker, and M.J. Kilby. 1997. Assessing the risk of cotton transgene escape into wild Australian Gossypium species. pp. 83–94. In: G.D. McLean, P.M. Waterhouse, G. Evans, and M.I. Gibbs (eds.), The commercialisation of transgenic crops: Risk, benefit and trade considerations. Bureau of Resource Sciences, Kingston, ACT, Australia. Garvin, D.F., A.H.D. Brown, and J.J. Burdon. 1997. Inheritance and chromosome locations of novel scald resistance genes derived from Iranian and Turkish wild barleys. Theor. Appl. Genet. 94:1087–1091. Roulin, S., P. Xu, A.H.D. Brown, and G.B. Fincher. 1997. Expression of specific (1!3)-bGlucanase genes in leaves of near-isogenic resistant and susceptible barley lines infected with the leaf scald fungus (Rhynchosporium secalis). Phys. Mol. Plant Path. 50:245–261. Garvin, D.F., J.E. Miller-Garvin, E.A. Viccars, J.V. Jacobsen, and A.H.D. Brown. 1998. Identification of molecular markers linked to ant28, a mutation that eliminates proanthocyanidin in barley seeds. Crop Sci. 38:1250–1255. Prober, S.M., L.H. Spindler, and A.H.D. Brown. 1998. Conservation of the grassy white box woodlands: Effects of remnant population size on genetic diversity of the outcrossing, allotetraploid herb, Microseris lanceolata. Conservation Biol. 12:1279–1290. Young, A.G. and A.H.D. Brown. 1998. Comparative analysis of mating systems in the rare woodland shrub Daviesia suaveolens and its congener D. mimosoides. Hered. 80: 374–381. Brown, A.H.D. 1999. The genetic structure of crop landraces and the challenge to conserve them in situ on farms. pp. 29–48. In: S.B. Brush (ed.), Genes in the field: Conserving plant diversity on farms. Lewis Publishers, Boca Raton, FL. Brubaker, C.L., A.H.D. Brown, J. McD. McStewart, M.J. Kilby, and J.P. Grace. 1999. Production of fertile hybrid germplasm with diploid Australian Gossypium species for cotton improvement. Euphytica 108:199–213.

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Burdon, J.J.P.H. Thrall, and A.H.D. Brown. 1999. Resistance and virulence structure in two Linum marginale—Melampsora lini host-pathogen metapopulations with different mating systems. Evolution 53:704–716. Doyle, J.J., J.L. Doyle, and A.H.D. Brown. 1999. Incongruence in the diploid B-genome species complex of Glycine (Leguminosae) revisited: Histone H3-D alleles vs. chloroplast haplotypes. Molec. Biol. Evol. 16:354–362. Doyle, J.J., J.L. Doyle, and A.H.D. Brown. 1999. Origins, colonization, and lineage recombination in a widespread perennial soybean polyploid complex. Proc. Nat. Acad. Sci. USA 96:10741–10745. Marshall, D.R. and A.H.D. Brown. 1999. Sampling wild legume populations. pp. 78–89. In: S.J. Bennett and P.S. Cocks (eds), Genetic resources of Mediterranean pasture and forage legumes. Kluwer Acad. Press, Dordrecht. Young, A.G., A.H.D. Brown, and F.C. Zich. 1999. Genetic structure of fragmented populations of the endangered grassland daisy Rutidosis leptorrhynchoides. Conservation Biol. 13:256–265. Young, A.G. and A.H.D. Brown. 1999. Paternal bottlenecks in fragmented populations of the grassland daisy Rutidosis leptorrhynchoides. Genet. Res. 73:111–117. Abbott, D.C., J.J. Burdon, A.H.D. Brown, B.J. Read, and D. Bittisnich. 2000. The incidence of barley scald in cultivar mixtures. Aust. J. Agric. Res. 51:355–360. Brown, A.H.D. and C.L. Brubaker. 2000. Genetics and the conservation and use of Australian wild relatives of crops. Aust. J. Bot. 48:297–303. Brown, A.H.D. and C.M. Hardner. 2000. Sampling the gene pools of forest trees for ex situ conservation. pp. 185–196. In: A. Young, T. Boyle, and D. Boshier (eds.), Forest conservation genetics: Principles and practice. CSIRO, Melbourne, Australia. Brown, A.H.D. and A.G. Young. 2000. Genetic diversity in tetraploid populations of the endangered daisy Rutidosis leptorrhynchoides and implications for its conservation. Hered. 85:122–129. Doyle, J.J., J.L. Doyle, A.H.D. Brown, and B.L. Pfeil. 2000. Confirmation of shared and divergent genomes in the Glycine tabacina polyploid complex (Leguminosae) using histone H3-D sequences. Syst. Bot. 25:437–448. Garvin, D.F., A.H.D. Brown, H. Raman, and B.J. Read. 2000. Genetic mapping of the barley Rrs14 scald resistance gene with RFLP, isozyme and seed storage protein markers. Plant Breed. 119:193–196. Brown, A.H.D. and C.L. Brubaker. 2001. Indicators for sustainable management of plant genetic resources—how well are we doing? pp. 249–262. In: J.M.M. Engels, V. Ramanatha Rao, A.H.D. Brown, and M T. Jackson (eds.), Managing plant genetic diversity. CAB International, Wallingford, Oxon, UK. Lin, J.-Z., A.H.D. Brown, and M.T. Clegg. 2001. Heterogeneous geographic patterns of nucleotide sequence diversity between two alcohol dehydrogenase genes in wild barley (Hordeum vulgare ssp. spontaneum). Proc. Nat. Acad. Sci. USA 98:531–536. Teshome, A., A.H.D. Brown, and T. Hodgkin. 2001. Diversity in landraces of cereal and legume crops. Plant Breed. Rev. 21:221–261. Brown, A.H.D., J.L. Doyle, J.P. Grace, and J.J. Doyle. 2002. Molecular phylogenetic relationships within and among diploid races of Glycine tomentella (Leguminosae). Aust. Syst. Bot. 15:37–47. Bleeker, W., A. Franzke, K. Pollman, A.H.D. Brown, and H. Hurka. 2002. Phylogeny and biogeography of southern hemisphere high mountain Cardamine species (Brassicaceae). Aust. Syst. Bot. 15:575–581.


19

Doyle, J.J., J.L. Doyle, A.H.D. Brown, and R.G. Palmer. 2002. Genomes, multiple origins, and lineage recombination in the Glycine tomentella (Leguminosae) polyploid complex: histone H3-D gene sequences. Evolution 56:1388–1402. Rauscher, J.T., J.J. Doyle, and A.H.D. Brown. 2002. Internal transcribed spacer repeat– specific primers and the analysis of hybridization in the Glycine tomentella (Leguminosae) polyploid complex. Molec. Ecol. 11:2691–2702. Brubaker, C.L. and A.H.D. Brown. 2003. The use of multiple alien chromosome addition aneuploids facilitates genetic linkage mapping of the Gossypium G genome. Genome 46:774–791. Genger, R.K., A.H.D. Brown, W. Knogge, K. Nesbitt, and J.J. Burdon. 2003. Development of SCAR markers linked to a scald resistance gene derived from wild barley. Euphytica 134:149–159. Genger, R.K., K.J. Williams, H. Raman, B.J. Read, H. Wallwork, J.J. Burdon, and A.H.D. Brown. 2003. Leaf scald resistance genes in Hordeum vulgare and Hordeum vulgare ssp. spontaneum: parallels between cultivated and wild barley. Aust. J. Agric. Res. 54:1335–1342. Doyle, J.J., J.L. Doyle, J.T. Rauscher, and A.H.D. Brown. 2003. Diploid and polyploidy reticulate evolution throughout the history of the perennial soybeans (Glycine subg. Glycine). New Phytologist 161:121–132. Murray, B.R., A.H.D. Brown, and J.P. Grace. 2003. Geographic gradients in seed size among and within perennial Australian Glycine species. Aust. J. Bot. 51:47–56. Rau, D., A.H.D. Brown, C.L. Brubaker, G. Attene, V. Balmas, E. Saba, and R. Papa. 2003. Population genetic structure of Pyrenophora teres Drechs., the causal agent of net blotch in Sardinian landraces of barley complex (Hordeum vulgare L.). Theor. Appl. Genet. 106:947–959. Doyle, J.J., J.L. Doyle, J.T. Rauscher, and A.H.D. Brown. 2004. Evolution of the perennial soybean polyploid (Glycine subgenus Glycine): A study of contrasts. Biol. J. Linnean Soc. 82:583–597. Joly, S., J.T. Rauscher, S.L. Sherman-Broyles, A.H.D. Brown, and J.J. Doyle. 2004. Evolution of the 18S-5.8S-26S nuclear ribosomal gene family and its expression in natural and artificial Glycine allopolyploids. Molec. Biol. Evol. 21:1409–1421. Murray, B.R., A.H.D. Brown, C.R. Dickman, and M.S. Crowther. 2004. Geographical gradients in seed mass in relation to climate.J. Biogeography 31:379–388. Rauscher, J.T., J.J. Doyle, and A.H.D. Brown. 2004. Multiple origins and nrDNA internal transcribed spacer homoeologue evolution in the Glycine tomentella (Leguminosae) allopolyploid complex. Genetics 166:987–998. Cervantes-Martinez, T., H.T. Horner, R.G. Palmer, T. Hymowitz, and A.H.D. Brown. 2005. Calcium oxalate crystal macropatterns in leaves of species from groups Glycine and Shuteria (Glycininae; Phaseoleae; Papilionoideae; Fabaceae). Can. J. Bot. 83:1410–1421 Genger, R.K., K. Nesbitt, A.H.D. Brown, D.C. Abbott, and J.J. Burdon. 2005. A novel barley scald resistance locus: Genetic mapping of the Rrs15 scald resistance gene derived from wild barley, Hordeum vulgare ssp. spontaneum. Plant Breed. 124: 137–141. Rau, D., F.J. Maier, R. Papa, A.H.D. Brown, V. Balmas, E. Saba, W. Shafer, and G. Attene, 2005. Isolation and characterization of the mating-type locus of the barley pathogen Pyrenophora teres frequencies of mating-type idiomorphs within and among fungal populations collected from barley landraces. Genome 48:855–869. Pfeil, B.E., L.A. Craven, A.H.D. Brown, B.G. Murray, and J.J. Doyle. 2006. Three new species of northern Australian Glycine (Fabaceae, Phaseolae) G-gracei, G. montis-dougle and G. syndetika. Aust. Syst. Bot. 19:245–258.

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Brown, A.H.D. and T. Hodgkin. 2007. Measuring, managing and maintaining crop genetic diversity on-farm. pp. 13–33. In: D. Jarvis, C. Paddoch, and D. Williams (eds.), Managing biodiversity in agricultural ecosystems. Columbia University Press, New York. Jarvis, D.I., A.H.D. Brown, V.I. Imbruce, J. Ochoa, M. Sadiki, E. Karamura, P. Trutmann, and M.R. Finckh. 2007. Managing crop disease in traditional agroecosystems: The benefits and hazards of genetic diversity. pp. 292–319. In: D. Jarvis, C. Paddoch, and D. Williams (eds.), Managing biodiversity in agricultural ecosystems. Columbia University Press, New York. Triono, T., M.D. Crisp, A.H.D, Brown, and J.G. West. 2007. A phylogency of Pouteria (Sapotaceae) from Malesia and Australasia. Aust. Syst. Bot. 20:107–118. Rau, D., G. Attene, A.H.D. Brown, L. Nanni, F.J. Maier, V. Balmas, E. Saba, W. Schaefer, and R. Papa. 2007. Phylogeny and evolution of mating-type genes from Pyrenophora teres, the causal agent of barley ‘‘net blotch’’ disease. Current Genetics 51:377–392. Jarvis, D.I., A.H.D. Brown, et al. 2008. A global perspective of the richness and evenness of traditional crop-variety diversity maintained by farming communities. Proc. Nat. Acad. Sci. USA 105:5326–5331.

2 Brassica and Its Close Allies: Cytogenetics and Evolution Shyam Prakash National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012 India S. R. Bhat National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012 India C. F. Quiros Department of Vegetable Crops University of California Davis, California 95616 USA P. B. Kirti Plant Science Department University of Hyderabad Hyderabad, 500046 India V. L. Chopra National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012 India

I. INTRODUCTION II. CYTOGENETICS A. Cytogenetic Architecture of Brassica Coenospecies B. Crop Species


22

III.

IV.

V. VI. VII.

VIII. IX.

X.

S. PRAKASH, S. R. BHAT, C. F. QUIROS, P. B. KIRTI, AND V. L. CHOPRA 1. Nature of Diploid Species 2. Nature of Alloploid Species 3. Nuclear DNA 4. Karyotypes 5. Pachytene Chromosomes 6. Satellite Chromosomes and rDNA Loci 7. Archetype and Evolution of Genomes GENOME MANIPULATION A. Resyntheses of Natural Allopolyploid Brassica spp. B. Agronomic Potential of Synthetics C. Diploidization of Allopolyploid Species D. Raphanobrassica E. Higher Allopolyploids in U Triangle Species through Protoplast Fusion WIDE HYBRIDIZATION A. Sexual Hybrids B. Somatic Hybrids C. Introgression of Genes CYTOPLASMIC SUBSTITUTION AND MALE STERILITY GENOME DISSECTION AND DEVELOPMENT OF CHROMOSOME ADDITION LINES MITOCHONDRIAL GENOME A. Organization B. Gene Content C. Mitochondrial Plasmids PLASTID GENOME POTENTIAL ROLE OF ARABIDOPSIS THALIANA IN BRASSICA IMPROVEMENT A. A. thaliana as a Model Crucifer B. Cytology and Possible Origin of the A. thaliana Genome C. Synteny Conservation D. Synteny-Based Gene Discovery and Cloning E. Arabidopsis Knowledge–Based Gene Discovery and Brassica Improvement 1. Understanding Domestication 2. Understanding Metabolism 3. Testing for Gene Function by Complementary Transformation CHLOROPLAST GENOMES AND THEIR PHYLOGENETIC IMPLICATIONS A. Subtribe Brassicinae 1. Brassica 2. Diplotaxis 3. Erucastrum 4. Sinapis 5. Trachystoma 6. Hirschfeldia incana 7. Sinapidendron 8. Coincya 9. Eruca B. Subtribe Raphaninae C. Subtribe Moricandiinae D. General Considerations

2. BRASSICA AND ITS CLOSE ALLIES: CYTOGENETICS AND EVOLUTION

23

XI. EVOLUTION OF MORPHOLOGICAL CHARACTERS A. Cotyledons B. Adult Leaves C. Fruits D. Isthmus Concept XII. CONCLUDING REMARKS ACKNOWLEDGMENTS LITERATURE CITED

ABBREVIATIONS ACO ADH AFLP Ag-NOR BACs BTL CAGs cp CP CMS Cytodeme DAPI ESTs FISH GISH GOT GSL IDH ISSR ITC ITS LAP MDH mt NOR PrBn 6 PGD 6 PGDH PGI PGM QTL

Aconitase Alcohol deshydrogenase Amplified fragment length polymorphism Silver-stained nucleolus organizer region Bacterial artificial chromosomes Binary trait loci Conserved Arabidopsis genome sequences Chloroplast Condensation pattern Cytoplasmic male sterility Crossing group 40 ,6-diamidino-2-phenylindole Expressed sequence tags Fluorescence in situ hybridization Genomic in situ hybridization Glutamate oxaloacetate transaminase Glucosinolate Isocitric dehydrogenase Inter-simple sequence repeats Isothiocynanates Internal transcribed spacers of nuclear ribosomal DNA genes Leucine amino-peptidase Malate dehydrogenase Mitochondria Nucleolus organizer region Pairing regulator Brassica napus 6-phosphogluconase dehydrogenase 6-phosphogluconate deshydrogenase Phosphoglucoisomerase Phosphoglucomutase Quantitative trait loci

24

RAPD RFLP rDNA rRNA SDH SSR TE TPI

S. PRAKASH, S. R. BHAT, C. F. QUIROS, P. B. KIRTI, AND V. L. CHOPRA

Randomly amplified polymorphic DNA Restriction fragment length polymorphism Ribosomal DNA genes Ribosomal RNA Shikimic acid dehydrogenase Simple sequence repeats Transposable element Triose phosphate isomerase

I. INTRODUCTION Brassica species, Brassicaceae (Cruciferae), provide an important component of human diet as major sources of edible oil and vegetables (Table 2.1). The antiquity of crops belonging to the genus Brassica is manifest from references in ancient literature of the Indian, Chinese, Greek, and Roman civilizations (Prakash and Hinata 1980; Go´mezCampo and Prakash, 1999). A number of taxonomic treatments of this family are available since 1700. Prominent among these are by Tournefort (1700), Linnaeus (1753), deCandolle (1821), Hooker (1862), Baillon (1871), and Prantl (1891). However, the most comprehensive one is by Schulz (1919, 1936), a German schoolteacher (Hedge 1976; Prakash and Hinata 1980; Gomez-Campo 1999b). A recent molecular account of the family has been provided by Beilstein et al. (2006). Brassiceae is one among the 19 tribes recognized by Schulz in the family and is divided into 7 to 9 subtribes (Go´mez-Campo 1980, 1999b). Brassica is the core genus in the subtribe Brassicinae. Several members of related subtribes, such as Raphaninae and Moricandiinae, exhibit close genetic affinities with Brassica. However, morphological distintiveness of these three subtribes is not well substantiated and molecular data provide scanty support for their independent status. A majority of the species related to Brassica are wild and weedy. They possess, however, useful genes that may confer agronomic advantages and/or enhance the quality and utility of crop species. In fact, genetic enrichment of crop species with genes from wild allies is a major approach for many crop improvement programs. Such gene transfer can be achieved both by conventional plant breeding methods and through biotechnology. Cytogenetic investigations on Brassica, initiated in the second decade of the 20th century, were confined to determining the chromosome numbers and studying chromosome pairing in interspecific hybrids, which finally led to unraveling the genetic architecture of crop species. Small size of chromosomes, scarcity of distinctive cytogenetic landmarks,


25

Table 2.1. Taxonomic components of Brassica and related genera and their usage. Botanical name B. nigra B. oleracea var. acephala var. capitata var. sabauda var. gemmifera var. gongylodes var. botrytis var. italica var. fruticosa var. alboglabra B. rapa spp. oleifera var. brown sarson var. yellow sarson var. toria ssp. rapifera ssp. chinensis ssp. pekinensis ssp. nipposinica ssp. parachinensis B. carinata B. juncea B. napus spp. oleifera spp. rapifera Eruca sativa Raphanus sativus Sinapis alba

Common name

Usage

black mustard

condiment (seed)

kale cabbage savoy cabbage brussels sprouts kohlrabi cauliflower broccoli branching bush kale Chinese kale

vegetable, fodder (leaves) vegetable (head) vegetable (terminal buds) vegetable (head) vegetable, fodder (stem) vegetable (inflorescence) vegetable (inflorescence) fodder (leaves) vegetable (stem, leaves)

turnip rape brown sarson yellow sarson toria turnip bok choi Chinese cabbage — — Ethiopian mustard mustard

oilseed oilseed oilseed oilseed fodder, vegetable (root) vegetable (leaves) vegetable, fodder (head) vegetable (leaves) vegetable (leaves) vegetable, oilseed oilseed, vegetable

rapeseed rutabaga, swede rocket, taramira radish white mustard

oilseed fodder vegetable, nonedible oilseed vegetable, fodder oilseed

and not being amenable to pachytene investigations were major deterrants to cytogenetical analyses. Advances in tissue culture techniques, including ovary and embryo rescue and protoplast fusion, since the 1950s made varied cytogenetic material available to investigate genome homologies and facilitated introgression of useful nuclear genes even across conventional generic boundaries. Such investigations require reliable markers for chromosome identification. A significant step toward this development has been the extensive use of molecular markers. Molecular biology in Brassica started with the determination of female parents of allopolyploid species using chloroplast DNA RFLPs by Palmer et al. (1983a). Use of genomic and fluorescence in situ hybridization (GISH and FISH respectively) methodology, in combination with ribosomal DNA markers have given new directions in genome analysis

26


and characterization of parental genome components as well as precise identification of the individual chromosomes and location of gene sequences directly on the chromosomes. These investigations led to the generation of first FISH-based molecular karyotypes (Fukui et al. 1998). At the same time, chloroplast and mitochondrial DNA RFLPs have been used extensively to elucidate phylogeny of Brassica and related genera. Molecular markers also have been identified to tag important agronomic traits. This research has not only substantiated some of the already existing concepts but also proposed several new ones. Potential sources of germplasm have been identified outside of the conventional boundaries, thus increasing the range of available germplasm relevant to Brassica improvement. Genomic studies on Arabidopsis, a crucifer and closely related to brassicas, has given a new direction in evolutionary studies of the family Brassicaceae and in particular the members of the genus Brassica. Inferences from comparative genomics between Arabidopsis and Brassica species have elucidated evolutionary processes. Arabidopsis has become a model plant in the field of experimental biology because of its several unique features: short life span, autogamy, and ease of tissue culture. Its entire genome has recently been sequenced (Arabidopsis Genome Initiative 2000). Biologically, Brassica and allied taxa have been grouped collectively and referred to as Brassica coenospecies (Harberd 1972). This review is an attempt to synthesize available literature and developments in Brassica coenospecies from classical to molecular cytology and application of genomic information to throw light on genome organization, genome manipulation, and phylogeny in Brassica and related genera. Informative reviews dealing with some of these aspects are given in Biology of Brassica Coenospecies (Go´mez-Campo 1999a) and Biotechnology in Agriculture and Forestry: Brassica (Pua and Douglas 2004). II. CYTOGENETICS A. Cytogenetic Architecture of Brassica Coenospecies Brassica coenospecies is not a taxonomic but a biological unit. More appropriately it is a cytogenetical concept. It is defined as that part of tribe Brassiceae of Schulz whose members are closely related to crop Brassica species and are potentially capable of exchanging genetic material with them. Harberd (1972) was the first to propose this concept on the basis of his extensive cytotaxonomical investigations on Brassica crop and related wild species. Harberd’s focus was on subtribe


27

Brassicinae of Schulz comprising of 11 genera and also on a part of related subtribe Raphaninae. His investigations involved studies on chromosome pairing and extent of fertility of hybrids. Harberd proposed that nine genera from subtribe Brassicinae—Brassica, Coincya, Diplotaxis, Eruca, Erucastrum, Hirschfeldia, Sinapis, Sinapidendron, and Trachystoma—and two genera from subtribe Raphaninae—Enarthrocarpus and Raphanus—constitute Brassica coenospecies. Harberd (1972) involved a wide spectrum of species in his hybridization program and studied this germplasm biologically rather than taxonomically to classify it into cytodemes or crossing groups to resolve the confusion about their species and generic status. A cytodeme is defined as a group consisting of any number of species or genera that have the same chromosome number, and crosses between them always yield fertile hybrids. Harberd (1972) established 38 cytodemes in the coenospecies. This number was further extended by Takahata and Hinata (1983). In fact, Harberd’s results revealed, for the first time, extensive genome homoeology across species and generic boundaries, implying that Brassica coenospecies constitutes a large gene pool and thus opening the possibilities of transferring agronomically desirable traits to crop species. The boundaries of coenospecies have further expanded with developments in molecular biology that have resulted in massive incongruities with established taxonomy. Chloroplast DNA RFLP studies on members of other related subtribes also suggest that delemitation of genera and species by Schulz does not fully reflect the natural boundaries (Warwick and Black 1991; Pradhan et al. 1992). These investigations strongly support the inclusion of not only Raphanus and Enarthrocarpus in the coenospecies as suggested by Harberd (1972, 1976), but also of three more genera—Moricandia, Pseuderucaria, and Rytidocarpus—from the related subtribe Moricandiinae (Warwick and Black 1997). At present, 63 cytodemes are recognized in coenospecies that spread over 14 taxonomically defined genera, as shown in Fig. 2.1 and Table 2.2 (Prakash et al. 1999). Determination of chromosome number of B. campestris syn. B. rapa (Takamine 1916) was the beginning of cytogenetic research on Brassica and in the Brassicaceae. Chromosome numbers for many species were determined between 1928 and 1932 (Jaretzky 1932; Manton 1932). Manton (1932) did pioneering work on cytology of Brassicaceae, publishing new chromosome reports and reviewing the work on the whole family. Credit for initiating cytogenetical researches on crop species goes to Karpechenko of Russia, Morinaga and U of Japan. Mizushima of Japan deserves the credit for work on wild germplasm. These researchers investigated different aspects and elucidated genomic homoeologies in a

28


Table 2.2. Cytodemes in Brassica coenospecies. Chromosome no. (n) 7

8

9

10

11

12

13 14

Principal species Brassica deflexa Boiss. Diplotaxis erucoides (L.) DC. Erucastrum virgatum C. Presl. Erucastrum varium Durieu Sinapis aucheri (Boiss.) O.E. Schulz Hirschfeldia incana (L.) Lagreze-Fossat Pseuderucaria spp. O.E. Schulz Brassica nigra (L.) Koch Brassica fruiticulosa Cyr. (þ maurorum þ spinescens) Diplotaxis siettiana Maire Erucastrum abyssinicum (A. Rich.) O.E. Schulz Erucastrum nasturtiifolium (Poiret) O.E. Schulz (þ leucanthum) Erucastrum strigosum (Thunb.) O.E. Schulz Trachystoma spp. Brassica oleracea L. and 8 wild Mediterranean allied species Brassica oxyrrhina Coss. Diplotaxis assurgens (Del.) Gren. Diplotaxis catholica (L.) DC. Diplotaxis tenuisiliqua Del. Diplotaxis virgata (Cav.) DC. Diplotaxis berthautii Braun-Blanq. and Maire Erucastrum cardaminoides Webb and Berth. (þ canariense þ ifniense) Raphanus L. all species and subspecies Sinapis arvensis L. (þ allioni) Sinapis pubescens L. Brassica tournefortii Gouan. Brassica barrelieri (L.) Janka Brassica gravinae Ten. Brassica repanda (Willd.) DC. (þ desnottesii) Brassica rapa L. (þ many cultivated subspecies) Diplotaxis siifolia G. Kunze Diplotaxis viminea (L.) DC Enarthrocarpus spp. Sinapidendron spp. Brassica souliei Batt. Diplotaxis acris (Forsk.) Boiss. Brassica elongata Ehrh. Diplotaxis tenuifolia (L.) DC. (þ pitardiana) Eruca spp. Mill. Coincya spp. (syn. Hutera and Rhynchosinapis) Sinapis alba L. Sinapis flexuosa Poir. Diplotaxis harra (Forsk.) Boiss. (þ several subsps.) Erucastrum virgatum C. Presl. (subsp. pseudosinapis) Moricandia arvensis (L.) DC. Moricandia moricandioides (Boiss.) Heywood Rytidocarpus moricandioides Coss.

2. BRASSICA AND ITS CLOSE ALLIES: CYTOGENETICS AND EVOLUTION Table 2.2. (Continued) Chromosome no. (n) 15

Principal species Erucastrum gallicum (Willd.) O.E. Schulz Erucastrum elatum (Ball.) O.E. Schulz Brassica cossoniana (Boiss. & Reut.) (4x) North African subspecies Brassica balearica Pers. Erucastrum nasturtiifolium (Poiret) O.E. Schulz (4x) Erucastrum abyssinicum (A. Rich.) O.E. Schulz (4x) Brassica carinata A. Braun Brassica juncea (L.) Czern & Coss. Brassica napus L. Brassica gravinae Ten. (4x) Diplotaxis muralis (L.) DC. Brassica dimorpha Coss. & Dur. Coincya spp. (4x) Moricandia suffruticosa (Desf.) Coss. & Dur. Moricandia spinosa Pomel Brassica repanda (Willd.) DC. (High Atlas)

16

17 18 19 20 21 22 24 28 42 80?

Source: From Prakash et al. 1999; C. Go´mez-Campo, personal communication.

Family

Brassicaceae

Tribe

Brassicaceae

Subtribe

Brassicinae

Genera

Brassica (20) Coincya (1) Diplotaxis (13) Eruca (1)

Raphaninae

Enarthrocarpus (1) Raphanus (1)

Moricandiinae

Moricandia (1) Pseuderucaria (1) Rytidocarpus (1)

Erucastrum (11) Hirschfeldia (1) Sinapis (5) Sinapidendron (1) Trachystoma (1) Fig. 2.1. Archetecture of Brassica coenospecies (number of cytodemes in brackets. (Source: From Prakash et al. 1999.)

29

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series of publications. Extensive taxonomical investigations on wild germplasm have been carried out by Go´mez-Campo (1999b). The lowest chromosome number in coenospecies, n ¼ 7, is characteristic of seven cytodemes. Harberd (1972) was of the view that cytodeme with n ¼ 14 or higher chromosome numbers should be attributed to polyploidy. According to this view, 43 cytodemes are diploids where every chromosome number from n ¼ 7 to n ¼ 13 is represented. However, variations in isozyme numbers of a vast range of taxa in the tribe Brassiceae suggest that genera with n ¼ 14 to 18 are not necessarily polyploids of n ¼ 7 to 13 genomes (Anderson and Warwick 1998). Around 50% of the cytodemes have gametic chromosome number n ¼ 9 and n ¼ 10. Polyploidy also played a role as both auto- and allopolyploids are represented by 20 cytodemes (Table 2.3). The majority are tetraploids. This polyploidy level is exceeded only in some accessions of Moricandia spinosa (2n ¼ 84, x ¼ 6) and Brassica repanda (2n ¼ 160, x ¼ 8) (Prakash et al. 1999). The genus Moricandia seems to be exclusively polyploid (Al-Shebaz 1984). B. Crop Species Genome analysis in crop species, pioneered by Morinaga (1928; 1929a, b,c; 1931; 1933; 1934a,b) was based on hybridizing high-chromosome species with low-chromosome species and interpreting the chromosome pairing behavior of the hybrids. This research led Morinaga (1934) to propose that crop brassicas comprise six species. Of these, three are low-chromosome monogenomic diploids—B. nigra (n ¼ 8), B. oleracea (n ¼ 9), and B. rapa (syn. B. campestris, n ¼ 10)—and three are high-chromosome digenomics—B. carinata (n ¼ 17), B. juncea (n ¼ 18), and B. napus (n ¼ 19), which evolved in nature through convergent alloploid evolution between any two of the diploid species. Morinaga also assigned genome symbols to these species. U (1935) represented this cytogenetical relationship diagramatically, in what is now commonly referred to as U triangle (Fig. 2.2). These relationships have, in recent years, been substantiated by cytogenetics, molecular analysis of nuclear and chloroplast DNA, and by genomic and fluorescence in situ hybridization (Snowdon et al. 2003; Snowdon 2007). This complex of diploids and allopolyploids is now considered a model system for investigations on polyploidy in crop plants (Lukens et al. 2006; Pires et al. 2006). 1. Nature of Diploid Species. The three basic diploid species, B. nigra, B. oleracea, and B. rapa, represent an ascending aneuploid series


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Table 2.3. Polyploid cytodemes in Brassica coenospecies. Allopolyploids Brassica Brassica Brassica Brassica

carinata, n ¼ 17 juncea, n ¼ 18 napus, n ¼ 19 balearca, n ¼ 16

Diplotaxis muralis, n ¼ 21 Erucastrum gallicum, n ¼ 15 Erucastrum elatum, n ¼ 15 Tentative autopolyploids Moricandia arvensis, n ¼ 14 Moricandia moricandiodes, n ¼ 14 Rytidocarpus moricandiodes, n ¼ 14 Erucastrum virgatum (subsp. pseudosinapis), n ¼ 14 Brassica cossoneana, n ¼ 16 Erucastrum abyssinicum, n ¼ 16 Erucastrum nasturtiifolium, n ¼ 16 Brassica gravinae, n ¼ 20

Diploid progenitors B. B. B. B.

nigra, B. oleracea rapa, B. nigra oleracea, B. rapa oleracea group another species D. viminea, D. tenuifolia

E. leucanthum sp.? Hirschfeldia incana Erucastrum littoreum Diploid homolog

Reference U 1935 U 1935 U 1935 Snogerup and Persoon 1983 Harberd 1976; Mummenhoff et al. 1993: Ueno et al. 2006 Harberd 1976 Go´mez-Campo 1983; Sańchez-Ye´lamo 1992 Reference

unknown

Harberd 1976

unknown

Harberd 1976

unknown

Harberd 1976

E.virgatum B. maurorum, n ¼ 8 E. abyssinicum, n ¼ 8 E. nasturtiifolium, n ¼ 8

Harberd 1976 Pradhan et al. 1992 Harberd 1976 Harberd 1976

B. gravinae, n ¼ 10

Takahata and Hinata 1983 Go´mez-Campo 1980 Harberd 1976 Sobrino-Vesperinas 1980 Sobrino–Vesperinas 1980 Galland 1988

Brassica dimorpha, n ¼ 22 Coincya spp., n ¼ 24 Moricandia suffruticosa, n ¼ 28 Moricandia spinosa, n ¼ 42

B. soullei, n ¼ 11 Coincya sp., n ¼ 12 Moricandia sp., n ¼ 14 Moricandia sp., n ¼ 14

Brassica repanda, n ¼ 80

B. repanda, n ¼ 10

Source: From Prakash et al. 1999.

(Manton 1932) and are regarded as secondary polyploids. Evidence for this conclusion were adduced from chromosome associations at meiosis in their respective haploids (Thompson 1956; Prakash 1974b; Armstrong and Keller 1981). Investigations on pachytene chromosome analysis (Ro¨bbelen 1960; Venkateswarlu and Kamla 1971), isozyme markers, and rDNA genes (Quiros et al. 1987) suggested that these species originated from a now-extinct archetype with a probable basic chromosome number of x ¼ 6. It was believed that selective doubling of some chromosomes in this archetype led to the evolution of the three diploid genomes. However, results of recent investigations on nuclear,

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B. carinata n = 17, bc

B. oleracea

B. nigra

n = 9, c

n = 8, b

B. napus

B. rapa

n = 19, ac

n = 10, a

B. juncea n = 18, ab

Fig. 2.2. Cytogenetic relationships of crop brassicas (U, 1935). Solid and broken lines in the allopolyploids represent female and male parents. respectively) (Source: According to Palmer et al. 1983a).

mitochondrial, and chloroplast DNA (Palmer 1988; Song et al. 1988a; Warwick and Black 1991; Pradhan et al. 1992) have discounted this theory of monophyletic origin and have instead suggested their origin from two linages: B. oleracea and B. rapa originating from one archetype and B. nigra evolving from the other. Nevertheless, these genomes share close homologies, as revealed by cytogenetical (Mizushima 1950a; Prakash and Hinata 1980; Attia and Ro¨bbelen 1986) and molecular studies (Hosaka et al. 1990; Teutonico and Osborn 1994; Truco et al. 1996; Parkin et al. 2003). Cytogenetical investigations in digenomic and trigenomic interspecific hybrids involving the three basic species showed high frequency of bivalents and multivalents. A good number of these were suggested to arise due to allosyndesis. GISH analysis confirmed three allosyndetic bivalents between B and A/C (Ge and Li 2007) and five bivalents between A and C genomes (Liu et al. 2006). All three genomes contain similar genetic information with many duplications (Slocum et al., 1990; Chyi et al. 1992; Jackson et al. 2000; Parkin et al. 2003); just the organization and distribution on chromosomes is different (Truco et al. 1996). Chromosome differentiation and repatterning has occurred mainly through duplications and translocations (Quiros et al. 1988; Hosaka et al. 1990; McGrath et al. 1990; Truco and Quiros 1994) and also deletions (Hu and Quiros 1991). These changes were tolerated and adjusted because of the secondary


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balanced nature of these genomes (Kianian and Quiros 1992a). Also, a large number of rearrangements separated the B genome from the A or C genome. In comparison, A and C genomes are less differentiated (Lagercrantz 1998). Genomes A and C are also cytogenetically very close (Mizushima 1950a; Olsson 1960b), a fact substantiated by: (1) FISH mapping of two families of repetitive DNA that are common to pericentromeric regions of most chromosomes of A and C genomes but are absent in the B genome (Harrison and Heslop-Harrison 1995); (2) structural analysis of rDNA intergenic spacers (Bhatia et al. 1996); (3) colinearity between them as revealed by comparative analysis (Scheffler et al. 1997); and (4) extent of homoeologous pairing detected by GISH (Snowdon et al. 1997a; Ge and Li 2007) and FISH and molecular markers (Nicolas et al. 2007). Interestingly, RFLP analysis of rDNA reveals, on the contrary, closer affinities between B and C genomes (Hasterok and Maluszynska 2000a). Also, as detected by microsatellites (Bornet and Blanchard 2004), the C genome is more conserved than A or B. Among the three basic species, two types of cytoplasm exist: the B type found in B. nigra and the A/C type occurring in B. rapa and B. oleracea. The A and B types are quite distinct although they retain homology to a large extent (Palmer et al. 1983a; Yanagino et al. 1987; Warwick and Black 1991; Pradhan et al. 1992).

2. Nature of Alloploid Species. The three high-chromosome species, B. carinata, B. juncea, and B. napus, originated through convergent alloploid evolution involving different combinations of the diploid species. This is amply demonstrated by several sets of evidence including taxonomy, artificial syntheses, molecular analysis, and chromosome mapping. The allopolyploids have attained a balance forming regular bivalents and are devoid of any homoeologous pairing. Information from Fraction-1 protein (Uchimiya and Wildman 1978) and chloroplast and mitochondrial DNA restriction patterns (Palmer et al. 1983a; Ichikawa and Hirai 1983; Erickson et al. 1983; Yanagino et al. 1987; Warwick and Black 1991; Pradhan et al. 1992; Cunha et al. 2004) have revealed directionality of natural hybridizations. It has conclusively been established that B. nigra and B. rapa contributed the cytoplasm to B. carinata and B. juncea, respectively. However, patterns of chloroplast DNA restriction of B. napus were different from both B. rapa and B. oleracea but approached that of B. oleracea (Erickson et al. 1983; Palmer et al. 1983a). Studies on comparative chloroplast and mitochondrial RFLP patterns strongly implicated B. montana, belonging to B. oleracea group, as the cytoplasm donor of B. napus (Song and

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Osborn 1992). However, plastid SSRs analysis does not support this view; on the contrary, it suggests that B. rapa is the more likely plastid genome donor of B. napus (Flannery et al. 2006). Various investigations also suggest that while chloroplast genomes have been conserved in B. carinata and B. juncea since their origin, the chloroplast genome of B. napus has gone through evolutionary alteration (Palmer et al. 1983a). Both mitochondrial and chloroplast genomes in all the three species have been coinherited over generations (Palmer 1988). It has also been observed that cytoplasm has exerted considerable influence on the evolution of nuclear genomes of alloploid species. When the parental diploid species of allopolyploid has highly differentiated cytoplasm, as in B. juncea and B. carinata, the nuclear genomes contributed by the male parents were altered considerably more compared to the nuclear genomes of female parents (Song et al. 1988a, 1995). Thus in B. juncea, nuclear genome A has remained mostly intact while nuclear genome B has changed considerably; B. carinata has B nuclear genome unchanged but considerably altered C genome. In B. napus, both A and C genomes have undergone a similar extent of changes. However, ISSR marker analysis suggests that B-genome-carrying allopolyploid species B. carinata and B. juncea underwent more modifications than B. napus. Also, in B. napus, A genome has changed more than C genome; A genome in B. juncea and C genome in B. carinata are drastically modified; and B. juncea and B. carinata have relatively conserved B genome (Liu and Wang 2006). Strong evidence suggests rapid and extensive structural rearrangements of chromosomes in these alloploid species since their evolution (Slocum et al. 1990; Song et al. 1991, 1995; Kianian and Quiros 1992a,b; Poulsen et al. 1993; Harrison and HeslopHarrison 1995). In fact, chromosomal rearrangements arising from homoeologous recombination between A and C genomes are widespread. Referred to as interstitial homoeologous reciprocal translocations they are a cause for origin of morphological variants in B. napus (Osborn et al. 2003; Udall et al. 2005) and are considered a progressive step in evolution (Nicolas et al. 2007). Morphological variants of nonhomologous origin have been obtained in B. juncea (Prakash 1973b). Occurrence of such events was earlier inferred from analysis of segregating populations employing molecular markers (Parkin et al. 1995; Sharpe et al. 1995). However, comparative analysis of RFLP marker-based chromosome maps of natural and synthesized B. juncea does not support Song et al. (1995) and indicate that B. juncea has remained largely unchanged since its origin (Axelsson et al. 2000). In spite of incorporation of these changes, the average chromosome size has remained unaltered (Parkin and Lydiate 1997).


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3. Nuclear DNA. Nuclear DNA content and nuclear volume were first estimated by Yamaguchi and Tsunoda (1969) in B. rapa, B. oleracea, and naturally occurring and synthetic strains of B. napus. They observed that the values for synthetic B. napus were the sum of the constituent parents. However, there was an appreciable reduction in total DNA content in natural forms. These authors proposed that nuclear DNA had been lost subsequent to evolution of allotetraploids. Verma and Rees (1974) further investigated this problem by estimating the amount of DNA in diploids and their allotetraploid derivatives in root meristem nuclei at the GI phase. No significant intraspecific variation in nuclear DNA amount was observed. However, differences exist at the interspecific level. In spite of the fact that values for allotetraploids were very close to the sum values of their constituent parents, reduction from the expected values for every species was observed. They postulated that the lower values in tetraploids result from underestimation of DNA due to higher nuclear density. It was also suggested that values observed by Yamaguchi and Tsunoda (1969) were based on dense nuclei and were underestimations; thus, when corrected, the values showed no significant deviations from those anticipated. Therefore, decrease in the amount of DNA was not associated with allopolyploidy. One significant observation by Verma and Rees (1974) was a remarkable reduction in nuclear size in natural allotetraploids, which suggested condensation of chromosomal material that probably reflected an adaptive switching off of redundant gene copies. In several recent investigations, DNA values have been estimated afresh (Arumugunathan and Earle 1991; Narayan 1998; Bennet and Leitch 2005; Johnston et al. 2005). A general observation is the evolution of DNA content from low to high in the genus. These studies also support the earlier observations of Yamaguchi and Tsunoda (1969) that there has been a decrease in DNA content in the present-day alloploid species. A decrease of 6% was observed by Narayan (1998), and the values for B. napus, B. juncea, and B. carinata are 0.095, 0.094, and 0.049 pg less respectively than the sum of their parental species (Johnston et al. 2005, Table 2.4). 4. Karyotypes. Brassica chromosomes are very small in size, as indicated by haploid 1C genome size (Bennet and Leitch 2005). They are also poorly differentiated in structure without distinctive cytological landmarks. Consequently, to date no precise nomenclature is available. Karyotype studies were initiated as early as 1934 by Catcheside and were followed by Alam (1936), Richharia (1937), and Sikka (1940). These investigations were confined to observations on the frequency of various chromosome types, number of satellites, and

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Table 2.4. 1c nuclear DNA content and genome size in Brassica species. Species B. B. B. B. B. B.

nigra oleracea rapa carinata juncea napus

1c nuclear DNA content (pg se)

Genome size (1 x) (Mbp)

0:647 0009 0:710 0:002 0:539 0:018 1:308 0:018 1:092 0:001 1:154 0:006

632 696 529 642 534 566

Source: From Johnston et al. 2005.

nucleoli in different genomes. They distinguished chromosomes into long, medium, small, and very small with median, subterminal and terminal constrictions. In recent years, karyotypes, particularly of diploid species, based on mitotic (Olin-Fatih and Heneen 1992; Cheng et al. 1995a; Fukui et al. 1998; Hasterok and Maluszynska 2000a; Hasterok et al. 2005a) and meiotic chromosome (Cheng et al. 1994b; Mackowiak and Heneen 1999; Koo et al. 2004) phenotypes, have been constructed using different staining. Mitotic prometaphase and meiotic diakinesis offer better possibilities for characterizing individual chromosomes and constructing karyotypes. Since early 1990s, use of FISH with ribosomal DNA probes has further helped in generating chromosome markers. Molecular karyotypes based on FISH have been generated, enabling more reliable identification of individual chromosomes. Maluszynska and Heslop-Harrison (1993), Snowdon et al. (1997a) and Fukui et al. (1998) employed FISH with a 45S rDNA probe for individual chromosome identification. However, simultaneous probing with 45S rDNA and 5S rDNA in B. napus, Sinapis alba and Raphanus sativus (Schrader et al. 2000) and in all the six species of U triangle (Hasterok et al 2001) proved to be more informative as they provided numerous signals on somatic chromosomes revealing new landmarks. Fukui (1998) developed a system for computer imaging of plant chromosomes that led to the definition of a new parameter, the ‘‘condensation pattern’’ (CP) for chromosome analysis. It is an effective and reproducible parameter and very useful in identification of small chromosomes. It is a general observation that somatic chromosomes of A and C genomes are morphologically very similar and difficult to distinguish (Olin-Fatih and Heneen 1992) although their condensation patterns differ in prometaphase chromosomes (Cheng et al. 1995a). However, making use of FISH and GISH, it has been possible to identify individual chromosomes of A, B, and C genomes and also to match chromosomes with corresponding counterparts in alloploid species


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with considerable reliability (Snowdon et al. 1997b, 2002; Kamisugi et al. 1998; Maluszynska and Hasterok 2005). Also, these investigations are very helpful in integrating genetical maps, emerging from analysis of molecular markers, with physical maps based on morphometric analysis. Although many papers describing the karyotypes of these species have appeared since 1937, overall conclusions can be summarized in this way: Chromosomes of A genome are morphologically most diverse, B genome chromosomes are much more uniform and difficult to identify individually, and C genome chromosomes are poorly differentiated in morphology and size, and undergo variable degree of condensation of hetero- and euchromatin in the chromosome arms (Olin-Fatih 1994). Discrepencies in nomenclature and numbering of chromosomes have occurred due to polymorphisms in the rDNA sites and contraction rates of the chromosomes. Brassica nigra. Hasterok and Maluszynska (2000a) observed that B. nigra chromosomes are more or less similar in size, ranging from 2.47 to 3.57 mm, and are morphologically undistinguishable. Only two types of chromosomes are present: median (no. 1–4) and submedian (no. 5–8). Chromosomes 7 and 8 contain secondary constriction and satellite on the short arm. Earlier, Mackowiak and Heneen (1999) presented a karyotype based on diakinesis bivalents wherein each chromosome exhibited a specific pattern of chromatin condensation or darkly stained regions. The eight bivalents were classified into three groups. Group 1, comprising pairs 1 and 2, has darkly stained median position signifying pericentric chromatin. Pair 2 was the smallest. Group 2 comprised pairs 3 to 5 with a submedian-subterminal, darkly stained region that represent pericentric chromatin. Group 3 comprised pairs 6 to 8 with relatively large-size subterminal-terminal darkly stained region. Pair 6 was larger than pairs 7 and 8. Pairs 6 to 8 were the satellited nucleolar chromosomes. Brassica oleracea. Cheng et al. (1995a) described a karyotype for B. oleracea where the absolute length ranged from 2.8 to 4.5 mm. The genome is comprised of three median group (1–3), four submedian group (4–7), and two subterminal group chromosomes with a nonsatellite pair (8) and a satellite pair (9). Armstrong et al. (1998) reported partial karyotype of B. oleracea var. alboglabra based on cytological landmarks (centromere locations) provided by FISH localization of three repetitive DNA sequences. Five of the nine chromosomes could be identified with precision.

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Chromosomes 2 and 4 carry 45S rDNA clusters. Chromosome 2 is also submetracentric. Chromosomes 4, 6, 8, and 9 are very much similar. In all, there are seven metacentric/submetacentric and two pairs of satellited acrocentric chromosomes. Hasterok and Maluszynska (2000a) studied a B. oleracea karyotype where chromosomes are poorly differentiated and are in the range of 2.17 to 2.97 mm. The karyotype consists of 1–4 median and 5–8 submedian chromosomes. Chromosome 9 has a secondary constriction and satellite on the short arm. Brassica rapa. Karyotypes of B. rapa, in the earlier studies, have been constructed on Giemsa-stained mitotic metaphase or pro-metaphase chromosomes (Nishibayashi 1992; Olin-Fatih and Heneen 1992; OlinFatih 1994; Cheng et al. 1995a). Making use of condensation pattern profiles and FISH patterns employing a 45S rDNA probe, Fukui et al. (1998) generated a karyotype of B. rapa. Subsequently, Snowdon et al. (2002) and Lim et al. (2005) also used FISH for chromosomal localization of rDNA loci and generated a karyotype. Cheng et al. (1995a) observed four large-sized chromosome pairs (1–4) in the median group, four relatively small-size pairs (5–8) in the submedian group, and a satellited (9) and a nonsatellited pair (10) in the subterminal group. The absolute length ranged from 2.1 to 5.2 mm. In general, chromosome 1 is the largest; chromosomes 2 and 4 are of similar size; chromosome 5 is the large one in submediann group; chromosome 6 has a less submedian centromere than chromosome 7. Chromosome 7 has a submedian centromere and is larger than chromosome 8. Chromosome 9 has a satellite on the short arm. However, Kim et al. (1998) reported six median, two submedian, and two subterminal chromosomes. Hasterok and Maluszynska (2000a) characterized individual chromosomes in the A genome morphologically. The absolute length ranged from 1.77 to 3.73 mm. Based on centromere position, there are five median (1–5), three submedian (6–8), and two subterminal pairs (9–10). Chromosome 10 has a secondary constriction and a prominent satellite on the short arm. Recently, Koo et al. (2004) developed a molecular cytogenetic map of B. rapa ssp. pekinensis. The average length of mitotic metaphase chromosomes is in the range of 1.46 mm to 3.30 mm. Earlier, Kim et al. (1998) observed the range 1.01 to 2.06 mm for B. rapa. A map of B. rapa var. pekinensis was developed of mitotic metaphase chromosomes using 45S and 5S rDNA and pericentromeric tandem repeats as probes (Lim et al. 2005). Centromere index varied from 50.7% to 32.9%, indicating that the chromosomes are metacentric or submetacentric. The average total length of the haploid complement was 32.47 mm. Chromosomes 1 and 2 were the longest (4.5 and 5.83 mm respectively),


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the latter one being a nucleolus chromosome including the satellite and NOR. The short arm of this chromosome possessed 45S and 5S rDNA sites. Chromosomes 1, 3, 4, and 5 had 45S rDNA loci in their long arm. Chromosome 10 was the shortest (2.85 mm), and its short arm occupied a 5S rDNA site. The number of rDNA sites in the interphase nuclei varied from 4 to 10. The three high-chromosome allotetraploid species have numerous chromosomes, making karyotype formation very difficult when based just on morphometric features. In recent investigations, Hasterok and Maluszynska (2000b) and Kulak et al. (2002) have presented karyotypes of B. carinata, B. juncea, and B. napus on features combining morphometric information and multicolor FISH. Brassica carinata. Its karyotype consists of fairly uniform chromosomes, both in morphology and in length, ranging from 1.56 to 2.40 mm. Two groups of chromosomes can be distinguished: median (1–6) and submedian (7–15). There are two pairs of satellite chromosomes (16–17) with distinct secondary constrictions (Kulak et al. 2002). Brassica juncea. The chromosome length in B. juncea ranges from 1.38 to 3.25 mm, and the karyotype comprises of median (1–6) and submedian groups (7–15).Two chromosomes (17–18) are NOR-bearing with prominent secondary constrictions in the short arm. Chromosome 16, although NOR bearing, does not have a distinct secondary constriction/satellite region (Kulak et al. 2002). The extent of variations in chromosome size and morphology is due to A genome chromosomes. Brassica napus. This karyotype has been studied extensively (OlinFatih and Heneen 1992; Skarzhinskaya et al. 1998; Hasterok and Maluszynska 2000b; Snowdon et al. 2000a, 2002; Wei et al 2005) and is comprised of relatively small and poorly differentiated chromosomes. According to Hasterok and Maluszynska (2000b) and Kulak et al. (2002), B. napus chromosomes range from 1.52 to 3.3 mm in length and are small and similar in shape. They occur in three groups: median/ metacentric (1–4), submedian/submetacentric (5–17), and subterminal/subtelocentric (18–19), which carry satellites. Although chromosome condensation patterns in B. oleracea and B. rapa differ in prometaphase chromosomes (Cheng et al. 1995a), Kamisugi et al. (1988) were successful in identifying B. rapa–type and B. oleracea– type chromosomes in B. napus. A direct association with their homologs, however, could not be made. Distribution of rRNA genes on

40


chromosomes can be helpful in this regard. For example, A genome chromosomes are characterized by pericentromeric localization, and C genome chromosomes have terminally distributed rRNA genes (Hasterok and Maluszynska 2000b). These were clearly identified using rDNA hybridization and DAPI staining by Snowdon et al. (2002). Another B. napus karyotype has been constructed based on Cot-1 DNA FISH banding patterns by Wei et al. (2005). Their results agreed with the earlier reports. It was demonstrated that this technique can be used with precision to identify individual chromosomes and would be very helpful in recognizing homologous and nonhomologous chromosome pairing. 5. Pachytene Chromosomes. The cytologically difficult nature of material has restricted investigation on pachytene chromosome morphology to only a few attempts. Ro¨bbelen (1960), for the first time, analyzed pachytene chromosomes in the three basic species: B. nigra, B. oleracea, and B. rapa. The chromosomes revealed differentiation into proximal heterochromatic and distal euchromatic segments. Individual chromosomes within the genomes were identified by the number, size, and distribution pattern of the heterochromatic segments near the centromeres. The chromosomes were classified on the basis of their absolute length and were distinguished into five different types: very short (up to 20 mm); short (20–25 mm); medium (25–30 mm); long (30–40 mm); and very long (more than 40 mm). The B genome of B. nigra consists of one very long, four long, and three medium chromosomes of which five are metacentric; chromosome 3 (type C) has a satellite. B. oleracea, representing the C genome, has one very long, five long, two medium, and one short chromosomes. One chromosome is subtelocentric (nucleolus organizing chromosome No. 1, type A), two are submetacentric, and the rest are metacentric. The A genome B. rapa is characterized by one very long, four long, three medium, one short, and one very short chromosomes. Four chromosomes have subterminal and two have median centrometres. Nucleoli are attached to chromosomes 1 and 2 (both of type A) in B. rapa, to chromosomes 1 and 4 (types A and C) in B. oleracea, and to 1 and 3 (types A and C) in B. nigra. B. nigra genome has higher content of heterochromatin as compared to B. oleracea and B. rapa. Ziolkowski and Sadowski (2002) presented a high-resolution pachytene karyotype of B. oleracea using FISH. Each bivalent had a pericentromeric, heterochromatic region devided by a centromere. Up to 11 chromatin blocks corresponding to 9 pericentromeric heterochromatic


41

regions and 2 nucleolus organizer regions were observed. Two NORassociated chromosomes were acrocentric, containing heterochromatin blocks at the ends of their short arm, and were designated C4 and C7. A prominent chromomere was present on the long arm of a submetacentric chromosome. Submetacentric chromosome C2 displayed three 5S rDNA loci on the same arm with medium (M), strong (S) and weak (W) FISH intensities. While locus M was very close to centromere, the two adjacent loci, S and W, were more distal. These three loci offer prominent landmarks for C2 chromosome of B. oleracea. Pachytene chromosome karyotype of B. rapa generated by Koo et al. (2004) was based on multicolor FISH and comprised of two metacentric (nos. 1, 6), five submetacentric (nos. 3, 4, 5, 9, and 10), two subtelocentric (nos. 7 and 8), and one acrocentric (no. 2) chromosomes. Their corresponding centromeric index ranges were 38.8% to 41.0%, 29.5% to 36.7%, 17.4% to 20.2%, and 9.38% respectively. The mean lengths varied from 23.7 to 51.3 mm with a total of 385 mm. As compared to mitotic metaphase chromosome length (1.46–3.30 mm), it is 17.5-fold higher. DAPI staining revealed variable length of heterochromatic blocks in the pericentromeric regions of all the chromosomes. Also, small heterochromatic regions, with a total length of 38.2 mm and approximately 10% of the total length of pachytene chromosomes, were observed on the long arm of chromosomes 3, 4, 5, and 7. FISH indicated 5S rDNA loci on pericentromeric regions of the short arms of chromosomes 2 and 10 and the long arm of chromosome 7. Similarly, 45S rDNA loci were observed on pericentromeric regions of short arms of chromosomes 1, 2, 4, and 5 and the long arm of chromosome 7. A 5S rDNA locus, observed on the long arm of bivalent no. 7, had not been detected on mitotic metaphase chromosomes in any earlier investigations. Ro¨bbelen (1960) recognized six basic types of chromosomes in each genome based on absolute length, symmetry of arms, and shape of heterochromatic centromeric region. These six types are: ‘‘A’’ with a distal heterochromatic satellite involved in nucleolus organization ‘‘B’’ with two heterochromatic segments of equal size near the centromere ‘‘C’’ with a small chromomere and two heterochromatic segments near the centromere ‘‘D’’ with three heterochromatic segments distributed in a 1:2 pattern near the centromere

42


‘‘E’’ with four or more heterochromatic segments ‘‘F’’ with two unequal heterochromatic segments. Based on these observations, Ro¨bbelen (1960) proposed the genetic constitution of the three basic species. B. rapa has two chromosome types, A and D, in tetrasomic and type F in hexasomic condition and the constitution AABCDDEFFF. B. nigra is tetrasomic for chromosomes D and F and has the constitution ABCDDEFF. B. oleracea is a triple tetrasomic for three chromosome types B, C, and E, with the constitution ABBCCDEEF. Venkateswarlu and Kamala (1971) arrived at a conclusion very similar to that of Ro¨bbelen (1960). They also identified six basic types of chromosomes. However, their observations regarding the type of chromosomes present in disomic or tetrasomic condition differed. According to them, the A genome has the genetic constitution AABCDDEFFF; B genome has the constitution ABCDEEFF; and C genome has the constitution ABCCDDEEF. These authors opined that basic genomes originated from loss of different sets of chromosomes from an allotetraploid (2n ¼ 20) rather than from duplication of different chromosomes, as proposed by Ro¨bbelen (1960). Generating karyotypes based on meiotic chromosome preparations rather than mitotic ones has a number of advantages although the clumping of pericentromeric heterochromatin makes the resolution of individual chromosomes difficult. Thus, combining pachytene and metaphase chromosome analysis for efficient physical mapping by FISH would be advantageous (Ziolkowski and Sadowski 2002). 6. Satellite Chromosomes and rDNA Loci. The small size of Brassica somatic chromosomes makes it difficult to identify all satellite chromosomes. In recent years, these have been identified with precision with silver staining and FISH, by detecting active nucleolar organizer regions (NORs) and nucleolar activity of ribosomal RNA (Table 2.5). Cheng and Heneen (1995), for the first time, used silver staining to detect the number and sites of nucleolar organizer regions and the number of nucleoli in Brassica species. Giemsa staining was used to characterize the satellite chromosomes. Their studies at prometaphase/early metaphase revealed that while one pair of chromosomes has secondary constriction and satellites in B. rapa, three such pairs exist in B. nigra, the last being substantiated by Hasterok et al. (2005a). The silver-stained nucleolar organizing regions were localized at the secondary constrictions of the satellite chromosomes. B. rapa revealed two NORs and two nucleoli; six NORs and six nucleoli were


43

Table 2.5. Satellite chromosomes in Brassica and allied genera. Species B. nigra

No. satellite chromosomes 4

6 B. oleracea

2

B. rapa

2

B. carinata B. juncea

4 6

B. napus

4 2 4

Raphanus sativus Sinapis alba Sinapis arvensis

4 6 6

Reference Sikka 1940; Ro¨bbelen 1960; This 1990; Lan et al. 1991; Hasterok and Maluszynska 2000a Cheng and Heneen 1995; Mackowiak and Heneen 1999; Hasterok et al. 2005a Sikka 1940, Wang and Luo 1987; Olin-Fatih and Heneen 1992; Cheng et al. 1995a; Armstrong et al. 1998; Hasterok and Maluszynska 2000a; Ziolkowski and Sadowski 2002; Hasterok et al. 2005a,b Sikka 1940; Nishibayashi 1992; Olin-Fatih and Heneen 1992; Cheng and Heneen 1995; Cheng et al. 1995a; Hasterok and Maluszynska 2000a; Hasterok et al. 2005a,b; Lim et al. 2005 Kulak et al. 2002 Sikka 1940; Maluszynska and Hasterok 2005 Kulak et al. 2002 Olin-Fatih and Heneen 1992; Olin-Fatih 1994, 1996; Skarzhinskaya et al. 1998 Snowdon et al. 1997a; Hasterok and Maluszynska 2000b; Kulak et al. 2002 Mukharjee 1979 Geber and Schweizer 1988 Cheng and Heneen 1995

observed in B. nigra in early and late telophase. The two Ag-NORs in B. rapa represent one pair of active rDNA loci. Maluszynska and Heslop-Harrison (1993), Snowdon et al. (1997a), and Fukui et al. (1998), on the contrary, reported five pairs of rDNA loci following in situ hybridization. These represent both active and inactive rDNA sites while silver staining reveals only the sites with active rDNA. It appears that four pairs of these sites are inactive in nucleolus formation in B. rapa (Cheng and Heneen 1995). The observations on B. nigra having six pairs do not correspond with the earlier investigations of Sikka (1940) and Lan et al. (1991), where only four satellite chromosomes were observed. Ro¨bbelen (1960) also observed that only four chromosomes were associated with nucleoli at pachytene of meiosis in B. nigra. This et al. (1990) also assigned rDNA markers to two pairs of B. nigra

44


chromosomes using B. nigra–specific rDNA RFLP markers. Closely related species Sinapis arvensis has five distinct satellited chromosome while one chromosome has a faint satellite. It is one of the two homologs that are heteromorphic to the size of satellite (Cheng and Heneen 1995). Six Ag-NORs were observed. The Ag-bands varied in size between and within pairs: two large, three intermediate, and one less distinct and small. These authors also suggested that higher number of NORs in B. nigra and Sinapis arvensis as compared to B. rapa and B. oleracea implies that the former two species have been more conservative during evolution. Brassica oleracea possesses only one pair of chromosomes exhibiting secondary constrictions and satellites as revealed by mitotic analysis (Sikka 1940; Wang and Luo 1987; Olin-Fatih and Heneen 1992; Cheng et al. 1995a). Giemsa staining also revealed one pair associated with secondary constriction and satellites (Cheng et al. 1995a). However, at pachytene of meiosis, two pairs of chromosomes are associated with nucleoli (Ro¨bbelen 1960; Kamala 1976). This fact gets support from the occurrence of four nucleoli—two major and two minor—observed by Cheng et al. (1995a). These authors also suggested that one satellited chromosome pair (no. 9) contains major active rRNA loci leading to the formation of major nucleoli and the nonsatellite pair (no. 8) carries minor active rRNA gene loci responsible for micronuclei formation. The size difference between major and minor pairs of rRNA gene loci probably suggests a reduction of most copies of rRNA genes on chromosome pair 8 during and/or after the speciation of B. oleracea. In all the three diploid species, silver staining confirmed that active rRNA genes are located at the secondary constriction of NOR chromosomes. Allopolyploid species have either the sum of or fewer satellite chromosomes than the diploids. B. carinata and B. juncea both have two pairs of satellite chromosomes (nos. 16–17 and 17–18, respectively) with distinct secondary constrictions (Kulak et al. 2002). In B. juncea, chromosome no. 16 is also a NOR-bearing type, but it lacks a distinct secondary constriction/satellite region. B. napus karyotype revealed one pair of satellited chromosomes based on C-banding (Olin-Fatih and Heneen 1992; Olin-Fatih 1994, 1996) and giemsa staining (Skarzhinskaya et al. 1998), However, after in situ hybridization with rDNA, two pairs (nos. 18–19) with distinct secondary constrictions and satellites were observed (Hasterok and Maluszynska 2000b; Snowdon et al. 2000a; Kulak et al. 2002). Both pairs of NORs are similar in size and morphology. The related species Raphanus sativus has two pairs (Mukharjee 1979) while Sinapis alba and S. arvensis carry two pairs each of NORs (Geber and Schweiger 1988; Cheng and Heneen 1995).


45

The knowledge of relative physical location and the number of rDNA gene loci is relevant for construction of physical maps of chromosomes and for phylogenetic studies. Two types of ribosomal RNA genes—5S rDNA and 45S rDNA—encoding for 18S-5.8S-25S ribosomal RNAs have been studied extensively in Brassica through FISH to find their locations on chromosomes. These genes also provide excellent cytological markers for karyotype analysis, particularly where the chromosomes are very small. FISH was first used in Brassica by Maluszynska and Heslop-Harrison (1993), who investigated all the six species of U triangle to determine the number of rDNA loci. They observed that the diploid species have more than one pair of rDNA loci (B. nigra 2, B. oleracea 3, and B. rapa 5), and the derived alloploids have fewer rDNA loci than the sum of parental species (Table 2.6). However, chromosome location of hybridization sites was generally unclear. The first precise localization of 45S rDNA sequence on mitotic chromosomes was determined in B. rapa, B. oleracea, and B. napus by Snowdon et al. (1997a). Subsequently, Fukui et al. (1998) and Hasterok and Maluszynska (2000a) analyzed rDNA sites followed by 5S rDNA sites (Schrader et al. 2000; Snowdon et al. 2000a; Hasterok et al. 2001; Kulak et al. 2002; Koo et al. 2004; Hasterok et al. 2005a). Recently two comprehensive investigations on number and physical localization of rDNA loci in a large number of genera and species in Brassica coenospecies have been carried out using both rDNA probes. Ali et al. (2005) studied karyotypes of 37 species in five tribes of Brassicaceae, many of which belong to Brassica coenospecies, and also compiled corresponding data of others (Table 2.6). In the investigation of Hasterok et al. (2006), a major focus was polymorphism number and also localization of rDNA loci in many accessions of Brassica species and related genera. These investigations clearly demonstrated the efficacy of rDNA markers in identifying the chromosomes and construction of karyotypes. Wide variations in the number and localization of rDNA sites have been observed among species in these studies (Table 2.6). The karyotypes of diploid species—B. nigra, B. oleracea, and Eruca sativa—carry the minimum number of two 5S rDNA sites. The highest number of sites was reported in B. rapa, ranging from 6 (Hasterok et al. 2001) to 10 (Hasterok et al. 2005a,b, 2006; Lim et al. 2005). The lowest number of two 45S rDNA loci among the diploids was observed in B. tournefortii while the highest was in B. rapa (10). Among the allopolyploids, the number of 5S rDNA sites varied from 4 in B. carinata to a high of 14 in B. napus (Ali et al. 2005). 45S rDNA sites are in the range of 8 to 10 in B. carinata, 12 to 16 in B. juncea, and 10 to 14 in B. napus (Hasterok et al. 2006). Regarding the

46

4 6–10

4 4 (i)y

B. oxyrrhina (18) B. rapa (20)

B. tournefortii (20) B. carinata (34)

2 8–10

4 10 (i)y

4 4 2 þ 2 minor 4 þ 2 minor (i)y (two 5S rDNA sites on the same chromosome)

B. fruticulosa (16) B. oleracea (18)

4–6

with 45S rDNA

2 (t)

z

with 5S rDNA

B. nigra (16)

Taxon (2 n)

2 —

2 4–8

2 —

—

with 5S and 45S rDNA

Number of Chromosomes

Reference Maluszynska and Heslop-Harrison 1993 (only 45S rDNA); Fukui et al. 1998 (only 45S rDNA); Hasterok and Maluszynska 2000a (only 45S rDNA); Hasterok et al. 2001; Ali et al. 2005; Hasterok et al. 2006 Hasterok et al. 2006 Maluszynska and Heslop-Harrison 1993 (only 45S rDNA); Snowdon et al. 1997a (only 45S rDNA); Armstrong et al. 1998; Fukui et al. 1998 (only 45S rDNA); Hasterok and Maluszynska 2000a (only 45S rDNA); Hasterok et al. 2001; Ziolkowski and Sadowski 2002; Snowdon et al. 2002; Ali et al. 2005; Hasterok et al. 2005a, 2006 Hasterok et al. 2006 Maluszynska and Heslop-Harrison 1993 (only 45S rDNA); Snowdon et al. 1997a (only 45S rDNA); Fukui et al. 1998 (only 45S rDNA), Hasterok and Maluszynska 2000a (only 45S rDNA); Hasterok et al. 2001; Snowdon et al. 2002; Koo et al. 2004; Ali et al. 2005; Hasterok et al. 2005a, 2006; Lim et al. 2005 Hasterok et al. 2006 Maluszynska and Heslop-Harrison 1993 (only 45S rDNA); Hasterok and Maluszynska 2000c (only 45S rDNA); Hasterok et al. 2001; Kulak et al. 2002; Ali et al. 2005; Hasterok et al. 2006

Table 2.6. Number and position of 5S and 45S rDNA sites in Brassica and allied genera.

47

8 6 6–7 8 þ 4 minor 4 6 5–6

4 (i)y 6 2 (i)y 4 (i)y 4 2 (i)y 4–5 (i)y

8 4 (t)

8 4 (t)z 6 22

Diplotaxis siifolia (20) Diplotaxis muralis (42) Eruca sativa (22) Erucastrum gallicum (30 ) Hirschfeldia incana (16) Moricandia arvensis (28) Raphanus sativus (18)

R. sativus (tetraploid) (36) Sinapis alba (24)

S. alba (tetraploid) (48) Sinapis arvensis (18) Sinapidendron frutescens (20) Orychophragmus violaceus (24)

4 2 2 8

4 2

2 — 3

2 2 2

5–8

6

Maluszynska and Heslop-Harrison 1993 (only 45S rDNA); Hasterok and Maluszynska 2000c (only 45S rDNA); Hasterok et al. 2001; Kulak et al. 2002; Ali et al. 2005; Hasterok et al. 2006 Maluszynska and Heslop-Harrison 1993 (only 45S rDNA); Snowdon et al. 1997a (only 45S rDNA); Kamisugi et al. 1998; Hasterok and Maluszynska 2000c (only 45S rDNA); Schrader et al. 2000; Snowdon et al. 2000a; Hasterok et al. 2001; Kulak et al. 2002; Snowdon et al. 2002; Ali et al. 2005; Hasterok et al. 2006 Ali et al. 2005 Hasterok et al. 2006 Ali et al. 2005; Hasterok et al. 2006 Ali et al. 2005 Ali et al. 2005 Ali et al. 2005 Schrader et al. 2000; Ali et al. 2005; Hasterok et al. 2006 Hasterok et al. 2006 Schrader et al. 2000; Ali et al. 2005; Hasterok et al. 2006 Hasterok et al. 2006 Ali et al. 2005; Hasterok et al. 2006 Ali et al. 2005 Li et al. 2005

y

z

t ¼ terminal/subterminal position of 5S rDNA. i ¼ interstitial position of 5S rDNA exclusively; interstitial and terminal position of 45S rDNA. Source: Compiled from Ali et al. 2005; Hasterok et al. 2006.

16 6 6 8

8 6–8

10–14 (i)y

9–14 (two 5S rDNA sites on the same chromosome)

B. napus (38)

12–16 (i)y

8–10

B. juncea (36)

48


location of these rDNA sites, B. rapa has 4 25S rDNA loci near the centromere of metacentric chromosomes 1, 4, 5, and 7. Chromosome 2 bears NOR and contains the fifth largest 25S rDNA locus extending over NOR and satellite. Chromosome 7 has a large 25S locus located interstitially and colocalized with a large 5S rDNA locus. Short arms of chromosome 2 and 10, the largest and smallest acrocentric chromosomes, respectively, in B. rapa genome have two more 5S loci (Snowdon et al. 2002). Koo et al. (2004) and Hasterok et al (2005a) also observed the same number of 45S and 5S rDNA loci at the same locations. Of these 10 rDNA loci, only 2 are active, distributed on the secondary constriction of chromosome 10 (Hasterok and Maluszynska 2000a). Koo et al. (2004) studied pachytene bivalents and observed 5S rDNA loci on pericentromeric region of short arm of chromosomes 2 and 10 and the long arm of chromosome 7. The long arm of chromosome 7 exhibited another 5S rDNA site, which was not detected in mitotic metaphase. These authors believe that two closely linked 5S rDNA loci could not be detected in earlier investigations because of lower resolution of FISH on mitotic chromosomes. Localization of 45S rDNA loci was revealed on pericentromeric regions of the short arm of chromosomes 1, 2, 4, and 5 and the long arm of chromosome 7. Brassica oleracea genome has two 18S-5.8S-25S rDNA sites subtelomerically on the short arms of two satellited acrocentric chromosomes (nos. 4 and 7). The third one occurs adjacent to the centromere on the short arm of chromosome 2, which is submetacentric. On the long arm of this chromosome, 5S rDNA sequences are located with closely adjacent major and minor loci (Armstrong et al. 1998). These results match those of Hasterok et al. (2001, 2005a) and Snowdon et al. (2002), who observed 5S rDNA genes in two closely adjacent loci on the long arm of a single large submetacentric chromosome 4. Two acrocentric satellite-possessing chromosomes (nos. 2 and 7) have 25S loci at the terminal ends of their short arm, which extends over the satellite. A novel 5S rDNA locus was also detected by Ziolkowski and Sadowski (2002). B. nigra has three pairs of 25S loci; one pair is located on the short arm of chromosome 6 and two pairs are located at the secondary constriction and satellite of chromosome pairs 7 and 8. Only these two pairs of loci are transcriptionally active (Hasterok and Maluszynska 2000c). The derived alloploids have fewer rDNA loci than the sum of their parents. B. carinata has four 5S and eight 25S rDNA sites, and no chromosome carries both the genes closely linked or colocalized. Ten sites of 5S and 16 sites of 25S rDNA are observed in B. juncea.


49

Chromosomes 4, 10, and 16 have colocalized both the gene sites (Kulak et al. 2002). Employing imaging methods in combination with FISH, Kamisugi et al. (1998) detected 25S/18S rDNA loci in the centromeric and distal regions of seven chromosomes in B. napus. While eight 5S rDNA loci were observed on five chromosomes mainly in the centromeric regions, two chromosomes carried both 25S/18S and 5S rDNA loci in close proximity. Regarding their localization, according to Snowdon et al. (1997a, 2000a), the largest site covers the satellite and short arm of the largest NOR-carrying chromosome. The second largest is located on a telomeric NOR-like structure on the short arm of a large subtelocentric pair, and the smallest locus is at the telomere on the short arm of a smaller submetacentric chromosome. The three other loci are localized at or near the centromeres of metacentric chromosomes. About the origin of these rDNA sites carrying chromosomes, Snowdon et al. (1997a) inferred that the two largest noncentromeric signal blocks and the NOR-carrying chromosomes closely resemble those of B. rapa and B. oleracea. Similarly, the three largest centromerically located loci in B. napus match to those of B. rapa. Schrader et al. (2000) indicated that in B. napus, one of the pair with 5S rDNA gene sites belongs to B. oleracea. Additionally, two submetacentric chromosomes having two closely adjacent 5S DNA clusters belong to B. oleracea. The other four pairs probably derived from the B. rapa progenitor. A comparison of chromosome sets of B. napus, B. oleracea and B. rapa revealed that B. napus chromosomes carrying rDNA loci could be matched with those of constituent parents (Snowdon et al. 2002). Chromosomes possessing rDNA loci could be identified based on size and centromere position. The chromosomes belonging to A and C genomes could clearly be distinguished with minor discrepancies. In general, these observations closely correspond to those of Kamisugi et al. (1998). Maluszynska and Hasterok (2005), using two-color GISH, successfully discriminated partaking genomes in B. juncea and assigned chromosomes to A and B genomes. Molecular analysis also indicated that in allopolyploids, B. nigra rRNA genes are dominant over those of B. rapa, which are in turn dominant over B. oleracea (Chen and Pickard 1997; Ge and Li 2007). However, according to Hasterok and Maluszynska (2000c), the number of Ag NORs in the alloploid species is equal to the sum of active NORs in diploid parental species, clearly indicating an absence of nuclear dominance in root meristematic cells. Views have been proposed regarding the evolutionary loss of rDNA in B. napus. It might be the result of genome organization due to translocation events between A and C genome chromosomes (Sharpe

50


et al. 1995). However, B. napus is of very recent origin, and we also have the conflicting views that the partaking A and C genomes are unaltered to a large extent (Parkin et al. 1995). In fact, Delseny et al. (1990) have reported that rDNA-carrying chromosomes of B. oleracea have not undergone any major structural changes since the evolution of B. napus. Earlier, Bennet and Smith (1991) suggested that there has been a large reduction in copy number of rDNA in present-day B. napus as compared to ancestral forms, which is due to a reduction in B. oleracea–type rDNA in existing B. napus forms from a total copy number of 1,500 to one of 800, while B. rapa–type rDNA is unaltered. Maluszynska and HeslopHarrison (1993) are of the view that a C-genome locus has been lost in B. napus due to reduction in B. oleracea–type rDNA. However, Snowdon et al. (1997a, 2000a) believe that both ancestral B. oleracea loci are still present, with reduced rDNA copy numbers, as suggested earlier by Bennet and Smith (1991). They also proposed that the smallest and relatively insignificant rDNA locus from B. rapa is absent in B. napus. On the whole, since substantial C-genome rDNA has been lost, it appears that A-genome rDNA is of greater genetic importance than C-genome rDNA in B. napus. In the species belonging to related genera Sinapis alba and Raphanus sativus, Schrader et al. (2000) detected 5S and 25S rRNA genes using double FISH. Sinapis alba possesses four 5S rDNA loci on the short arm of two submetacentric chromosome pairs and six 25S rDNA loci on the short arm of six chromosomes. In one of the chromosome pairs, both rDNA genes were closely linked on the short arm. These results were confirmed by Ali et al. (2005). Schrader et al. (2000) suggested that of six sites of 25S rDNA genes in S. alba, three represent active and three inactive loci. Raphanus sativus displays four sites each of 5S and 25S rDNA gene loci. Both rDNA genes were localized distally on the short arm (25S) and near the centromere on the long arm (25S) on one of the longest chromosome pair. The other 5S or 25S rDNA genes were separately located on the long arm of two chromosome pairs (Scharder et al. 2000; Ali et al. 2005). Investigations on 15 species in Brassica and allied genera (Table 2.6) reveal that 5S rDNA loci occupied interestitial positions in four species—Eruca sativa, Erucastrum gallicum, B. carinata, and Raphanus sativus—and terminal/subterminal and also interestitial positions in other species (Ali et al 2005). Hirschfeldia incana, Sinapis arvensis, S. alba, Diplotaxis siifolia, Sinapidendron frutescens, Eruca sativa, and Raphanus sativus have one chromosome pair carrying both loci. Sinapis arvensis and Raphanus sativus have these on opposite arms of chromosomes; in others these are located adjacently on the same arm.


51

A comparative analysis clearly revealed the occurrence of polymorphism in number and chromosomal distribution of rDNA loci among different ecotypes of a species and their population (Hasterok et al. 2006). Inter- and/or intravarietal polymorphism was evident in B. oleracea, B. rapa, B. carinata, B. juncea, B. napus, and Raphanus sativus. It was also observed that Brassica species carrying A genome—B. rapa, B. juncea, and B. napus—are highly polymorphic and contain high numbers of rDNA sites (Hasterok et al. 2001).

7. Archetype and Evolution of Genomes. Several investigators (Thompson 1956; Ro¨bbelen 1960; Prakash and Hinata 1980) have proposed that basic diploid species are polyploids that evolved from a common ancestor in the ancient past. While evidence are presented that this archetype was a smaller genome, views on the genetic constitution of the ancestral genome are variable, proposing the ancestral basic numbers x ¼ 37: Catcheside (1934, 1937), basing his conclusion on secondary chromosome associations in B. napus and B. oleracea, suggested x ¼ 6 as the basic number. He also believed that the primitive haploid number in Brassicaceae is x ¼ 7 from which x ¼ 6 arose by fusion of two chromosomes. Alam (1936) and Haga (1938) supported Catcheside’s views after observing secondary pairing in the three basic diploid species. Sikka (1940) proposed a basic number x ¼ 5, from the study of secondary bivalent associations in three species—B. monensis (syn. Coincya monensis, 2n ¼ 24), B. sinapistrium (syn. Sinapis arvensis, 2n ¼ 18), and B. nigra—and suggested the evolution in Brassica toward tetraploidy, citing the series 2n ¼ 30, 60, 90, and 120 in the genus Crambe, all multiples of x ¼ 5. However, the pachytene chromosome analysis in diploid species revealing six basic types of chromosomes led Ro¨bbelen (1960) to suggest that the archetype had x ¼ 6 as the basic constitution. This is supported by meiotic chromosome pairing in haploids of B. nigra and B. tournefortii (Prakash 1974a,b) and substantiated by GISH in trigenomic ABC hybrids (Ge and Li 2007). An entirely different number of x ¼ 3 was proposed by Hussein and Abobakr (1976) based on secondary bivalent associations in B. oleracea. This view found support from: (1) Chen and Heneen (1991) and Cheng and Heneen (1995), who observed three pairs of satellited chromosomes with active NORs in B. nigra and S. arvensis; (2) three pairs of chromosomes carrying 25S rDNA gene loci in B. nigra (Fukui et al. 1998), B. oleracea (Maluszynska and Heslop-Harrison 1993; Snowdon et al. 1997a; Armstrong et al. 1998; Ali et al. 2005; Hasterok et al. 2006), S. alba (Schrader et al. 2000), and S. arvensis (Ali et al. 2005);

52


and (3) three copies of the gene encoding acyl-CoA-binding protein in B. rapa and B. oleracea (Hills et al. 1994). It is inferred that the number of three chromosome pairs carrying the 25S rDNA gene is basic for the family Brassicaceae. The earlier view was that the three basic diploid species evolved from a common archetype following duplication of whole chromosomes (i.e., aneuploidy) accompanied by differentiation following structural changes. The mapping data, in contrast, clearly discounts the role of polysomy or duplication of whole chromosomes (Quiros 1999). Recent information emerging from use of molecular markers firmly disproves the theory of monophyletic origin and instead suggests a biphyletic origin of the diploid species. It concludes that B. oleracea/ B. rapa originated from one archetype while B. nigra originated from the other (Song et al. 1990; Warwick and Black 1991; Pradhan et al. 1992). Cytogenetical investigations preceeded in predicting this genetic divergence between B. nigra and B. oleracea/B. rapa based on chromosome pairing in hybrids (Mizushima 1950a; Prakash and Hinata 1980). Subsequently, molecular analysis has been very revealing. The first evidence came from nuclear RFLPs by Song et al. (1988a), which was substantiated by other investigations. Information from nuclear, chloroplast, and mitochondrial DNA RFLPs has established that the primitive genome diversified into two lineages and all the taxa in subtribe Brassicinae fall in these two lineages (Warwick and Black 1991; Pradhan et al. 1992). This view also gets support from a comparative study of molecular markers (Lagercrantz 1998) and rDNA intergenic spacer (Bhatia et al. 1996). The evolutionary divergence is also reflected in their cytoplasm (Palmer 1988; Warwick and Black 1991; Pradhan et al. 1992). B. oleracea and B. rapa cytoplasms are closer to each other than either is to B. nigra (Palmer 1988). Evidence also indicates that the A genome was derived in the distant past from an already existing C genome, as these two genomes have extensive genomic regions of conserved homology (Slocum 1989). Many investigations during the past 15 years have dealt with comparative mapping of various genera and species, which throws light on the constitution of archetype and history of polyploidy. Truco et al. (1996) suggested a range of x ¼ 5 to 7 of this ancestral archetype, which was further narrowed down to x ¼ 4 by Lagercrantz (1998). Evidence cited in support was that several species of the related subtribe Lepidieae also have n ¼ 4 chromosome number, the lowest in Brassicaceae family (Mulligan 1964). Henry et al. (2006), based on recent genome sequencing data, also support n ¼ 4 as the basal


53

genomic number of the family, followed by tetraploidization before the separation of the Brassica and Arabidopsis lineages. An interesting hypothesis proposed by Lagercrantz and Lydiate (1996), Lagercrantz (1998), and O’Neil and Bancroft (2000) envisages that (i) Arabidopsis shares common ancestry with Brassica crop species, and (ii) three ancestral species with x ¼ 5 and whose genomes were similar to Arabidopsis genome gave rise to a hexaploid following hybridization between them. This was the ancestral archetype of A, B, and C genomes from which the basic genomes evolved through reduction in chromosome number by extensive chromosome fusion. This view, known as the triplication theory, gets support from the fact that some loci are triplicated as detected by molecular markers (Cavell et al. 1998; O’Neil and Bancroft 2000; Parkin et al. 2002, 2003, 2005; Rana et al. 2004; Lysak et al. 2005, 2007; Park et al. 2005; Yang et al. 2005; Lim et al. 2006; Matthew and Lydiate 2006; Nelson and Lydiate 2006; Ziolkowski et al. 2006; Yang et al. 2006) and also that diploid Brassica genomes contain approximately three times the DNA of Arabidopsis genome (Arumugnathan and Earle 1991). The event of hexaploidy occurred around 7.9 to 14.6 million years ago (Lysak et al. 2005). However, the occurrence of a large proportion of heterochromatin, repetitive DNA (Gupta et al. 1990, 1992; Iwabuchi et al. 1991), transposable elements (TE) (Zhang and Wessler 2004; Gao et al. 2005; Lim et al. 2007), and the ancestral role of Arabidopsis would argue against this hypothesis. Lukens et al. (2004) also found no strong evidence of the role of the ancestral hexaploid genome. Furthermore, considering that the ancestral species to the Brassicaceae was a tetraploid of 2n ¼ 4x ¼ 16 (Henry et al. 2006), it already explains the origin of species such as B. nigra, also with 2n ¼ 16, without the need to invoke another round of polyploidization or hexaploidy. Another hypothesis referred to as cyclic amphiploidy was proposed by Truco et al. (1996) and Quiros (1999), which suggests that hybridizations occurred among different species with x ¼ 4 and/or x ¼ 5 chromosome constitution. These species were derived from an ancestral Brassica archetype genome with x ¼ 4 or 5. Each pair of species crosses gave rise to A, B, and C genomes and are partial allopolyploids (Fig. 2.3). Also sequidiploidy resulting from hybridization of allotetraploids to ancestral diploids could have been originated genomes of various chromosome numbers. Structural chromosomal modifications, mostly reciprocal translocations, played a pivotal role initially in the origin of these genomes (Quiros 1999). Recent evidence also supports further and constant genome modification by insertion and movement of TE (Zhang and Wessler 2004; Gao et al. 2005;

54


Ancestral genome x =4, 5 ?

Chromosomal changes Geographic isolation Derived genomes x=4, 5

Chromosomal changes Z1

TE Z2

1st cycle of allopolyploidy

Diploid species genomes

Z4

Z3

Zn

Aneuploidy B

A

C

Structural changes 2nd cycle of allopolyploidy

Cultivated allopolyploids

BC

AB

AC

Fig. 2.3. Cyclic amphidiploidy and the origin of Brassica diploid and allopolyloid species solid and broken lines represent female and male parents respectively in allopolyploids. (Source: After Quiros 1999.)

Lim et al. 2007). Surprisingly, the genome size of these basic species has remained practically unaltered in spite of changes in chromosome numbers and structure. Based on marker arrangement conservation, Truco et al. (1996) proposed a model of genome evolution and phylogenetic relationships among the chromosomes of the three basic species considering two assumptions: that (1) A and C genomes are closely related, and possibly C genome is the predecessor of A genome; and (2) the genus Brassica is of biphletic origin. It envisages that the ancient genome possessed at least five and no more than seven chromosomes. B and/or C genome chromosomes evolved from six ancestral chromosomes (W1 to W6) (Fig. 2.4). C genome chromosomes also gave rise to A genome chromosomes. Two intermediate chromosomes Bx and Cx originated from W1. Bx produced B1, B2, B4, and B8 chromosomes and the Cx


A10

A5

C5

55

A4

B6

C1

B3

A1

B5 W3

C4 A6

C7

W5

W4

C×

W2

A3

C3

W1

W6

C6

A7

C8

B×

B1

A8 C9

B8 B7

B4

A9

B2 C2

A2

Fig. 2.4. Hypothetical ancestral genome of six chromosomes (W1 to W6) originating specific A, B and C genome chromosomes deduced by homoeologous relationships. Bx and Cx are intermediate chromosomes. Broken lines indicate tentative homologies. (Source: From Truco et al. 1996.)

chromosome gave rise to A7. Chromosomes Bx and C1 were similar in their genetic content. Chromosomes B7 and C9 might have originated from W6 or independently, one from W6 and other from a seventh ancestral chromosome, W7. These two chromosomes, B7 and C9, do not share homology with any other group. In spite of their biphyletic origin, the three basic genomes still share regions of homology, as determined by Truco et al. (1996), expressed in cM by adding the distance of chromosome segments sharing homology between the two genomes following the comparison of linkage maps of these species. The lowest homology is between A and B genomes, which share 92.7 and 219.5 cM of their genomes respectively and results in up to six bivalents in hybrids between them (Prakash 1973a,b). Homology between B and C genomes is intermediate with 223 and 365.7 cM respectively and form up to four bivalents between them (Mizushima

56


1950a; Song et al.1993). The highest homology is observed between A and C genomes where they share 337.2 and 487.2 cM respectively and form up to nine bivalents (Olsson 1960b). ISSR data also reflected these relationships, as observed by Liu and Wang (2006). The average genetic distance between B. rapa and B. oleracea is 0.499, indicating close homology; between B. rapa and B. nigra, 0.528; and between B. oleracea and B. nigra, 0.615 showing clearly the divergence between A/C and B genomes. In fact, the genomic contents of A and C genomes are equivalent, and rearrangements are the cause of difference in their chromosome number (Parkin et al. 2003). To summarize, basic Brassica genomes evolved and differentiated from an originally smaller genome. Chromosome arrangements due to homoeologous recombination and hybridizations were the major factors in their stabilization. These three species are, in fact, secondary polyploids with regions of shared ancestry. As expected, duplications are widespread in these genomes.

III. GENOME MANIPULATION A. Resyntheses of Natural Allopolyploid Brassica spp. Soon after the proposals of Morinaga (1928–1934) on genome analysis, Nagaharu U (1935) presented the cytogenetical relationships among different Brassica crop species. He is also credited with substantiating it by experimental synthesis of B. napus. A little later Ramanujam and Srinivasachar (1943) successfully produced B. juncea. Simultaneously, Frandsen (1943, 1947) synthesized all the three allopolyploid species. These early academic reports were primarily aimed at verifying Morinaga’s proposals. Subsequently, the activities shifted to enhancement of genetic variability of breeding value utilizing extensive genetic and phenotypic range of variation available in constituent parents. It is now believed that intensive breeding, particularly in B. napus, which is a relatively younger species, and also in B. juncea, has led to a narrowing of the genetic base. In contrast, experimentally resynthesized strains have considerably broadened the gene pool for use by the breeders. These synthetics are also valuable in heterosis breeding programs; several investigations report high heterosis in crosses between synthetics and crop cultivars (Pradhan et al. 1993; Jain et al 1994; Girke et al. 1999; Seyis et al. 2003). Literature on sexually and parasexually produced versions of naturally occurring Brassica species is summarized in Tables 2.7 and 2.8.


57

Table 2.7. Major investigations on artificial synthesis of natural allopolyploid species B. carinata, B. juncea, and B. napus through sexual hybridization. Species

Reference

B. carinata (B. nigra B. oleracea and reciprocals) B. juncea (B. rapa B. nigra and reciprocals)

B. napus (B. rapa B. oleracea and reciprocals) Oil rape

Forage rape Rutabaga

Heading form

Frandsen 1943; Mizushima 1950b; Pearson 1972; Prakash et al. 1984; Song et al. 1993 Frandsen 1943; Ramanujam and Srinivasachar 1943; Olsson 1960a; Prakash 1973a,b; Campbell et al. 1990, 1991; Song et al. 1993; Srivastava et al. 2001, 2004; Se´guin-Swartz et al. 2004

U 1935; Karpechenko and Bogdanova 1937; Frandsen 1947; Rudorf 1950; Hoffmann and Peters 1958; Olsson 1960b; Gland 1982; Prakash and Raut 1983; Chen et al. 1988a,b; Chen and Hennen 1989; Akbar 1989; Hossain et al. 1990; Mithen and Magrath 1992; Song et al. 1993; Ozminkowski and Jourdan 1994a,b; Beschorner et al. 1995; Heath and Earle 1996; Girke et al. 1999; Lu et al. 2001; Rahman et al. 2001; Zhang et al. 2002; Happastadius et al. 2003; Luhs et al. 2003; Seyis et al. 2003; Niu et al. 2004; Zhang et al. 2004; Abel et al. 2005; Rahman 2005; Zhou et al. 2007; Wen et al. 2008 Hosoda 1950, 1953, 1961; Feng 1955; Sarashima 1967, 1973; Hosoda et al. 1969; Nishi et al. 1970 Olsson et al. 1955; Olsson 1960b; Hosoda et al. 1963, 1969; Namai and Hosoda 1967, 1968; Kato et al. 1968 Shinohara and Kanno 1961

Table 2.8. Major papers on synthesis of natural allopolyploid species through protoplast fusion. Brassica species carinata juncea napus

Reference Narasimhulu et al. 1992; Jourdan and Salazar 1993 Campbell et al. 1990, 1991; Se´guin-Swartz et al. 2004; Bhat et al. unpubl. Schenck and Ro¨bbelen 1982; Sundberg and Glimelius 1986; Taguchi and Kameya 1986; Robertson et al. 1987; Sundberg et al. 1987; Terada et al. 1987; Rosen et al. 1988; Jourdan et al. 1889b; Hossain and Asahira 1992; Yamagishi et al. 1992; Ozminkowski and Jourdan 1993, 1994a,b; Hansen and Earle 1994; Heath and Earle 1995, 1996, 1997; Ren et al. 2000; Lian and Lim 2001

58


The objectives in these allopolyploid syntheses vary, from purely academic (e.g., Song et al. 1993; Srivastava et al. 2001, 2004), to developing agricultural forms that include early and productive B. carinata forms for Indian conditions (Prakash et al. 1984), highseed-yielding B. juncea (Olsson 1960a; Prakash 1973a), early-maturing B. napus suitable for the Indian subcontinent (Prakash and Raut 1983; Akbar 1989), productive oil seed B. napus (Olsson 1960b; Seyis et al. 2003), fodder forms of B. napus (Namai and Hosoda 1967, 1968; Ellerstro¨m and Sjo¨din 1973), root-forming sweeds or rutabagas (Olsson et al. 1955; Kato et al. 1968; Namai and Hosoda 1968), and a new headforming vegetable form (Shinohara and Kanno 1961; Takeda 1986). A major objective in B. napus syntheses in recent years has been the modification of oil and meal quality (Lu et al. 2001; Lu¨hs et al. 2003; Seyis et al. 2005) and incorporation of yellow seed coat color (Shirzadegan and Ro¨bbelen 1985; Liu and Gao 1987; Chen et al. 1988; Tang et al. 1997; Meng et al. 1998; Baetzel et al. 1999; Rahman 2001; Wen et al. 2008). Initially these studies were carried out chiefly in Japan, Sweden, Germany, and India, and later in other countries. In fact, resynthesis has been widely attempted for improvement of B. napus (Olsson and Ellerstro¨m 1980; Chen and Hennen 1989b; Lu¨hs et al. 2002; Friedt et al. 2003). Synthetic alloploids of all three species have been obtained through sexual hybridizations from reciprocal crosses. In some combinations, hybrids are difficult to obtain because of postfertilization barriers, which are generally overcome by sequential ovary culture or embryo rescue. Failure of normal endosperm development following fertilization is the major cause for hybrid embryo abortion (Inomata 1976; Wojciechowski 1985). Highly significant influence of maternal genotype on crossability, particularly in the synthesis of B. napus, has been observed (Diederichen and Sacristań 1994; Lu et al. 2001; Abel et al. 2005). Similarily, the role of cytoplasm has also been implicated in the success of hybridization (Song et al. 1993). It is observed that hybrids with the same cytoplasm as natural alloploids are easier to synthesize than others (Song et al. 1993). Synthetic B. juncea with B. nigra cytoplasm and B. napus with B. oleracea cytoplasm are difficult to obtain. In general, synthesis of B. juncea and B. carinata is easier than synthesis of B. napus (Prakash and Chopra 1991; Song et al. 1993; Stewart 2004). However, in vitro techniques, such as embryo and ovary culture, have made possible the generation of a large number of synthetic alloploids of choice. Protoplast fusion technology for Brassica became available in 1980s and enabled synthesis of somatic hybrids of desired combinations. Besides


59

enlarging nuclear variability, this methodology generates novel combinations of cytoplasmic organelles and has received considerable attention in recent years. Synthesis of somatic hybrids of B. napus by Schenck and Ro¨bbelen (1982) was the earliest success. Most reports on somatic hybridization relate to synthesis of B. napus. Ozminkowski and Jourdan (1994a,b) and Heath and Earle (1996) reconstructed B. napus both sexually and following somatic cell fusion. B. napus allohaploids have also been synthesized by fusing pollen protoplasts of B. oleracea var. italica and haploid mesophyll protoplasts of B. rapa; Fan et al. (2007) present the first report about a hybrid formation between two haploid protoplasts. Hybrids could be obtained faster through somatic fusion because of avoidance of chromosome doubling in F1 sexual hybrids for restoring fertility. Somatic hybrids have also been obtained involving B. carinata and B. juncea (Table 2.8). These investigations on somatic hybridizations represent significant breakthroughs in interspecific hybridizations. Synthetics obtained following sexual hybridization and chromosome doubling have the sum of the parental chromosome number. The somatic hybrids also have, in general, these summations. However, in some somatically produced B. napus, there were deviations, where the plants possessed variable chromosome number ranging from 33 to 57 (Table 2.9). These resulted from triple fusions, such as one B. oleracea and two B. rapa protoplasts (2n ¼ 58) or vice versa, resulting in digenomic hexaploid AAAAACC (2n ¼ 58) and AACCCC (2n ¼ 56) plants (Terada et al. 1987; Heath and Earle 1996). Aneuploids with somatic chromosome number 33, 49, 54, 57 were also recorded. These probably originated by chromosome elimination during regeneration and subsequent development of plants. Somatic hybrid plants of B. carinata had the normal chromosome number of 34 (Narasimhulu et al. 1992; Jourdan and Salazar 1993). Campbell (1993), Se´guin-Swartz et al. (2004), and Bhat et al. (unpublished) also observed the normal somatic chromosome number in B. juncea somatic hybrid plants (Table 2.9). Meiosis in the synthetic allopolyploids obtained from either sexual or somatic hybridizations was disturbed in early generations, primarily due to the preponderance of univalents and multivalents. However, it was more normal in somatic hybrids. Up to four quadrivalents in B. carinata (Mizushima 1950b; Narasimhulu et al. 1992) and up to three quadrivalents and two trivalents occurred in B. napus (Sarashima 1973; Prakash and Raut 1983; Wen et al. 2008). Higher associations were rare or absent in B. juncea (Olsson 1960a; Prakash 1973a). The number of multivalents and univalents decreased with the

60

B. napus 12 4 10 23

5

B. juncea 3

38, 36, 54 38 38, 33, 36, 49, 56, 57 38, 50–68

36, 18 II–16 II þ 4 I

36, 18 II

34, 56

34, 17 II, 11 II þ 3 IV

B. carinata 74

64

Chromosome no. pairing at M1

No. somatic hybrids obtained

Rapa (2) Nig (1) Nig (3) Rec (2)

Ole (5) Nig (18) Novel (1)

Ole (1) Nig (3)

CP

Rapa (5) Ole (6) ND (12)

Rapa (3) Nig (2)

Rapa (3)

Ole (6) Nig (18)

Ole (1) Nig (2) Rec (1)

MT

Organelle genome composition

6–70

4–98

36–87

Pollen

very poor

very poor

19

Seed

Fertility (%)

Table 2.9. Summary of results of somatic hybrids of B. carinata, B. juncea, and B. napus.

Schenck and Ro¨bbelen 1982 Taguchi and Kameya 1986 Terada et al. 1987 Sundberg et al. 1987

Bhat et al. unpubl.

Campbell 1993

Jourdan and Salazar 1993

Narasimhulu et al. 1992

Reference

61

38 38, 56, 58 Rapa (30) Mix (9) Rapa (38) Rapa (8) Ole (16)

Rapa (3) Ole (2) 38, 58, aneuploid also Rec (12) Rapa (7) Rapa (1) Rec (10) 38,58

36–38 38, 19 II–18 II þ 2 I

z

ND ¼ not determined. Rec ¼ recombined. Number in parentheses denotes the number of plants.

38 24

109 39

72

11

34

1 5

Rapa (12) Ole (10) Rec (2)

Ole (34) Rapa (5) Ole (6)

Ole (3)

NDz

0–97

fertile –—

ND

good

low – high

sterile very poor

Cardi and Earle 1997

Heath and Earle 1997

Ozminkowski and Jourdan 1993, 1994a,b Heath and Earle 1995 Heath and Earle 1996

Jourdan et al. 1989 Sundberg and Glimelius 1991a

Robertson et al. 1987 Rosen et al. 1988

62


advancing generations, and meiotic stabilization with regular bivalent formation was achieved by the amphidiploid 3 (A3) generation. Sexually obtained synthetics had reduced pollen and seed fertility in early generations, sometimes as low as 6% in B. juncea (Olsson 1960a). With stabilization of meiosis and selection, fertility improved considerably. By the A5 generation, the attained fertility was much higher. A7 generation plants had fertility comparable to naturally occurring forms (Table 2.10). Somatic hybrids also had very low pollen fertility and seed set. Pollen was ineffective in producing seeds on selfing or on pollinations to natural forms of B. carinata (Jourdan and Salazar 1993). Similar observations were recorded for B. napus by Rosen et al. (1988), Sundberg et al. (1987), and Heath and Earle (1996, 1997). Since the plants had more or less regular meiosis, the reasons for a high degree of sterility are unknown. The organellar constitution of somatic hybrids does not follow any pattern; all possible combinations of mitochondria and chloroplast genomes are observed in addition to frequent intergenomic mitochondrial recombination. A majority of B. napus somatic hybrids contain B. rapa chloroplast; some have both chloroplast types (heteroplastidic); and only a few have B. oleracea chloroplasts. They contain mostly B. rapa and recombinant mitochondria. Some plants also have a mix of mitachondria and chloroplast genomes of B. rapa and B. oleracea. Most B. carinata plants contain both chloroplast and mitochondrial genomes from B. nigra, but some combine these from both the parents. A similar phenomenon is observed with in B. juncea, where combinations of chloroplast and mitchondria parental genomes have been obtained as shown in Table 2.9 (Bhat et al. unpublished). B. Agronomic Potential of Synthetics Attempts to synthesize allopolyploids and use them either directly or as useful germplasm have been made in several countries. The general observation has been that synthetics do not compete for productivity with cultivated forms but are a rich reservoir of diversity, as revealed by allozyme and molecular marker studies (Chen et al. 1989; Song et al. 1993; Becker et al. 1995; Seyis et al. 2003; Srivastava et al. 2004; Liu and Yang 2004). Their value is particularly relevant for such traits as productivity, disease resistance, and quality parameters and are utilized in backcross programs to introgress these novel characters. Hybridization between synthetics and elite breeding lines have resulted in many high-yielding cultivars because of high genetic distance between them.

63

— 17 II þ 4 IV þ 9 II

17 II–1 4 II þ 6 I

—

18 II mostly 16 II þ 4 I–18 II

—

19 II 2 IV þ 15 II–19 II 19 II mostly

19 II mostly

17 II16 II þ 2 I 17 II þ 4 IV þ 9 II

17 II þ 4 IV þ 9 II

12 II þ 10 I

18 II16 II þ 4 I 18 II14 II þ 8 I

12 II þ 12 I

19 II18 II þ 2 I 2 IV þ 2 III þ 7 II 3 IV þ 9 II þ 8I

19 II mostly

B. carinata

B. napus

B. juncea

A2

A1

Alloploid

Chromosome pairing

—

— — —

18 II 18 II mostly —

— 17 II mostly 17 II mostly —

A3

poor

7–43 8.8 7–29

poor

6.2 26.1

poor

22–31

very poor poor

A1

poor

89 24 1952

poor

26.6 45.8

poor

26–69

very low 21.3

A2

—

96 36 5892

3164 6469

5482

— 27–53

A3

—

96 59 81–94

43–92 96

87

— 47–62

A4

Seed fetility (%)

—

96 79 96

96 96

94

— 87

A5

—

96

96

96 96

04

— 92

A6

Olsson 1960b Sarashima 1973 Prakash and Raut 1983 Song et al. 1993

Song et al. 1993

Olsson 1960a Prakash 1973a

Song et al. 1993

Frandsen 1947 Mizushima and Katsuo 1953 S. Prakash unpubl.

Reference

Table 2.10. Chromosome pairing and seed fertility in sexually synthesized Brassica allopolyploids: B. carinata, B. juncea, and B. napus.

64


Desirable characteristics in synthetic B. carinata include dwarf stature, early flowering, and high pod density (Prakash and Chopra 1996). A derivative of a synthetic ‘Pusa Gaurav’ has been released for cultivation in India. B. juncea synthesized from leafy vegetable forms has vigorous vegetative growth with more biomass and has been identified as preferred fodder. Synthetics originating from oleiferous forms of B. rapa are high yielders and contain more oil. Some of these are also early maturing (Prakash 1973a; Prakash and Chopra 1996). High-yielding cultivars released in India, such as ‘Pusa Jagannath’ and ‘Pusa Agrani’, have originated from synthetics. Several oil-yielding forms of B. napus that are derivatives of synthetics have been marketed in Europe. These include ‘Svalo¨f Panter’, a high-oil-yielding form suitable for low-temperature conditions; ‘Svalo¨f Norde’, a high-seed and oil-yielding form with considerable resistance to Peronospora and Verticillium; and ‘Brink’ and ‘Jupiter’, which combine high seed yields with very low erucic acid content (Olsson 1986). Several synthetic zero-erucic acid lines possess a high degree of resistance to Verticillium longisporum (Rygulla et al. 2007). A number of hybrids between synthetics and cultivars in B. napus have yield potential as high as 40 q/ha (Seyis et al. 2003,2006). Some of the synthetic strains that are early maturing and productive have been found to be promising for cultivation in India (Prakash and Raut 1983) and Bangladesh (Zaman 1989), where the photo- and thermo-sensitive strains from Europe and Canada remain vegetative and do not flower. Early-flowering (RC) and vigorous strains were also obtained by Hansen and Earle (1994). Among synthetics are strains that exhibit a high degree of resistance to diseases such as black leg (Mithen and Magrath 1992), clubroot (Diederichsen and Sacristań 1996), bacterial soft rot (Ren et al. 2000, 2001), Verticillium wilt (Happastadius et al. 2003), turnip yellows luteovirus (TuVV) (Paetsch et al. 2003), and pod shatter (Child et al. 2003). Winter oilseed rape varieties ‘Mendal’ and ‘Tosca’ combining clubroot resistance and cv. ‘Surpass’ carrying resistance to black leg were marketed in Europe in the last two decades. Synthesis of yellow-seeded B. napus has been the objective of many investigations because of the association of yellow seededness with increased oil and lower fiber content and also the improved feed quality of meal (Shirzadegan and Ro¨bbelen 1985; Chen et al. 1988; Chen and Hennen 1992; Tang et al. 1997; Meng et al. 1998; Baetzel et al. 1999; Rahman 2001; Wen et al. 2008), and such yellow-seeded synthetic forms have been obtained. A super-long silique form of resynthesized B. napus has been developed in China (Niu et al. 2004). Genetic variants for low glucosinolate content (Gland 1982) and


65

modified fatty acid composition, particularly low erucic acid, have been created through resynthesis in B. napus (Chen and Hennen 1989b; Lu et al. 2001; Lu¨hs et al. 2002, 2003; Seyis et al. 2005). Synthetic rapeseed with high erucic acid content for industrial use has also been produced (Chen et al. 1989a; Lu¨hs and Friedt 1994, 1995a,b; Weir et al. 1997; Lu¨hs et al. 1999a,b; Han et al. 2001). Heath and Earle (1996, 1997) introduced a nonshattering trait and large-size seeds in somatic hybrids of B. napus. These authors also obtained B. napus somatic hybrids that were low in linolenic acid (Heath and Earle 1997) and high in erucic acid content (Heath and Earle 1995). Synthetic selfincompatible B. napus lines have recently been obtained through sexual hybridizations for developing commercial F1 hybrids (Rahman 2005). Fodder forms of B. napus have been synthesized in Japan and Sweden using leafy and root-forming forms of B. rapa—ssp. chinensis, pekinensis, narinosa, nipposinica, and rapa. Hosoda (1950) bred a fodder rape ‘CO’, which was very popular in Japan because of its vigorous growth and winter hardiness. A novel synthetic head-forming vegetable type has been developed in Japan from the cross B. oleracea var. capitata B. rapa ssp. pekinensis. It is a popular vegetable form released in 1968 under the name ‘Hakuran’. It has soft leaves, fewer fibers, tastes like heady lettuce, and possesses high degree of resistance to soft rot (Shinohara and Kanno 1961; Takeda 1986). Such a type has also been produced using the same parents through protoplast fusion (Taguchi and Kameya 1986). As has been pointed out by many investigators, the germplasm pool of cultivated alloploid species is narrow. The genetic base can be enlarged by synthesizing polyploids with specifically chosen types of constituent parents. Two of the diploid constituent species—B. rapa and B. oleracea—are highly polymorphic, encompassing enormous physiological and morphological variations. These are of relatively recent origin and have not participated in the evolution of natural alloploid forms. Utilization of this variation will generate novel variability not available in the conventional germplasm. At present, resynthesis is routinely carried out using in vitro techniques following reciprocal hybridizations to gain the advantage of reciprocal cytoplasms, and by protoplast fusion to obtain various combinations of cytoplasmic organelles and mitochondrial recombination. Further variability in these synthetics also arises from intergenomic recombination between the chromosomes of constituent parents during early generations of allopolyploidy (Prakash 1973b). The desirable attributes of synthetics, such as disease resistance and quality characters, must

66


be integrated into high-yielding varieties either by developing semisynthetic forms or in backcross breeding programs (Kraling 1987; Friedt et al. 2003). Many studies have suggested that heterosis for seed yield in intervarietal hybrids is positively correlated with genetic distance (Jain et al. 1994; Ali et al. 1995; Diers et al. 1995; Seyis et al. 2003; Shen et al. 2003; Burton et al. 2004). The variability and genetic distance of synthetics from cultivars in cultivation can be usefully exploited for generating both highly productive hybrids and genetically enhanced cultivars. Seyis et al. (2006) demonstrated the potential of synthesized B. napus for developing experimental hybrids having high yields. C. Diploidization of Allopolyploid Species As discussed earlier, the three basic genomes of Brassica crops share considerable homoeology among their chromosomes as evidenced from frequent occurrence of bivalents in diploid hybrids AB, AC, and BC. As a consequence, intergenomic allosyndetic pairing is expected in allopolyploids. However, the extant Brassica allopolyploids exhibit diploidlike meiosis and a completely bivalent forming regime. This diploidization of polyploids is an important step in their evolution and successful colonization. Several mechanisms including structural alterations in chromosomes, incorporation of adaptive mutations that prevent homoeologous chromosome pairing, and the silencing of duplicate genes have contributed this diploidization. It has been proposed that diploidlike meiosis is genetically regulated in Brassica and its related genera (Prakash 1974c; Harberd 1976; Attia and Ro¨bbelen 1986; Eber et al. 1994; Sharpe et al. 1995; Jenczewski et al. 2003). Prakash (1974c) proposed a genetic mechanism in B. juncea that is similar to the one that operates in wheat, where a gene is responsible for diploidlike meiosis and, when, absent allows paring of homoeologous chromosomes disturbing normal meiosis. His observations are based on the high extent of chromosome pairing in plants originating from a translocation heterozygote. These plants represent duplicationdeficiency types in which a very small segment presumably carrying the pairing regulator gene(s) is missing and which accounts for normal bivalents regime. However, because of the absence of monosomic series in alloploid Brassica, it has not been possible to validate this proposal experimentally. Attia and Ro¨bbelen (1986) observed low meiotic pairing in hybrids when B. nigra was one of the parents—for example, B. rapa B. nigra and B. nigra B. oleracea—and suggested a genetic meiotic suppressor in B. nigra genome that restricts the homoeologous


67

pairing. However, this view was later discounted (Busso et al. 1987). By observing frequent intergenomic recombination in a B. rapa–B. alboglabra monosomic addition line but not in trigenomic AAC hybrids, Chen et al. (1992) proposed that it could be due to a pairing control mechanism. Jenczewski et al. (2002) postulated a pairing regulator gene for diploidlike meiotic regime in an induced autotetraploid of B. oleracea. Later, a hypothesis that envisions the presence of a major gene PrBn (Pairing regulator Brassica napus) in alloploid B. napus was proposed by Jenczewski et al. (2003). These authors studied the chromosome pairing in low- and high-bivalent-forming haploids of B. napus and observed that chromosome pairing patterns are inherited in a Mendelian way, indicating the presence of a major gene for restricting the homoeologous pairing. It was also suggested that since regular bivalents are observed in all B. napus accessions, regardless of bivalent frequency in their haploids, PrBn could contribute to the regularity of chromosome pairing. It could be ineffective at hemizygous stage or at least less efficient as compared to at the diploid state (Jenczewski and Alix 2004). PrBn gene has been mapped on a C genome chromosome and displays complete penetrance. Additionally, three to six minor QTL/BTL have slight additive effect on pairing without any interaction with PrBn. However, a number of other loci interact epistatically with PrBn (Liu et al. 2006). The other mechanism implicated in stabilization of alloploids is genic changes. Song et al. (1988a), following studies on nuclear RFLPs of diploid and alloploid species observed that nuclear DNA composition of alloploid species is closer to their cytoplasmic donor species than to the male parent donors. They concluded that when constituent diploid species of alloploids have highly differentiated cytoplasm, the nuclear genomes of male parent undergo substantial changes as compared to nuclear genome contributed by cytoplasmic donor. As mentioned earlier, two divergent types of cytoplasms occur in basic species: the B type as present in B. nigra and the A/C type as present in B. rapa and B. oleracea and quite diverged from the B type. As revealed by cp DNA studies (Erickson et al. 1983; Palmer et al. 1983a), B. juncea and B. napus have A type cytoplasm, while B. carinata has B type cytoplasm. During their evolution, nuclear genomes of male parents in B. juncea and B. carinata have undergone extensive changes while the nuclear genomes of female parents remained intact. In B. napus, both the genomes A and C have evolved with similar rates of change (Song et al. 1988a). Such changes are brought about by interaction between nuclear and cytoplasmic genomes and play an important role in stabilization of raw allopolyploids. The cytoplasm exerts selection

68


pressure on alien nuclear genome and brings about a harmoneous interaction between cytoplasm and both the nuclear genomes in the new environment. To understand the process of changes in the genomes, Song et al. (1993) developed a series of synthetic alloploids of B. carinata, B. juncea, and B. napus following reciprocal hybridizations and characterized them for RFLP patterns of nuclear and cytoplasmic genomes. It was observed in subsequent generations that frequency of genome changes are associated with genetic divergence of constituent diploid parents: the more the genetic divergence, the higher the frequency of changes (Song et al. 1995). These changes could have resulted from chromosome rearrangements, point mutations, gene conversion, and DNA methylation. Intergenomic homologous recombination could lead to chromosome rearrangements and provide opportunities for gene conversion–like events (Osborn 2004; Pires et al. 2004, 2006). It has been suggested that extensive genome changes occur during early generations of polyploidy, and this accelerates the evolutionary processes (Song et al. 1995; Lukens et al. 2006). Also, intergenomic heterozygosity and epigenetic changes give rise to new variations crucial to their ecological success (Schranz and Osborn 2004). Another factor in stabilizing the chromosome pairing may be the role of rRNA genes. It has been reported in a number of allopolyploids that rRNA genes from only one parent are transcribed while the transcription of such genes of the other parent is suppressed: a phenomenon referred to as nucleolar dominance. A hierarchy of nucleolar dominance has been demonstrated to be B. nigra > B. rapa > B. olerace in three Brassica allotetraploids (Chen and Pikaard 1997; Pikaard 2000; Ge and Li 2007). These results suggest that nucleolar dominance may contribute decisively in preferential stabilization of chromosomes from rRNAsdonor parent. D. Raphanobrassica Raphanobrassica does not occur in nature but is a man-made curiosity—a hybrid between Raphanus sativus and B. oleracea. There is a long history of hybridizing Raphanus with B. oleracea. A sterile hybrid was obtained as early as 1826 by Sageret. However, the first fertile Raphanobrassica was synthesized by the Russian botanist Karpechenko (1927) following chromosome doubling of the hybrid R. sativus and B. oleracea. The classical Raphanobrassica has 36 somatic chromosomes representing full chromosome complements of R. sativus and B. oleracea. Subsequently, a number of researchers


69

attempted its synthesis including Kakizaki (1927), Richharia (1937), Howard (1938), Kondo (1942), Crescini (1942), Honma and Heeckt (1962), Moskov and Makarova (1969), and McNaughton (1973). Details are presented by Prakash and Hinata (1980). More recently, it has been synthesized from the cross Raphanus sativus B. oleracea var. alboglabra (Chen and Wu 2007). All these investigations involved sexual hybridization where Raphanus was always the female parent. The reciprocal sexual hybridization was not successful. The idea in later investigations was to develop fodder forms of Raphanobrassica incorporating the enormous diversity of B. oleracea. Hybrids were obtained by crossing diploids or tetraploids; however, the success was more at the tetraploid level. In recent years strains of Raphanobrassica have also been synthesized following protoplast fusion (Hagimori et al. 1992; Yamanaka et al. 1992). Although Raphanobrassica plants were intermediate between parents, in vegetative characters, B. oleracea traits are predominant. Flowers, however, have close resemblance with radish—larger in size with white petals and violet venations. The siliqua has a nondehiscent Raphanus–like apical portion and a dehiscent Brassica–like basal portion. Considerable variation in chromosome pairing has been observed by different investigators. Karpechenko (1927) found regular meiosis with 18 bivalents. However, Richharia (1937) and Howard (1938) reported frequent quadrivalents and trivalents. McNaughton (1973) observed that while regular 18 bivalents were predominant, occasionally 2 univalents were also present. A majority of Karpechenko’s plants were pollen and seed fertile, with seed fertility ranging from 51% to 66%. However, a high degree of seed sterility was reported by Richharia (1937; he found only 0.2 to 1.3 seeds per siliqua), Howard (1938), and McNaughton (1973). In a recent investigation, repeated selections resulted in highly fertile 10th-generation plants with 14.9 seeds per siliqua (Chen and Wu 2008). Somatic hybrids had only low levels of fertility (Hagimori et al. 1992; Yamanaka et al 1992). The low fertility in Raphanobrassica has been ascribed to chromosome irregularities, but mainly genetic imbalance led to embryo abortion following endosperm deficiency (Ellerstro¨m and Zagorcheva 1977; Iwasa and Ellerstro¨m 1981). As compared to sexual hybrids where Raphanus contributed both mt and cp genomes, in somatic hybrids radish chloroplast genomes dominated (90%). The mitochondrial genome was contributed either by Raphanus or B. oleracea and some also possessed recombinant mitochondria (Hagimori et al. 1992; Yamanaka et al. 1992). Since the 1970s, the major objective has been to exploit Raphanobrassica as a possible new forage crop combining the disease resistance, fast growth rate, and high

70


quality of fodder radish with winter hardiness and high productivity of B. oleracea. In fact, several superior lines designated as Radicole were developed at Svalo¨f, Sweden (Olsson and Ellerstro¨m 1980) and the Scottish Plant Breeding Institute (McNaughton 1982). Some of the strains exceeded forage rape in fresh weight and dry matter yield by 20%. They also have resistance to clubroot and downy mildew. Somatic hybrids were generated to introgress clubroot resistance from radish to B. oleracea, and these did in fact posses high degree of resistance to clubroot (Hagimori et al. 1992; Yamanaka et al. 1992). Another synthetic alloploid involving radish is Raparadish (Brassicoraphanus, 2n ¼ 38). It was obtained from the cross B. rapa R. sativus primarily for determining the homoeology between the two genomes (Terasawa 1932; Mizushima 1950b). Later the synthesis aimed at developing a fodder type (Tokumasu and Kato 1976, 1988) and transferring resistance to beet cyst nematode from Raphanus to B. rapa (Dolstra 1982). Raparadish grows vigorously, combining the rapid growth with resistance to beet cyst nematode and clubroot (Lange et al. 1989). A detailed cytogenetical study on Brassicoraphanus synthesized for fodder has been carried out by Tokumasu and Kato (1976) and Matsuzawa et al. (2000), who recorded 15–19 II þ 08 I at M1 of meiosis, with occasional occurrence of a tri- or quadrivalent. However, the pollen fertility was low (0–89%) and the seed set was 0.01 to 0.1 seeds per siliqua after self- and open pollinations, respectively. Some of the A3 generation plants with yellow flowers showed considerably improved fertility. It was suggested that the genes for flower color are closely linked with those controlling embryo development. The genetic reconstitution due to intergenomic segmental exchange promotes development of embryos leading to higher fertility in yellowflowered plants (Kato and Tokumasu 1976; Tokumasu and Kato 1988). Matsuzawa et al. (2000) reported that two Raparadish lines had potential to be used as fodder. Further, they also obtained it from the reciprocal cross R. sativus B. rapa, which showed mostly regular meiosis with 19 bivalents. E. Higher Allopolyploids in U Triangle Species through Protoplast Fusion The optimum level of genome organization in crop brassicas is tetraploidy, as represented by naturally occurring digenomic tetraploids. However, it is possible to bring together all the three basic genomes in one combination. In the past, such combinations were created to determine chromosome homoeology among A, B, and C


71

genomes (see Prakash and Hinata 1980). In recent years, the three genomes have been brought together through protoplast fusion (Table 2.11) to make use of them as bridge species for transferring traits of agronomic importance, particularly resistance to fungal diseases, such as blackleg and clubroot caused by Phoma lingam and Plasmodiophora brassiceae, respectively. These are serious diseases on B. napus in Europe, Australia, and Canada. Genes conferring resistance are available in B. nigra and natural alloploid species containing B. nigra genome, specifically B. carinata and B. juncea (Sacristań and Gerdeman-Kno¨rck 1986; Sjo¨din and Glimelius 1989a,b; Zhu and Spanier 1991). The other objectives are incorporation of herbicide resistance and cytoplasmic male sterility (Kao et al. 1992; Hansen and Earle 1995; Arumugam et al. 1996).

IV. WIDE HYBRIDIZATION Hybridization in brassicas goes back to early 19th century when Sageret (1826) obtained intersub-tribal hybrid Raphanus sativus B. oleracea and Herbert (1847) produced interspecific hybrid B. napus B. rapa. Cytogenetical interest following determination of chromosome numbers gave a boost to wide hybridization. Initial attempts at hybridizations were for elucidating genomic homoeology. Later, attention shifted to utilizing wide hybridization for expanding genetic variability, introgressing nuclear genes that conferred desirable agronomic traits or cytoplasmic genes for inducing male sterility. Chromosome addition lines have also been generated to locate genes on specific chromosomes and for construction of genetic maps. During the last 30 years, in vitro techniques such as ovary and embryo culture and protoplast fusion have been employed successfully to obtain a large number of sexual and somatic hybrids. A. Sexual Hybrids Initial attempts at hybridizatization through conventional methods required very large numbers of pollinations to realize hybrids. Success was negligible for many combinations because of inability of pollen tubes to grow down the style to effect fertilization. Even after successful fertilization, hybrid embryos can abort due to genetic incompatibilities between the developing embryo and the endosperm or to partial sterilization of ovaries by alien pollen tube. In some instances, unilateral incompatibility has also been observed (Harberd 1976; Shivanna 1996).

72

52, BBCCCC 54, AABBCC

56, AACCCC

54, AABBCC 72, AABBCCCC 74, AAAABBCC

B. napus þ B. oleracea

B. juncea þ B. oleracea B. napus þ B. carinata B. napus þ B. juncea

Somatic chromosome number and genomes

B. carinata þ B. oleracea B. napus þ B. nigra

Somatic hybrids

Hygromycin resistance Club root resistance Atrazine resistance Polima CMS Not defined CMS Ogu, triazine tolerance Xanthomonas resistance, CMS Ogu, atrazine resistance Not defined Plastome transformation Tour CMS Black leg resistance Black leg resistance Male sterility

Disease resistance Black leg resistance

Trait to transfer

Table 2.11. Interspecific somatic hybrids within U triangle species.

Olin-Fatih et al. 1996 Nitovskaya et al. 2006a Arumugam et al. 1996 Sjo¨din and Glimelius 1989b Sjo¨din and Glimelius 1989b Szasz et al. 1991

Ryschka et al. 1996 Sjo¨din and Glimelius 1989a; Yamagishi et al. 1989; Sakhno et al. 1991 Sacristań et al. 1989 Gerdemann-Kno¨rck et al. 1994, 1995 Nitovskaya et al. 1988; Jourdan et al. 1989 Yarrow et al. 1990 Sundberg et al. 1991 Kao et al. 1992 Hansen and Earle 1995

Reference


73

Limited investigations have been undertaken to determine the details of postfertilization barriers. Lack of a functional endosperm or its early degeneration appear to be the major reasons for abortion of hybrid embryos. Ways devised to overcome these hybridization barriers include grafting, mixed pollination, bud pollination, and stump pollination (Hosoda et al. 1963; Sarashima 1964; Namai 1971). Kameya and Hinata (1970) succeeded in performing in vitro fertilization and obtained interspecific hybrids. A modified technique of placental pollination was used by Zenkteler (1990). Embryo rescue technique has been an effective technique for overcoming postfertilization barriers and is used extensively to obtain wide hybrids. Japanese scientists, particularly Nishi and his group (1959), pioneered it in Brassica in the late 1950s (Nishi et al. 1959). Sequential culture, which involves successive culture of ovaries, ovules, and seeds/embryos, is more effective than simple ovary or ovule culture (Shivanna 1996; Wen et al. 2008). Although wide hybridizations in Brassica have been carried out for a long time, here we define it in terms of hybridizing species of secondary and tertiary gene pools. A pioneer in this area was Mizushima (1950a,b, 1968) who attempted such hybridizations involving wild germplasm. Subsequent extensive investigations were by Harberd and McArthur (1980), who reported nearly 50 distant hybrids in which a majority were intergeneric. At present, hybridization between wild and crop species has become a routine. The last 20 years have witnessed a large number of sexual hybrids comprising interspecific, intergeneric, intersubtribal, and intertribal combinations. These hybrids and their meiotic behavior are listed in Table 2.12. Sexual hybrids are characterized by a highly disorganized meiosis, particularly when both parents are diploid. Chromosomes, due to the absence of a homologous partner, remain mostly as univalents but occasionally undergo pairing and also form bivalents in a very low frequency. Bivalents, when they occur, are mostly rod-shape monochiasmates and rarely ring shape with multiple chiasmata. Multivalents in diploid hybrids occur only rarely. However, a variable number of bivalents and frequent trivalent/quadrivalents are formed in triploid (tetraploid diploid) and tetraploid (tetraploid tetraploid species) combinations. Harberd and McArthur (1980) observed a close relationship between mean chromosome number and mean bivalent frequency at three ploidy levels (Table 2.13). Among the diploid hybrids, high chromosome pairing has been observed in several combinations:. Sinapis arvensis B. nigra (2n ¼ 17, 8 II, Mizushima 1950a), Diplotaxis erucoides B. nigra (2n ¼ 15, 6 II,

74 15 16 17

Brassica maurorum (n ¼ 8) Erucastrum varium (n ¼ 7) Brassica nigra (n ¼ 8)

Sinapis arvensis (n ¼ 9)

17 I–3 II þ 11 I

15 I–4 II þ 7 I 16 I–7 II þ 2 I

II þ 2 I II þ 11 I II þ 10 I II þ 4 I

10–24 I þ 1--7 II þ 0--1 IV

15 26 I–7 I–3 I–4 I–7

16 I–4 II þ 8 I

16

16 17 18 18

Harberd and McArthur 1980

15 I–5 II þ 5 I

15

16 17 18 18

Takahata and Hinata 1983

1 III þ 19 II þ 4 I

45

Erucastrum varium (n ¼ 7) Brassica nigra (n ¼ 8) Erucastrum virgatum (n ¼ 7) Sinapis pubescens (n ¼ 9) Hirschfeldia incana (n ¼ 7) Brassica nigra (n ¼ 8) Brassica napus (n ¼ 19) Brassica fruticulosa (n ¼ 8) Brassica nigra (n ¼ 8) Erucastrum cardaminoides (n ¼ 9) Brassica barrelieri (n ¼ 10) Brassica rapa (n ¼ 10)

Takahata and Hinata 1983 Takahata and Hinata 1983; Truco and Quiros 1991 Truco and Quiros 1991

Mizushima 1968; Truco and Quiros 1991 Takahata and Hinata 1983 Harberd and McArthur 1980 Mizushima 1968; Takahata and Hinata 1983; Nanda Kumar et al. 1988

Mattsson 1988 Kerlan et al. 1993

Quiros et al. 1988 Quiros et al. 1988 Harberd and McArthur 1980 Vyas et al. 1995 Vyas et al. 1995 Vyas et al. 1995 Quiros 1986a; Ringdahl et al. 1987; Delourme et al. 1989 Vyas et al. 1995

14 I–6 II þ 2 I 15 I–7 II þ 1 I 16 I–4 II þ 8 I 25 I–7 II þ 11 I 17 I–1 III þ 3 II þ 8 I 1 II þ 23 I–1 IV þ 6 II þ 9 I 1 IV þ 1 II þ 5 I–13 II þ 6--23 I

14 15 16 25 17 25 26

Diplotaxis erucoides (n ¼ 7) Hirschfeldia incana (n ¼ 7) Brassica nigra (n ¼ 8) Sinapis pubescens (n ¼ 9) Brassica oleracea (n ¼ 9) Brassica rapa (n ¼ 10) Brassica juncea (n ¼ 18) Brassica napus (n ¼ 19)

Reference

2n

Hybrids

Chromosome pairing at M1 of meiosis

Table 2.12. Wide hybrids in Brassica coenospecies and their meiotic behavior.

75

Takahata and Hinata 1983 Nandkumar and Shivanna 1993 Mizushima 1980 Quiros et al. 1988 Sarashima et al. 1980 Mizushima 1950a; Momotaz et al. 1998 Mizushima 1950a Harberd and McArthur 1980 Harberd and McArthur 1980; Momotaz et al. 1998 Li and Heneen 1999 Apel et al. 1984 Harberd and McArthur 1980

16 I–4 II þ 8 I 16 I–6 II þ 4 I 16 I–4 II þ 8 I 16 I–6 II þ 4 I

16–18 16 16 18 16 16 18 18 18 21 21

Orychophragmus violaceus (n ¼ 12) Moricandia arvensis (n ¼ 14) Diplotaxis muralis (n ¼ 21)

Orychophragmus violaceus (n ¼ 12) Brassica spinescens (n ¼ 8) Brassica nigra (n ¼ 8) Diplotaxis siettiana (n ¼ 8) Brassica nigra (n ¼ 8) Brassica rapa (n ¼ 10) Brassica oleracea (n ¼ 9) Diplotaxis erucoides (n ¼ 7) Hirschfeldia incana (n ¼ 7) Raphanus sativus (n ¼ 9) Sinapis arvensis (n ¼ 9) Sinapis pubescens (n ¼ 9) Coincya spp. (n ¼ 12) Sinapis alba (n ¼ 12) 3 III þ 4 II þ 4 I–21 I 1 II þ 28 I–6 II þ 18 I

30

I I II þ 7 I II þ 18 I

21

18 I–5 II þ 8 18 I–5 II þ 8 2 II þ 17 I–7 2 II þ 19 I–6

(continued)

Truco and Quiros 1991

3 III þ 4 II þ 4 I–21 I

16 16 17 17

1 II þ 15 I–8 II þ 1 I

Harberd and McArthur 1980 Quiros et al. 1988 Harberd and McArthur 1980; Prakash et al. 1982; Mattson 1988 Prakash et al. 1982 Prakash et al. 1982 Matsuzawa and Sarashima 1984 Mizushima 1950a; Harberd and McArthur 1980; Mattson 1988 Li and Heneen 1999

1 II þ 13 I–4 II þ 7 I 15 I–1 II þ 5 II þ 2 I 16 I–2 II þ 12 I

15 15 16

Brassica maurorum (n ¼ 8) Brassica spinescens (n ¼ 8) Raphanus sativus (n ¼ 9) Sinapis arvensis (n ¼ 9)

Takahata and Hinata 1983 Takahata and Hinata 1983

18 I–2 II þ 14 I 18 I–5 II þ 8 I

18 18

Brassica barrelieri (n ¼ 10) Brassica rapa (n ¼ 10) Brassica nigra (n ¼ 8) Erucastrum virgatum (n ¼ 7) Hirschfeldia incana (n ¼ 7) Brassica fruticulosa (n ¼ 8)

76

Erucastrum canariense (n ¼ 9) Brassica oleracea (n ¼ 9) Brassica rapa (n ¼ 10) Erucastrum cardaminoides (n ¼ 9) Brassica nigra (n ¼ 8) Brassica oleracea (n ¼ 9) Brassica rapa (n ¼ 10) Raphanus sativus (n ¼ 9) Brassica fruticulosa (n ¼ 8) Brassica nigra (n ¼ 8) Brassica oleracea (n ¼ 9) Brassica rapa (n ¼ 10) Brassica juncea (n ¼ 18) Brassica napus (n ¼ 19)

Brassica oxyrrhina (n ¼ 9) Brassica nigra (n ¼ 8) Brassica oleracea (n ¼ 9) Raphanus sativus (n ¼ 9) Sinapis pubescens (n ¼ 9) Brassica barrelieri (n ¼ 10) Brassica rapa (n ¼ 10) Brassica tournefortii (n ¼ 10) Diplotaxis catholica (n ¼ 9) Brassica rapa (n ¼ 10) Brassica juncea (n ¼ 18) Diplotaxis virgata (n ¼ 9) Brassica rapa (n ¼ 10) Brassica juncea (n ¼ 18)

Hybrids

Table 2.12. (Continued)

Harberd and McArthur 1980 Prakash et al. 2001; Bhasker et al. 2002 Chandra et al. 2004 Mohanty 1996 Chandra et al. 2004

1II þ 16 I–8 II þ 2 I 19 I–4 II þ 11 I 17 I–5 II þ 7 I 18 I–1 IV þ 1 III þ 1 II þ 9 I 19 I–5 II þ 9 I

18 I–1 III þ 6 II þ 3 I 1 II þ 17 I–7 II þ 5 I

18 19 17 18 19 17 17 18 19 27 28

8–20 I þ 4--10 II þ 0--1 IV

Takahata and Hinata 1983 Harberd and McArthur 1980 Inomata 2003

19 I–6 II þ 7 I 1 II þ 25 I–8 II þ 11 I IV þ 02 III þ 8--12 II þ 10--20 I

19 27 36

Bang et al. 1997 Matsuzawa and Sarashima 1984, 1986 McNaughton 1973; Chen and Wu 2008 Mizushima 1950a; Matsuzawa et al. 2000 Rhee et al. 1997 Kerlan et al. 1993

Mohanty 1996 Mohanty 1996

Prakash et al. 1982 Harberd and McArthur 1980 Matsuzawa et al. 1997; Kaneda and Kato 1999 Harberd and McArthur 1980 Mattsson 1988 Prakash and Chopra 1990 Mattsson 1988

Reference

2 II þ 15 I–1 III þ 3 II þ 10 I 13 I þ 7 II–2 III þ 7 II þ 7 I

19 I–4 II þ 11 I

2 II þ 14 I – 6 II þ 6 I 18 I–3 II þ 12 I 1 II þ 16 I–4 II þ 10 I


19 17

17 18 18 18 19 19 19

2n

77

Coincya pseuderucastrum (n ¼ 12) Sinapis alba (n ¼ 12) Orychophragmus violaceus (n ¼ 12) Moricandia arvensis ( n ¼ 14) Capsella bursa–pastoris (n ¼ 16) Brassica tournefortii (n ¼ 10) Brassica fruticulosa (n ¼ 8) Brassica nigra (n ¼ 8) 10–20 II þ 2--11 I 24 I–5 II þ 14 I 3 I–13 II

18 I–3 II þ 12 I

22 22 23–42 24 29 18 18

1 IV þ 11--15 II þ 4--12 I

20 I–4 II þ 12 I 20 I–8 II þ 5 I

19 I–5 II þ 9 I

(continued)

19 20 20 21

19 I–5 II þ 9 I

Prakash et al. 1982 Narain and Prakash 1972

Mizushima 1968 Mizushima 1968 Prakash et al. 1982 Harberd and McArthur 1980 Mithen and Herron 1991 Mizushima 1950a; Harberd and McArthur 1980; Matsuzawa et al. 2000 Mizushima 1950a Mattsson 1988 Prakash and Narain 1971 Mizushima 1950a; Harberd and McArthur 1980 Inomata 2004 Jandurova and Dolezel 1995 Li and Heneen 1999 Takahata and Takeda 1990 Chen et al. 2007

17 I–5 II þ 7 I 18 I–7 II þ 4 I

17 18 18 18 19 19

Sinapis arvensis (n ¼ 9) Brassica barrelieri (n ¼ 10) Brassica tournefortii (n ¼ 10) Eruca sativa (n ¼ 11)

Takahata and Hinata 1983 Takahata and Hinata 1983 Harberd 1972 Harberd and McArthur 1980 Takahata and Hinata 1983

18 I–4 II þ 10 I 18 I–4 II þ 10 I 9 II þ 1 I 19 I–6 II þ 7 I 20 I–6 II þ 8 I

18 18 19 19 20

1 II þ 16 I–5 II þ 8 I

Mizushima 1950a Momotaz et al. 1998 Mathias 1991

18 I–3 II þ 12 I

18 19 28

Sinapis arvensis (n ¼ 9) Raphanus sativus (n ¼ 9) Brassica rapa (n ¼ 10) Brassica napus (n ¼ 19) Brassica barrelieri (n ¼ 10) Brassica fruticulosa (n ¼ 8) Brassica nigra (n ¼ 8) Brassica oxyrrhina (n ¼ 9) Sinapis pubescens (n ¼ 9) Brassica rapa (n ¼ 10) Brassica rapa (n ¼ 10) Hirschfeldia incana (n ¼ 7) Brassica fruticulosa (n ¼ 8) Brassica spinescens (n ¼ 8) Erucastrum leucanthum (n ¼ 8) Brassica atlantica (n ¼ 9) Raphanus sativus (n ¼ 9)

78 Harberd and McArthur 1980 Mohanty 1996 Mohanty 1996 Gundimeda et al. 1992 Gundimeda et al. 1992 Harberd and McArthur 1980 Gundimeda et al. 1992 Gundimeda et al. 1992 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980

21 I–4 II þ 13 I 3 III þ 4 II þ 10 I 2 IV þ 1 II 2 II þ 14 I 19 I–1 III þ 4 II þ 8 I 20 I–2 III þ 4 II þ 6 I 6 II þ 14 I–10 II þ 6 I 1 III þ 13 II þ 8 I 29 I–IV þ 1 III þ 6 II þ 10 I 3 II þ 12 I–9 II 1 II þ 17 I–6 II þ 7 I 2 II þ 24 I–6 II þ 12 I

21 27 29 19 20 26 37 29 18 19 28

20 I–5 II þ 10 I

Ahuja et al. 2003 Batra et al. 1990 Batra et al. 1990

20

Brassica rapa (n ¼ 10)

19 I–5 II þ 9 I

Mizushima 1968; Narain and Prakash 1972 Mattsson 1988 Mizushima 1950a; Harberd and McArthur 1980 Mizushima 1950a; Harberd and McArthur 1980 Sikka 1940; Mizushima 1968; Choudhary and Joshi 2001

Reference

20 I–6 II þ 8 I 28 I–6 II þ 22 I

19

Sinapis arvensis (n ¼ 9)

19 I–7 II þ 5 I

19 I–3 II þ 32 I


20 28 29

19 19 19

Brassica oleracea (n ¼ 9) Brassica oxyrrhina (n ¼ 9) Raphanus sativus (n ¼ 9)

Diplotaxis siifolia (n ¼ 10) Brassica rapa (n ¼ 10) Brassica juncea (n ¼ 18) Brassica napus (n ¼ 19) Diplotaxis viminea (n ¼ 10) Diplotaxis tennuifolia (n ¼ 11) Brassica carinata (n ¼ 17) Brassica napus (n ¼ 19) Enarthrocarpus lyratus (n ¼ 10) Brassica oleracea (n ¼ 9) Brassica rapa (n ¼ 10) Erucastrum abyssinicum (n ¼ 16) Brassica carinata (n ¼ 17) Brassica napus (n ¼ 19) Sinapidendron frutescens (n ¼ 10) Brassica fruticulosa (n ¼ 8) Sinapis pubescens (n ¼ 9) Brassica juncea (n ¼ 18)

2n

Hybrids


79

Diplotaxis tennuifolia (n ¼ 11) Sinapis alba (n ¼ 12) Brassica oleracea (n ¼ 9) Brassica napus (n ¼ 19) Erucastrum laevigatum (n ¼ 14) Hirschfeldia incana (n ¼ 7) Moricandia arvensis (n ¼ 14) Brassica nigra (n ¼ 8) Brassica oleracea (n ¼ 9) Raphanus sativus (n ¼ 9) Brassica rapa (n ¼ 10) Brassica juncea (n ¼ 18) Bassica napus (n ¼ 19) Diplotaxis muralis (n ¼ 21) Moricandia nitens (n ¼ 14) Brassica napus (n ¼ 19)

Diplotaxis tennuifolia (n ¼ 11) Erucastrum virgatum (n ¼ 7) Hirschfeldia incana (n ¼ 7) Brassica nigra (n ¼ 8) Brassica oleracea (n ¼ 9) Raphanus sativus (n ¼ 9) Brassica rapa (n ¼ 10) Brassica elongata (n ¼ 11) Coincya spp. (n ¼ 12) Brassica juncea (n ¼ 18) Eruca sativa (n ¼ 11) Brassica oleracea (n ¼ 9) Raphanus sativus (n ¼ 9) Brassica rapa (n ¼ 10)

(continued)

Takahata and Takeda 1990 Takahata 1990 Bang et al. 1995 Takahata and Takeda 1990 Takahata et al. 1993 Takahata et al. 1993; Meng et al. 1997 Razmjoo et al. 1996

22 I–1 III þ 5 II þ 9 I 23 I–2 III þ 6 II þ 5 I 1 III þ 0--5 II þ 13--23 I 24 I–5 II þ 14 I

22 23 23 24 32 33 35

Meng et al. 1999


3 II þ 15 I–9 II þ 3 I

21

33

U et al. 1937; Wei et al. 2007 Ripley and Arnison 1990; Brown et al. 1997

21 I, 21 I–3 II þ 15 I 2 IV þ 6 II þ 11 I

21 31

21 II 22 I–5 II þ 12 I

1980 1980

1980

1980 1980

U et al. 1937; Matsuzawa and Sarashima 1986 Matsuzawa and Sarashima 1986 Agnihotri et al. 1988; Matsuzawa et al. 1999 Takahata and Hinata 1983

Harberd and McArthur Harberd and McArthur Salisbury 1989 Harberd and McArthur Bang et al. 2003 Salisbury 1989 Harberd and McArthur Harberd and McArthur Salisbury 1989

20 I–3 II þ 14 I

22 I–4 II þ 14 I 23 I–5 II þ 13 I

20 I–2 II þ 16 I

18 I–7 II þ 4 I 18 I–6 II þ 6 I

21 20 42 21 22

18 18 19 20 20 21 22 23 29

80 26 33 34

7 II þ 12 I–10 II þ 6I

1980

1980 1980 1980 1980;

Brassica rapa (n ¼ 10) Brassica carinata (n ¼ 17) Brassica juncea (n ¼ 18)

Harberd and McArthur Harberd and McArthur Harberd and McArthur Harberd and McArthur Rao et al. 1996 Harberd and McArthur Rao et al. 1996 Rao et al. 1996

II þ 9 I–9 II þ 5 I II þ 14 I –8 II þ 8 I II þ 14 I–10 II þ 4 I II þ 23 I–9 II þ 7 I

23 24 24 25

7 5 5 1


13 II þ 9 I–16 II þ 3 I

35

Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Takahata and Hinata 1983 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Batra et al. 1989 Batra et al. 1989 Snogerup and Persoon 1983 Snogerup and Persoon 1983

II þ 18 I–8 II þ 6 I II þ 20 I–7 II þ 8 I II þ 20 I–7 II þ 8 I II þ 13 I–10 II þ 3 I II þ 9 I–8 II þ 7 I II þ 18 I–8 II þ 8 I II þ 20 I–9 II þ 6 I II þ 16 I–9 II þ 6 I II þ 19 I–8 II þ 9 I II þ 17 I–8 II þ 9 I II þ 17 I–9 II þ 7 I III þ 1 II þ 22 I III þ 6 II þ 17 I

9 II þ 7 I

2 1 1 5 7 3 2 4 3 3 3 3 3

Reference

25 25

24 24 24 25 25 25 33 38

22 22 22 23

Erucastrum gallicum (n ¼ 15) Diplotaxis erucoides (n ¼ 7) Erucastrum virgatum (n ¼ 7) Hirschfeldia incana (n ¼ 7) Erucastrum nasturtiifolium (n ¼ 8)


Raphanus sativus (n ¼ 9) Sinapis arvensis (n ¼ 9) Sinapis pubescens (n ¼ 9) Brassica barrelieri (n ¼ 10) Enarthrocarpus lyratus (n ¼ 10) Sinapidendron frutescens (n ¼ 10) Brassica juncea (n ¼ 18) Brassica napus (n ¼ 19) Brassica balearica (n ¼ 16) Brassica oleracea. alboglabra (n ¼ 9) Brassica oleracea var. insularis (n ¼ 9) Brassica cossoneana (n ¼ 16) Brassica napus (n ¼ 19) Erucastrum abyssinicum (n ¼ 16) Erucastrum virgatum (n ¼ 7) Erucastrum leucanthemum (n ¼ 8) Erucastrum nasturtiifolium (n ¼ 8) Brassica oleracea (n ¼ 9)

2n

Hybrids


81

Sinapis pubescens (n ¼ 9) Brassica gravinae (n ¼ 10) Brassica tournefortii (n ¼ 10) Enarthrocarpus lyratus (n ¼ 10) Sinapis alba (n ¼ 12) Orychophragmus violaceus (n ¼ 12) Crambe abyssinica (n ¼ 45) Brassica napus (n ¼ 19) Lesquerella fendleri (n ¼ 6) Diplotaxis erucoides (n ¼ 7) Hirschfeldia incana (n ¼ 7)

Sinapis pubescens (n ¼ 9) Orychophragmus violaceus (n ¼ 12) Sinapis alba (n ¼ 12) Erucastrum gallicum (n ¼ 15) Brassica juncea (n ¼ 18) Diplotaxis virgata (n ¼ 9) Raphanus sativus (n ¼ 9) Sinapis arvensis (n ¼ 9)

Brassica carinata (n ¼ 17) Brassica fruticulosa (n ¼ 8) Diplotaxis assurgens (n ¼ 9) Diplotaxis tenuisiliqua (n ¼ 9) Diplotaxis virgata (n ¼ 9) Raphanus sativus (n ¼ 9) Sinapis arvensis (n ¼ 9)

Mixoploids 63 I Irregular 3–10 I I þ 6--20 I 0–4 IV þ 1--7 II þ 10--24 I

25–42 26 26

Du et al. 2008 Harberd and McArthur 1980 Kerlan et al. 1993 (continued)

Gundimeda et al. 1992 Mohapatra and Bajaj 1990 Li et al. 1998a,b Wang and Luo 1998

28 I–7 II þ 14 I

0–6 I I þ 15--27 I 28 I–1 8 II þ 2 I

7–10 I I þ 7--13 I

Inomata 2003 Fukushima 1945 Mizushima 1950a; Harberd and McArthur 1980 Inomata 1991 Nandkumar et al. 1989

Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Mizushima 1950a; Bing et al. 199; Momotaz et al. 1998 Harberd and McArthur 1980 Hua et al. 2006 Momotaz et al. 1998 Harberd and McArthur 1980

0–4 IV þ 0--2 III þ 7--12 II þ 12--20 I

5–12 I I þ 8--22 I

3–17 I I þ 12--20 I Mixoploids, mostly 17 II

3 II þ 19 I–4 II þ 17 I 3 II þ 20 I–10 II þ 6 I 1 II þ 24 I–10 II þ 6 I 4 II þ 18 I–11 II þ 4 I 2 II þ 22 I–9 II þ 8 I 26 I–8 II þ 10 I

27 28 28 28 30 30–36 63

27 27 27

26 17–35 29 32

25 26 26 26 26 26

82

Diplotaxis harra (n ¼ 12) Erucastrum gallicum (n ¼ 15) Brassica cossoneana (n ¼ 16) Capsella bursa–pastoris (n ¼ 16) Diplotaxis muralis (n ¼ 21) Brassica rapa (n ¼ 10) Sinapidendron frutescens (n ¼ 10) Diplotaxis harra (n ¼ 13) Erucastrum gallicum (n ¼ 15) Brassica napus (n ¼ 19) 31 31 34 36 40

26 28 28 28 28 39 30–38 35–37 31 34 35 29

Matthiola incana (n ¼ 7) Raphanus raphanistrum (n ¼ 9) Raphanus sativus (n ¼ 9) Sinapis arvensis (n ¼ 9) Sinapis pubescens (n ¼ 9) Brassica gravinae (n ¼ 10) Orychophragmus violaceus (n ¼ 12)

2n

Hybrids


31 I–5 II þ 21 I 1 II þ 29 I–5 II þ 21 I 34 I–6 II þ 22 I 3 II þ 3010 II þ 16 I

12–16 I I þ 3--11 I 1 III þ 9 II þ 8 I

16–18 II þ 3--1 I 0–2 III þ 2--10 II þ 12--28 I

0–2 III þ 0--10 II þ 6--28 I 0–1 III þ 2--11 II þ 5--24 I 39 I–10 II þ 19 I

0–4 IV þ 4--10 II þ 8--20 I


Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Harberd and McArthur 1980 Fan et al. 1985; Ringdhal et al. 1987

Peng et al. 2003 Kerlan et al. 1993; Lefol et al. 1997 Takeshita et al. 1980 Kerlan et al. 1993 Inomata 1991, 1994 Nandkumar et al. 1989 Li et al. 1995; Hua and Li 2006; Cheng et al. 2002 Inomata 2005 Lefol et al. 1997 Harberd and McArthur 1980 Chen et al. 2007

Reference


83

Table 2.13. Mean chromosome number and bivalent frequency at three ploidy levels in the tribe Brassiceae. Hybrid Diploids Triploids Tetraploids

Mean chromosome number, 2n 18.7 25.6 34.3

Mean bivalent frequency 2.9 6.2 10.2

Source: Harberd and McArthur 1980.

Quiros et al. 1988), B. fruticulosa B. nigra (2n ¼ 16, 7 II, Mizushima 1968), B. nigra Hirschfeldia incana (2n ¼ 15, 1 III þ 5 II, Quiros et al. 1988), Erucastrum canariense B. oleracea (2n ¼ 18, 8 II, Harberd and McArthur 1980), E. cardaminoides B. oleracea (2n ¼ 18, 1 IV þ 1 III þ 1 II, Mohanty 1996), and Enarthrocarpus lyratus B. rapa (2n ¼ 20, 2 III þ 4 II, Gundimeda et al. 1992). The triploid and tetraploid hybrids where higher associations have been observed include B juncea Diplotaxis virgata (1 IV/2 III, Inomata 2003), B. napus Hirschfeldia incana (1 IV, Kerlan et al. 1993), Diplotaxis viminea B. napus (2 IV, Mohanty 1996), and Diplotaxis erucoides B. napus (1 IV, Delourme et al. 1989). Hybrids between the diploids were absolutely pollen and seed sterile while triploid and tetraploid hybrids had a little pollen and seed fertility. Bivalents and higher associations may be interpreted to result from archaic homology within the chromosomes of the same genome (autosyndesis) or because of intergenomic homoeology (allosyndesis). However, it is difficult to interpret the pairing precisely in terms of auto- or allosyndesis since there is little information on the extent of autosyndesis observed through chromosome pairing in haploids. Mizushima (1950a, 1968, 1980) made some observations on the extent of allosyndesis between a limited number of genomes; Harberd and McArthur (1980) could not arrive at any definite conclusion. What can be stated safely is that intrageneric homoeology is not always higher than intergeneric homoeology. With the progress in GISH techniques, the degree of autoand allosyndesis can be ascertained precisely in wide hybrids. An interesting cytological phenomenon was observed in hybrids between Brassica species and Orychophragmus violaceus (2n ¼ 24). O. violaceus is cultivated in China as an ornamental plant and has desirable oil quality. Hybrids with all the six crop species have been obtained with O. violaceus always the pollen parent. Chromosomes remain unpaired as univalents in hybrid cells, and separation of parental genomes occurs regularly during mitotic and meiotic

84


divisions (Li et al. 1995, 1996, 1998a,b, 2003; Li and Heneen 1999). During mitosis, any of three situations may occur, and subsequently the chromosomes are doubled following chromosome duplications in daughter cells: 1. Complete separation of parental genomes results into cells with haploid and diploid complements of the two parents. 2. Partial separation leads to inclusion of some chromosomes of one parent with the haploid complement another genome producing hypo- and hyperdiploid cells. 3. During partial separation, chromosomes of either parent are included in the genomes resulting into substitution lines. Hybrids B. oleracea O. violaceus had the sum of parental chromosomes (2n ¼ 21) in mitotic and meiotic cells. B. rapa O. violaceus hybrids were mixoploid with somatic chromosome number ranging from 23 to 42 but cells with 2n ¼ 34 predominating. Partial separation of parental genomes occurred during mitosis, leading to the addition of some Orychophragmus chromosome to the B. rapa complement. Hybridization with B. nigra produced a majority of maternal type F1 plants (2n ¼ 16) and some mixoploids. Hybrids with the three tetraploid species showed variable chromosome numbers: B. carinata O. violaceus (2n ¼ 12--34), B. juncea O. violaceus (2n ¼ 30--42), and B. napus O. violaceus (2n¼12–38). Partial and complete separation was more frequent in B. juncea O. violaceus and B. carinata O. violaceus hybrids respectively. Somatic cells and PMCs with additional O. violaceus chromosomes often occurred in B. juncea O. violaceus and not in other two combinations. It was proposed that differences in the duration of somatic cell cycles of two parents cause partial or complete genome elimination. Based on cytological observations, Li and Heneen (1999) and Li et al. (2003) proposed that B genome accounts for complete and partial genome separation in B. carinata; both A and B genomes contribute to this separation in B. juncea; and A genome is more influential than C genome in B. napus during mitosis and meiosis. Genetic information from Orychophragmus has been introgressed into Brassica genomes as demonstrated by GISH (Hua and Li 2006). Employing these hybridizations, it may be possible to produce Brassica aneuploids and haploids and subsequently homozygous lines (see review by Li and Ge 2007). B. Somatic Hybrids The majority of the wild Brassica species belong to the secondary and tertiary gene pool, which renders their genes inaccessible for crop


85

improvement. As mentioned earlier, barriers to sexual hybridization are easily overcome through the somatic route. Recent years have seen spectacular developments in protoplast fusion technology, particularly in Brassicaceae. Also, Brassica and related genera are very amenable to tissue culture techniques. Cell fusion allows cytoplasmic substitutions and generation of novel cytoplasmic variability through organellar reassortment and DNA recombination, a phenomenon not possible during sexual hybridization. Because of these advantages, cell fusion has become a promising methodology for introgressing desirable alien genes in crop cultivars (see reviews by Glimelius 1999a; Christey 2004; Navra´tilova´ 2004; Liu et al. 2005). The first successful report of cell fusion in Brassicacea was by Kartha et al. (1974) and involved protoplasts of B. napus and Glycine max. A major breakthrough was made by Gleba and Hoffmann (1979, 1980) when, following fusion of B. rapa and Arabidopsis thaliana protoplasts, an intertribal hybrid was successfully regenerated. This event achieved the distinction of first somatic hybrid in Brassicaceae, although no offspring could be obtained from it. Subsequently, a large number of somatic hybrids have been obtained that combine crop species with taxonomically divergent wild germ pools (Tables 2.14, 2.15). These represent interspecific, intergeneric, and a substantial number of intertribal combinations from six different tribes— Sisymbrieae (Arabidopsis thaliana, Camelina sativa); Arabideae (Armoracia rusticana, Barbarea stricta, B. vulgaris); Drabeae (Lesquerella fendleri); Lepidieae (Capsella bursapastoris, Lepidium, Thlaspi caerulescens, T. perfoliatum); Lunarieae (Lunaria annua); and Hesperideae (Matthiola incana)—and a few subtribes— Raphananiae (Raphanus, Trachystoma), and Moricandiineae (Moricandia). Another species, Orychophragmus violaceus, previously included in subtribe Moricandiinae but now excluded from tribe Brassiceae (Go´mez-Campo, personal communication), has also been hybridized. The priorities have shifted to practical utilization and efforts are toward introgressing nuclear and cytoplasmic genes from wild relatives to crop species. The desirable traits targeted include: C3–C4 intermediate photosynthetic system (from Moricandia arvensis and M. nitens) Resistance to: club root (from Raphanus sativus); alternaria leaf spot (from Sinapis alba, Camelina sativa, Capsella bursa-pastoris); beet cyst nematodes (from Sinapis alba, C. bursa–pastoris); blackleg (from B. tournefortii, Sinapis arvensis, Arabidopsis thaliana) High nervonic acid content (from Thlaspi perfoliatum) High erucic acid content (from Crambe abyssinica)

86

Crambe abyssinica (n ¼ 45) þ B. napus

Armoracea rusticana (n ¼ 16) þ B. oleracea Barbarea vulgaris (n ¼ 8) þ B. oleracea Barbarea vulgaris (n ¼ 8) þ B. rapa Barbarea vulgaris (n ¼ 8) þ B. napus Barbarea stricta (n ¼ 8) þ B. rapa Camelina sativa (n ¼ 20) þ B. oleracea Camelina sativa (n ¼ 20) þ B. carinata Capsella bursa–pastoris (n ¼ 16) þ B. oleracea

Arabidopsis thaliana (n ¼ 5) þ B. rapa Arabidopsis thaliana (n ¼ 5) þ B. juncea Arabidopsis thaliana (n ¼ 5) þ B. napus

Arabidopsis thaliana (n ¼ 5) þ B. oleracea

Arabidopsis thaliana (n ¼ 5) þ B. nigra

Somatic hybrid

Experimental demonstration Phosphinothricin resistance Herbicide resistance, black leg resistance Transposable element Spm/dSpm Clubroot resistance Cold tolerance Cold tolerance Cold tolerance Cold tolerance Alternaria resistance Alternaria resistance Resistance to flea beetles, alternaria blight High erucic acid content, insect resistance

Resistance to flea beetles, cold tolerance, short life cycle Plastome transformation

Desirable trait to introgress

Table 2.14. Intertribal somatic hybrids in Brassiceae.

Wang et al. 2003,* 2004a*

Nitovskaya and Shahkhovskii 1998; Yamagishi and Nakagawa 2004; Nitovskaya et al. 2006a Gleba and Hoffmann 1979, 1980 Ovcharenko et al. 2004 Bauer-Weston et al.1993;* Forsberg et al. 1994, 1998;* Yamagishi et al. 2002* Ovcharenko et al. 2005* Navra´tilova´ et al. 1997 Ryschka et al. 1999 Oikarinen and Ryo¨ppy 1992 Fahleson et al. 1994b Oikarinen and Ryo¨ppy 1992 Hansen 1998; Sigareva and Earle 1999a Narasimhulu et al. 1994 Nitovskaya et al. 1998; Sigareva and Earle 1999b

Siemens and Sacristań 1995*

Reference

87

*Asymmetric. **Both symmetric and asymmetric.

Thlaspi perfoliatum (n ¼ 21) þ B. napus Thlaspi caerulescens (n ¼ 7) þ B. napus Thlaspi caerulescens (n ¼ 7) þ B. juncea

Lunaria annua (n ¼ 14) þ B. napus Matthiola incana (n ¼ 7) þ B. oleracea Orychophragmus violaceus (n ¼ 12) þ B. napus

Lepidium meyenii (n ¼ 32) þ B. oleracea Lesquerella fendleri (n ¼ 6) þ B. napus

Descurainia sophia (n ¼ 14) þ B. napus High linolenic acid content, cold tolerance Glucosinolate content High lesquerolic acid content, drought tolerance, Lesquerella chloroplasts High nervonic acid content Oil quality High linoleic and palmitic acid content, phosphinothrin resistance Chlorosis correction High nervonic acid content Zinc and cadmium tolerance High metal accumulation Vasilenko et al. 2003 Fahleson et al. 1994a** Brewer et al. 1999 Dushenkov et al. 2002

Ryschka et al. 2003 Skarzhinskaya et al. 1996,** 1998; Schro¨der-Pontoppidan et al. 1999 Nitovskaya et al. 2006b Craig and Millam 1995 Ryschka et al. 1999; Sheng et al. 2008 Hu et al. 1999; Hu et al. 2000b;* Sakhno et al. 2007

Guan et al. 2007

88

Moricandia arvensis (n ¼ 14) þ B. juncea Moricandia arvensis (n ¼ 14) þ B. napus Moricandia nitens (n ¼ 14) þ B. oleracea Moricandia nitens (n ¼ 14) þ B. rapa Moricandia nitens (n ¼ 14) þ B. napus Raphanus sativus (n ¼ 9) þ B. oleracea

Intergeneric Diplotaxis catholica (n ¼ 9) þ B. juncea Diplotaxis harra (n ¼ 13) þ B. juncea Diplotaxis harra (n ¼ 13) þ B. napus Diplotaxis muralis (n ¼ 21) þ B. juncea Eruca sativa (n ¼ 11) þ B. juncea Eruca sativa (n ¼ 11) þ B. napus Moricandia arvensis (n ¼ 14) þ B. oleracea

Interspecific Brassica spinescens (n ¼ 8) þ B. juncea Brassica tournefortii (n ¼ 10) þ B. napus

Somatic hybrids

3 IV þ 33 II 1 IV þ 27 II

27 II–2 IV þ 23 II 60, 62, 1–4 IV þ 23 II

25, 36, 62 36

64, 3 III þ 15 II þ 25 I 66 46, 74, 92

54, 58, 64 78, 58, 60 46

52, 26 II 58

2n and meiotic pairing

sterile — fertile 0–50 fertile — —

0–56 sterile — sterile 0–8 8–98

sterile 0–31 0–43

sterile — fertile 0–50 fertile very low very low

0–7 sterile — sterile very poor 0–28

sterile poor poor

Seed

Fertility (%) Pollen

Table 2.15. Interspecific and intergeneric somatic hybrids in coenospecies.

Kirti et al. 1995c Begum et al. 1995 Klimaszewska and Keller 1988 Chatterjee et al. 1988 Sikdar et al. 1990 Fahleson et al. 1988, 1997 Toriyama et al. 1987a; Sobrino-Vesperinas 1988; Ishikawa et al. 2003 Kirti et al. 1992b O’Neill et al. 1996 Meng et al. 1999 Meng et al. 1999 Meng et al. 1999 Kameya et al. 1989 Hagimori et al. 1992; Yamanaka et al. 1992

Kirti et al. 1991 Stiewe and Ro¨bbelen 1994 Liu et al. 1995

Reference

89

arvensis (n ¼ 8) þ arvensis (n ¼ 8) þ arvensis (n ¼ 8) þ alba (n ¼ 12) þ B.

B. nigra B. oleracea B. napus oleracea

Trachystoma ballii (n ¼ 8) þ B. juncea

Sinapis alba (n ¼ 12) þ B. juncea Sinapis alba (n ¼ 12) þ B. napus

Sinapis Sinapis Sinapis Sinapis

Raphanus sativus (n ¼ 9) þ B. juncea Raphanus sativus (n ¼ 9) þ B. napus

66–77 sterile

56–90, 30–44 II þ 5 I–10 I 62, 31 II 52, 26 II–2 II þ 22 II þ 2 I

sterile sterile — — low

sterile 43–63 — — 23–58

good sterile

sterile low very low

sterile sterile

sterile 5–7

sterile 0–20 0–3

60, 30 II

56, 26 ii 32 34 54, predominantly 27 II 42

54, 56, 70, 26–30 II

Muller et al. 2001 Sakai and Inamura 1990; Sundberg and Glimelius 1991; Lelivelt and Krens 1992 Muller et al. 2001 Wang et al. 2006b Toriyama et al. 1987b Toriyama et al. 1987b Hu et al. 2002a Bauer 1990; Ryschka et al. 1996; Hansen and Earle 1997; Sigareva et al. 1999 Gaikwad et al. 1996 Primard et al. 1988 Lelivelt et al. 1993 Chevre et al. 1994a Wang et al. 2005b Kirti et al. 1992a

90


High lesquerolic acid content (from Lesquerella fendleri) High linoleic and palmitic acid content (from Orychophragmus violaceus) Cold tolerance (from Barbarea vulgaris) Zinc and cadmium hyperaccumulation (from Thlaspi carulesens) Cytoplasmic genes for inducing male sterility from a number of wild species Somatic hybrids in several combinations—for example, Camelina sativa þ B. carinata (Narasimhulu et al. 1994), Camelina sativa þ B. oleracea (Hansen 1998) and Barbarea vulgaris þ B. napus (Fahleson et al. 1994b)—could not be established to viable field plants. It appears that although protoplast fusion removes fertilization barriers, genetic incompatibilities due largely to phylogenetic distances still prevail at the somatic level, affecting differentiation, growth, and development of normal plant parts, particularly floral organs, thus leading to sterility. However, other distant hybrids, particularly with Arabidopsis thaliana, probably could be established successfully, due to its small genome and also with little repetitive DNA, which promotes greater homoeology between the partaking genomes (Hansen 1998). Lunaria annua þ B. napus hybrid has been reported only up to callus stage (Craig and Millam 1995). Somatic hybrids have been identified and characterized by a range of techniques including morphological attributes, chromosome number, meiotic behavior, fertility, DNA content estimation, isozyme analysis, RFLP, AFLP, and cytoplasmic constitution. However, there are not many reports on chromosome cytology, and in several studies, the ploidy status has been determined by estimating DNA content. Somatic hybrids, in general, are intermediate in morphology between the fusion partner species. This expression is particularly relevant for leaves and frequently for flower characteristics. Floral abnormalities are also observed and include 6 to 8 petals and multiple carpellike structures in A. thaliana þ B. napus (Bauer-Weston et al. 1993); 1 or 2 petals in Thlaspi perfoliatum þ B. napus (Fahleson et al. 1994a); enlarged, distorted or globular pistils, and reduced or missing stamens in L. fendleri þ B. napus (Skarzhniskaya et al. 1996); stamens with stunted filaments in R. sativus þ B. napus (Lelivelt et al. 1992); and shorter, thicker pistils in D. harra þ B. napus (Klimaszewska and Keller 1988). Chromosome cytology revealed that somatic hybrids either have the full chromosome complements of both parents, which are derived mostly from symmetric fusions, or spontaneous chromosome elimination occurs leading to generation of asymmetric hybrids or


91

cybrids (Navara´tilova´ et al. 1997; Hu et al. 2002a). Sometimes the regenerants from the same fusion events have different chromosome numbers (Hoffman and Adachi 1981). Meiotic studies have been carried out in some of these hybrids which include intergeneric and a few intertribal combinations. Besides occurrence of normal bivalents as the sum of parental chromosomes at M1, higher associations such as tri- and quadrivalents, in addition to univalents, were also encountered (Table 2.15). Interestingly, the intersubtribal hybrid Moricandia arvensis þ B. juncea exhibits up to three quadrivalents (Kirti et al.1992b). Such higher associations suggest intergenomic chromosome homoeology. Post-metaphase-1 stages have not been investigated, but it appears that meiosis proceeds normally, as can be inferred from normal pollen formation in many of the hybrids. Intergenomic chromosome recombination due to allosyndesis has been documented in some somatic hybrids, such as Moricandia arvensis þ B. juncea, D. catholica þ B. juncea, Trachystoma ballii þ B. juncea. Genomic in situ hybridization has been used effectively to determine the alien chromosome status at mitosis in some somatic hybrids and their progeny, for example, in Eruca sativa þ B. napus (Fahleson et al. 1988, 1997), Lesquerella fendeleri þ B. napus (Skarzhniskaya et al. 1996), Crambe abyssinica þ B. napus (Wang et al. 2004a,b), and Sinapis alba þ B. napus (Wang et al. 2005a). GISH was also employed to dectect intergenomic homoeologous recombination in these hybrids. A majority of somatic hybrid plants were seed sterile when selfed. However, some fertile hybrids were also obtained, including intertribal hybrids Arabidopsis thaliana þ B. napus (Forsberg et al. 1994), Thlaspi perfoliatum þ B. napus (Fahleson et al. 1994a), Capsella bursa-pastoris þ B. oleracea (Sigareva and Earle 1999b), Orychophragmus violaceus þ B. napus (Hu et al. 2002b), and Moricandia arvensis þ B. oleracea (Ishikawa et al. 2003), and a few intergeneric and interspecific ones. Wherever pollen fertility was observed, it was quite low in A1 generation. With a few exceptions seed fertility was invariably very poor, and seeds could be obtained only on backcrossing. Only in rare instances were seeds obtained on selfing. A correlation between the presence of alien chromosomes and fertility was observed in somatic hybrids Lesquerrella fendleri þ B. napus (Skarzhinskaya et al. 1998) and Crambe abyssinica þ B. napus (Wang et al. 2004b); with the decreasing number of alien chromosomes, the fertility of the hybrids increased and plants possessing the entire alien chromosome complement were absolutely sterile. Similar correlations were obtained in asymmetric hybrids A. thaliana þ B. napus (Forsberg et al.

92


1998) and Orychophragmus violaceus þ B. napus (Hu et al. 2002b). In many instances, to produce reasonably fertile hybrids, irradiated protoplasts from wild species have been used, eliminating substantial amounts of alien DNA to obtain asymmetric hybrids that contain varying amounts of alien DNA from donor species. Somatic hybrids present three possibilities with respect to their cytoplasmic genomes: (1) parental genomes segregate to homogeneity during cell division, (2) both the parental genomes occur as a mixed population, and (3) novel genome constitution is generated when parental genomes undergo recombination. Segregation of chloroplasts is independent of mitochondrial segregation. In Brassiceae, mitochondrial recombination has been observed to occur frequently and is very well documented (Glimelius 1999a). In sharp contrast, intergenomic chloroplast recombination is rare. Two chloroplast types occurring in mixture is also rare, and there is no information about whether this mixture persists in subsequent generations, wherever it does occur. It is observed in interspecific, intergeneric, and intertribal somatic hybrids that chloroplast from crop species are generally favored. This biased segregation is attributed to genetic divergence, ploidy level differences between the parental species, and rate of chloroplast division (Sundberg and Glimelius 1991). Also, plastome-genome incompatibility may be a factor. A higher ploidy level of one of the parental species contributes a larger number of chloroplasts per cell (Butterfass 1989). Since in most of the hybrid alloploid species B. napus or B. juncea has been one of the parents, they contribute more chloroplasts to the fusion products than the wild diploid parent. However, it is not possible to predict which parental chloroplast will establish in hybrids. The intertribal somatic hybrid Lesquerella fendleri þ Brassica napus has been reported to have mixed chloroplasts (Skarzhinskaya et al. 1996), as has intergeneric hybrid Diplotaxis catholica þ B. juncea (Mohapatra et al. 1998). A report documented the occurrence of intergeneric chloroplast recombination in the somatic hybrid Trachystoma balli þ B. juncea (Baldev et al. 1998). where the recombination has occurred in a single copy region and remains stable over the generations. Also, it caused no imbalance in the recombinant plastomes in terms of chloroplast-related functions. In addition, choroplast recombination was also indicated in the somatic hybrid B. oleracea þ Raphanus sativus (Kanno et al. 1997). Mitochondrial genomes behave differently from plastid genomes. Besides segregation and mixture of parental types, intergenomic mitochondrial recombination is frequent in Brassiceae somatic hybrids. The recombinant mitochondrial genome contains DNA fragments of both the


93

parents or entirely new and unique ones not found in parental types (Belliard et al. 1979). While investigating seven sets of interspecific, intergeneric, and intertribal combinations, Landgren and Glimelius (1994b) observed that 43% to 95% of the hybrids had mt DNA rearrangements. Recombination hot spots have also been found; for example, Mohapatra et al. (1998) suggested that intergenomic recombination is preferred at specific sites in somatic hybrids Diplotaxis catholica þ B. juncea. The cox2 coding region may serve as an active site for inter- or intragenomic recombination (Stiewe and Ro¨bbelen 1994; Liu et al 1995). Conflicting views are reported regarding the segregation of mitochondria in somatic hybrids. According to Landgren and Glimelius (1990, 1994a,b), crop types are favored. In cybrids where CMS line is one of the parents, the mt segregation was slightly biased toward the CMS parent (Mukhopadhyay et al. 1994; Liu et al. 1996). However, many of the somatic hybrids have recombinant mitochondrial genomes, although a lack of recombination has also been documented, for example, S. alba þ B. napus (Lelivelt et al. 1993), Lesqurella fendleri þ B. napus (Skarzhinskaya et al. 1996), and Moricandia arvensis þ B. oleracea (Ishikawa et al. 2003). Somatic hybridization in Brassicaceae has crossed all the intergeneric and intertribal barriers. However, the results are not too encouraging because of a general high degree of sterility or severe intergenomic incompatibilities leading to many abnormalities. Asymmetric hybrids in such instances appear to be more promising as crop species, tolerating only a fraction of alien genetic content rather the whole genome for integrated functioning of the system. Such asymmetric fusions have been obtained by irradiating donor (wild) protoplasts to induce doublestrand DNA breaks. Most of intertribal hybrids are asymmetric and show improved fertility. One of the limiting factors in gene transfer from wild to crop species is the very low level or complete absence of intergenomic chromosome pairing, which implies that overall genome structures interfere with free gene flow across the generic boundaries. Nevertheless, several traits of agronomic importance have been observed in somatic hybrids and in some cases, the genes have been introgressed to crop species, as revealed by progeny plant analysis. Examples include: 1. Raphanus sativus þ B. napus: express resistance to beet cyst nematode—Heterodera schachtii (Lelivelt and Krens 1992) 2. Sinapis alba þ B. napus: possess high level of beet cyst nematode (Heterodera schachtii) resistance (Lelivelt et al. 1993) 3. Thlaspi perfoliatum þ B. napus: contain significantly greater amounts of nervonic acid (Fahleson et al. 1994a)

94


4. B. tournefortii þ B. napus: express resistance to blackleg (Phoma lingum) (Liu et al. 1995) 5. Moricandia arvensis þ B. napus: C3-C4 character is expressed at both the physiological and anatomical level (O’Neill et al. 1996) 6. Moricandia nitens þ B. oleracea: C3–C4 character expressed as transition between the parents (Yan et al. 1999) 7. Sinapis alba þ B. oleracea: exhibit resistance to Alternaria brassicicola and Phoma lingam (Ryschka et al. 1996; Hansen and Earle 1997; Sigareva et al. 1999) 8. Capsella bursa-pastoris þ B. oleracea: exhibit high degree of resistance to Alternaria brassicicola (Sigereva and Earle 1999b) 9. Thlaspi caerulescens þ B. napus: accumulate high levels of zinc and cadmium, which would have been toxic to B. napus (Brewer et al. 1999) 10. Camelina sativa þ B. oleracea: possess resistance to Alternaria (Sigareva and Earle 1999a ) 11. Lesquerella fendleri þ B. napus: contain high amount of erucic acid for industrial purpose (Glimelius 1999b; Schro¨der-Pontokpidan et al. 1999) 12. Arabidopsis thaliana þ B. napus: possess resistance to Leptosphaeria maculans (Bohman et al. 2002) 13. Orychophragmus violaceus þ B. napus: contain high content of palmitic and linoleic acid expressed in the progeny plants (Hu et al. 2002b; Ma and Li 2007) 14. Sinapis avensis þ B. napus: possess resistance to blackleg in the hybrids and progeny (Hu et al. 2002a) 15. Crambe abyssinica þ B. napus: progeny contain high amount of seed erucic acid (Wang et al. 2004b) Several CMS systems in B. napus and B. juncea have been obtained following protoplast fusion. These are based on Arabidopsis thaliana, Brassica tournefortii, Diplotaxis catholica, Eruca sativa, Moricandia arvensis, Orychophragmus violaceus, Raphanus, Sinapis arvensis, and Trachystoma ballii. C. Introgression of Genes A number of bacterial, fungal, and viral diseases cause severe infection on Brassica crops and inflict heavy losses to seed yield. Among them, black leg or stem canker (Leptosphaeria maculans/Phoma lingam),


95

alternaria leaf spot (Alternaria spp.), white rust (Albugo candida), black rot (Xanthomonas campestris pv. campestris), soft rot (Erwinia carotovora), and sclerotinia stem rot (Sclerotinia sclerotiorum) are important. Nuclear genes conferring resistance to these diseases have been transferred from related species and alien wild germplasm. Other desirable traits, particularly the fertility restoration for several CMS systems, have also been incorporated (Table 2.16). These genes have been introgressed, taking advantage of nonhomologous allosyndetic recombination in early backcross generations following sexual/somatic hybridizations and also through generating chromosome addition lines. In recent years, efforts have been made to identify alien introgressions to specific chromosomes through GISH and molecular markers. Results of GISH are not very encouraging primarily due to unusually low copy number of repeat sequences in chromosome arms, which form the basis of GISH signals. Nevertheless, examples of detecting introgressions include B. napus from Lesquerella fendleri (Skarzhinskaya et al. 1998), Raphanus sativus (Voss et al. 2000), Sinapis arvensis (Snowdon et al. 2000b), Crambe abyssinica (Wang et al. 2004b), and Orychophragmus violaceus (Li and Ge 2007).

V. CYTOPLASMIC SUBSTITUTION AND MALE STERILITY During the last 50 years, several investigations have reported the expression of a high degree of heterosis for seed yield in intervarietial hybrids of B. rapa, B. juncea, and B. napus (see Fu and Yang 1998). However, in earlier years, full potential of heterosis could not be exploited in B. juncea and B. napus as these are predominantly selffertilized crops. A suitable pollination control mechanism is required to produce commercial hybrid seed. A cytoplasmic male sterility (CMS) fertility restoration system is an excellent potential means to facilitate hybridization because it is easy to maintain. CMS, a maternally inherited inability to produce fertile pollen, is encoded in the mitochondrial genome and can arise spontaneously due to mutation in the genome (autoplasmy) or can be expressed following cytoplasmic substitutions due to nuclear-mitochondrial incompatibility (alloplasmy). A sterility inducing cytoplasm was identified in a wild population of Raphanus sativus in 1968 (Ogura 1968), and the first alloplasmic in Brassica was developed by introgressing this sterility inducing cytoplasm into B. napus and B. oleracea (Bannerot et al. 1974). Around that time, Pearson (1972) also developed male sterile broccoli

96

Black leg resistance

Club root resistance

Chlorosis correction Beet cyst nematode resistance

B. napus B. rapa B. juncea

Raphanus sativus

Raphanus sativus Sinapis alba

Enarthrocarpus lyratus

Raphanus sativus Raphanus sativus B. juncea B. tournefortii Trachystoma ballii Moricandia arvensis Erucastrum canariense

Fertility restoration

rapa juncea/B. carinata carinata rapa/B. carinata rapa/B. juncea

B. B. B. B. B.

Donor species

Yellow seed coat color

Trait napus napus napus napus napus

B. oleracea var. capitata B. napus B. napus

B. napus

CMS (Ogu?) B. napus CMS (Kosena) B. napus CMS (Polima) B. napus CMS (Tour) B. napus CMS (Trachy) B. juncea CMS (Moricandia) B. juncea CMS (Canariense) B. juncea CMS (Canariense) B. napus CMS (Lyratus) B. rapa CMS (Lyratus) B. juncea/ B. napus CMS (Ogu) B. napus B. napus

B. B. B. B. B.

Recepient species

Table 2.16. Introgression of nuclear genes conferring desirable traits.

Lelivelt and Krens 1992; Voss et al. 2000; Peterka et al. 2004; Budahn et al. 2006 Chiang et al. 1977, 1980 Gowers 1982 Roy et al. 1984; Sacristań and GerdemannKno¨rck 1986; Chevre et al. 1997a; Dixelius 1999

Paulmann and Ro¨bbelen 1988 Lelivelt et al. 1993

Chen and Heneen 1992 Rashid et al. 1994 Qi et al. 1995 Meng et al. 1998 Rahman et al. 2001; Potapov and Osipova 2003 Heyn 1978; Rousselle and Dosba 1985 Sakai et al. 1996 Fan et al. 1986 Stiewe and Ro¨bbelen 1994 Kirti et al. 1997 Prakash et al. 1998 Prakash et al. 2001 Banga et al. 2003b Deol et al. 2003 Banga et al. 2003a

Reference

97

Earliness

Altered oil quality

Black rot resistance Soft rot resistance

Alternaria leaf spot resistance

B. napus B. napus B. napus B. napus B. napus B. napus B. napus B. oleracea; B. rapa ssp. pekinensis B. napus B. napus B. napus

Arabidopsis thaliana

Sinapis arvensis

Coincya monensis B. rapa Sinapis alba

Diplotaxis erucoides B. juncea B. oleracea var. italica

Orychophragmus violaceus Lesquerella fendleri B. rapa

B. napus

B. nigra

Hu et al. 2000b; Hu and Li 2006 Du et al. 2008 Shiga 1970; Namai et al. 1980

Klewer et al. 2003 Tonguc and Griffiths 2004 Ren et al. 2000, 2001

Struss et al. 1996; Chevre et al. 1996, 1997b; Plieske et al. 1998; Dixelius 1999 Bohman et al. 2002; Ogbonnaya et al. 2003; Saal et al. 2004 Snowdon et al. 2000b: Hu et al. 2002a: Winter et al. 2003 Winter et al. 2003 Chevre et al. 2003; Leflon et al. 2007 Primard et al. 1988

98


(B. oleracea var. italica) by placing its nucleus in B. nigra cytoplasm. A large number of alloplasmics have been reported since. Brassica coenospecies is a rich repository of diverse mitochondrial genomes, as revealed by RFLP studies (Pradhan et al. 1992). By combining these cytoplasms with crop nuclei, a spectrum of alloplasmic lines of diverse origin expressing male sterility has been obtained, particularly in B. juncea (Table 2.17) (see reviews by Delourme and Budar 1999; Prakash 2001; Budar et al. 2004). Cytoplasmic male sterile lines have been developed following backcrossings of either sexually synthesized allopolyploids or somatic hybrids between wild and crop species. Somatic hybridization for the synthesis of an alloplasmic was attempted for the first time by Kameya et al. (1989) when they combined the nucleus of B. oleracea with Raphanus cytoplasm. Subsequently, it has been employed extensively to obtain new combinations. As expected, CMS originating from sexual hybridizations possess unaltered organellar genomes because of exclusive maternal inheritance. Since organelle assortment and intergenomic mitochondrial recombinant is of frequent occurrence in Brassiceae, the cytoplasmic constitution is entirely different in those originating from somatic hybrids, and various possible combinations of mitochondrial and chloroplast genomes have been reported in different CMS lines (Table 2.17). Alloplasmic CMS plants, in general, are similar to euplasmic plants in development and morphology. However, many of them exhibit abnormalities, both developmental and floral, which are manifestations of alien cytoplasms. Varying degrees of leaf chlorosis, ranging from mild to severe, occurs in several systems, such as Raphanus/Ogu, Oxyrrhina, Tournefortii, Moricandia, and Enarthrocarpus. Floral abnormalities are also common in male sterile plants and are expressed in several ways: petaloid anthers (Nigra, Muralis, Trachystoma, Raphanus, Tournefortii, Canariense); poor or absent nectaries (Tournefortii and Raphanus); crooked style (Tournefortii, Raphanus); thick pistil (Raphanus); and low seed fertility (Raphanus, Tournefortii, Enarthrocarpus and Trachystoma). Leaf chlorosis is an expression of incompatible interaction between alien chloroplasts and endogenous nucleus. Agronomically, it is an undesirable feature associated with reduced growth and adversely affects productivity. Similarly, it has clearly been demonstrated at least in Brassica that nuclearmitochondrial interaction is critical for flower development, normal morphology, and subsequent seed fertility (see Glimelius 1999a). Application of biotechnology, particularly protoplast fusion methodology, has created entirely new opportunities for improving these

99

B. rapa B. napus

B. rapa B. juncea B. napus

B. oleracea Somatic hybrid R. sativus var. capitata þ B. oleracea

Diplotaxis muralis

Brassica oxyrrhina

Raphanus sativus

Sexual alloploid B. oxyrrhina B. rapa

Sexual alloploid D.muralis B. rapa

Sexual alloploid B. nigra B. oleracea var. italica

B. olerecea var. italica

Spontaneous mutation in Raphanus mitochondrial genome

Brassica nigra

oleracea rapa juncea napus

Origin

B. B. B. B.

Recepient species

Raphanus sativus (Ogura)

Cytoplasmic donor

Table 2.17. Cytoplasmic substitution lines in Brassiceae.

(continued)

Kameya et al. 1989

Prakash and Chopra 1988, 1990; Kirti et al. 1993

Hinata and Konno 1979; Pellan-Delourme and Renard 1987

Pelletier et al. 1983; Jarl and Bornman 1988; Jarl et al. 1989; Kao et al. 1992; Kirti et al. 1995a Pearson 1972

Abnormalities removed following protoplast fusion.

Leaves green, flowers of two types: (1) petaloid anthers and absent nectaries; (2) rudimentary anthers. Poor seed fertility. cp and mt: B. nigra Flowers with narrow petals and occasionally with petaloid anthers. Nectaries very poorly developed. Female fertility low. cp and mt: D. muralis Flowers normal with nondeshicent anthers and excellent nectaries. Female fertility: 96%. Cp and mt: B. oxyrrhina. Leaf chlorosis in B. juncea rectified following protoplast fusion Plants similar to B. oleracea in floral morphology and female fertility. cp: R. ativus

Ogura 1968; Bannerot et al. 1974; Mathias 1985

Reference

Leaves highly chlorotic. Flowers with petaloid anthers or absent nectaries. Female fertility poor.

Characteristics

100

B. napus

B. napus

B. juncea B. napus

B. juncea

B. juncea B. napus

Brassica tournefortii

Diplotaxis siifolia

Trachystoma ballii

Moricandia arvensis

Recepient species

Raphanus sativus (Kosena)

Cytoplasmic donor

Table 2.17. (Continued) Characteristics

Plants green. Flowers with stunted filaments and aborted anthers. Female fertility normal. mt: recombined Somatic hybrid Leaves green. Flowers with normal B. tournefortii or without petals and reduced þ B. napus empty anthers. Female fertility normal. cp: B. napus, mt: recombined Sexual alloploid Leaves green. Flowers with reduced D. siifolia B. juncea anthers and well-developed nectaries. Female fertility 95 %. cp and mt: D. siifolia Somatic hybrid Leaves green. Flowers with 2 types T. ballii þ B. juncea; of anthers: petaloid and selender cp: B. juncea mt: reco nondehiscent. Female fertility 30–40%. cp: B. juncea; mt: recombined Somatic hybrid M. ‘arvensis Leaves highly chlorotic almost þ B. juncea yellowish. Delayed flowering. Normal flowers with slender anthers and excellent nectaries. Female fertility 96%. cp and mt: M. arvensis. Chlorosis rectified following protoplast fusion.

Somatic hybrid Raphanus sativus þ B. napus

Origin

Prakash et al. 1998 Kirti et al. 1998

Kirti et al. 1995b

Rao et al. 1994; Rao and Shivanna 1996

Stiewe and Ro¨bbelen 1994; Liu et al. 1996

Sakai and Inamura 1990

Reference

101

B. juncea

B. juncea

B. rapa

B. juncea B. napus

B. juncea

B. juncea

Raphanus sativus

Diplotaxis erucoides

Diplotaxis berthautii

Eruca sativa

Erucastrum canariense

Diplotaxis catholica

Diplotaxis catholica

Moricandia arvensis

Sexual hybrid M. arvensis R. sativus

Sexual hybrid D. catholica B. juncea

Somatic hybrid D. catholica þ B. juncea

Sexual alloploid E. canariense B. rapa

Sexual hybrid E. sativa B. rapa

Sexual hybrid D. berthautii B. rapa

Sexual hybrid D. erucoides B. rapa

Leaves normal green. Flowers with smaller slender anthers and excellent nectaries. Female fertility 96%. cp and mt: D. erucoides Leaves green, flowers with short indehiscent or petaloid anthers. Female fertility 95%. cp and mt: D. berthautii Leaves green. Abnormal flowers with petaloid, partial petaloid or slender anthers. Poorly developed nectaries. Female fertility low. cp and mt: E. sativa Leaves normal green. Flowers with slender anthers and excellent nectaries. Female fertility 95%. cp and mt: E. canariense Leaves green, flowers with small rudimentary anthers and excellent nectaries. Female fertility 96%. cp: B. juncea, mt: recombined Leaves green. Flowers with petaloid/tubular structures. Nectaries poorly developed. Abnormal style. Female fertility 40%. cp and mt: D. catholica Leaves light green. Flowers with well-developed anthers and nectaries. cp and mt: M. arvensis Bang et al. 2002

(continued)

Mohanty 1996; Pathania et al. 2003

Prakash 2001; Pathania et al. 2007

Prakash et al. 2001; Banga et al. 2003b

Matsuzawa et al. 1999

Malik et al. 1999; Bhat et al. 2008

Malik et al. 1999; Prakash 2001; Bhat et al. 2006

102

B. napus

B. napus

Arabidopsis thaliana

Orychophragmus violaceus Sinapis arvensis

B. napus

B. rapa B. juncea B. napus

Recepient species

Enarthrocarpus lyratus

Cytoplasmic donor


Somatic hybrid O. violaceus þ B. napus Somatic hybrid S. arvensis þ B. napus

Somatic hybrid A. thaliana þ B. napus

Sexual hybrid E. lyratus B. rapa

Origin Leaves pale green. Delayed flowering. Flowers small with narrow petals having petaloid or rudimentary anthers. Female fertility poor. cp and mt: E. lyraus Plants normal green. Two types of flowers: (1) feminized anthers and abnormal pistils and (2) reduced shrunken anthers. Normal nectaries. Female fertility normal. mt: recombined Plants green. Flowers with reduced anthers. Female fertility normal. Plants green. Flowers normal with slender anthers. Female fertility normal.

Characteristics

Mei et al. 2003; Hu et al. 2004

Mei et al. 2003

Leino et al. 2003

Deol et al. 2003; Banga et al. 2003a; Janeja et al. 2003

Reference


103

organelle associated traits, which are not likely to obtain through conventional sexual hybridization. Protoplast fusion allows the production of hybrid cytoplasm, commonly referred to as cybrid, that often has new and rearranged novel combinations of mitochondria and chloroplast genomes. Intergenomic mitochondrial recombination in Brassiceae somatic hybrids is a frequent phenomenon. Pelletier et al. (1983) were the first to demonstrate the efficacy of protoplast fusion for rectifying chlorosis through chloroplast substitution while retaining the male-sterility trait in CMS (Raphanus/Ogu) B. napus. Furthermore, some cybrids with better nectaries and atrazine resistance were obtained. This investigation induced researchers to transfer and combine sterility-inducing cytoplasm with chloroplast-encoded atrazine resistance and improve CMS systems through cytoplasmic hybridization in B. rapa, B. oleracea, and B. napus (Chetrit et al. 1985; Barsby et al. 1987; Robertson et al. 1987; Chuong et al. 1988; Jourdan et al. 1989a,b; Christey et al. 1991; Kao et al. 1992; Temple et al. 1992; Earle and Dickson 1994). Leaf chlorosis has been overcome in CMS (Raphanus/Ogu) B. napus (Pelleter et al. 1983; Menczel et al. 1987; Jarl and Bornman 1988; Jarl et al. 1989)), (Raphanus/Ogu) B. oleracea (Kao et al. 1992; Walters et al. 1992: Walters and Earle 1993), (Raphanus/ Ogu) B. juncea (Kirti et al. 1995a), (Oxyrrhina) B. juncea (Kirti et al. 1993; Arumugam et al. 2000), and (Moricandia) B. juncea (Kirti et al. 1998). In all these investigations, chlorosis correction was attributed to substitution of alien chloroplasts with those of crop chloroplasts. The cybrids are completely green; show normal growth and flowering; are fully male sterile and have normal female fertility. Because of maternal inheritance of the chloroplast genome, green character in CMS is stable in subsequent generations. Similarly, cold-tolerant CMS (Ogu) B. oleracea var. capitata was produced following protoplast fusion between cold-tolerant CMS broccoli and fertile cabbage (Sigareva and Earle 1997). In addition to these new cytoplasmic substitution lines, already existing sterile cytoplasms have been transferred following protoplast fusion. Examples include Polima CMS to broccoli (Yarrow et al. 1990), Nigra CMS to broccoli (Christey et al. 1991), and Tournefortii cytoplasm to rapid-cycling B. oleracea (Cardi and Earle 1997). It has been elegantly demonstrated in the Brassiceae that CMS plants with genetically reconstituted mitochondria obtained from protoplast fusion with less alien genetic content and least divergence from cultivated species have minimal floral abnormalities. Systems where such improved floral morphologies have been obtained include (Raphanus/Ogu) B. napus (Pelletier et al. 1983), B. oleracea (Kao et al. 1992), and B. juncea (Kirti et al. 1995a), (Tournefortii) B. napus (Stiewe

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and Ro¨bbelen 1994; Liu et al.1996), (Tournefortii) B. juncea (Arumugam et al. 1996), and (Arabidopsis) B. napus (Leino et al. 2003).

VI. GENOME DISSECTION AND DEVELOPMENT OF CHROMOSOME ADDITION LINES Chromosome addition lines have a major role in revealing genome organization and evolution, identifying gene linkage groups, assigning species-specific characters to a particular chromosome, and comparing gene synteny between related species. Localization of specific markers on individual chromosomes facilitates construction of genetic and cytogenetic maps. Their practical utilization lies in introgressing characters of agronomic value, particularly from alien species to crop cultivars. Several Brassica and related genomes— B. nigra, B. oleracea, B. rapa and B. oxyrrhina, Diplotaxis erucoides, Raphanus sativus, Sinapis alba, S. arvensis, Moricandia arvensis, Crambe abyssinica, Orychophragmus violaceus, and Arabidopsis thaliana—have been dissected using a series of monosomic addition lines (Table 2.18). Disomic additions have also been generated but only in a few instances for a specific chromosome as in (A. thaliana) B. napus–A. thaliana (Leino et al. 2004), B. napus–S. alba (Wang et al. 2005b) and B. napus–C. abyssinica (Wang et al. 2006a). The recently developed full set of nine disomic B. napus–R. sativus addition lines by Budhan et al. (2008) is the first complete disomic alien chromosome addition series in Brassicaceae. B. oleracea was the first genome to be dissected and is the most extensively studied. Addition lines generally do not show specific morphological phenotypes associated with a particular chromosome and are rarely distinguishable from one another, thus requiring additional markers for identification. It may well be that the recipient nuclear background masks the effect of added chromosome, or its effect is negated by homoeologous chromosome as these genomes evolved from a common archetype. Nevertheless, the added chromosomes sometimes exhibit peculiar morphological characters. For example, a radish chromosome addition in B. napus exhibits white flower color (Sernyk and Stefansson 1982). Chromosome 1 of Diplotaxis erucoides in B. napus was distinguished by light yellow color of their flowers (Chevre et al. 1994b). Also all additions of Sinapis alba in B. napus background possessed a long beak characteristic of S. alba (Wang et al. 2005b). Cytological investigations have been carried out in a number of studies. In general, the alien chromosome remained unpaired as a

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B. rapa B. rapa

B. rapa B. rapa

B. rapa B. rapa B. rapa Raphanus sativus B. napus B. napus

B. oleracea B. oleracea

B. oleracea var. alboglabra B. oleracea var. alboglabra

B. B. B. B.

B. B. B. B. B. B.

nigra nigra nigra nigra nigra nigra

B. nigra B. nigra

napus napus napus napus napus oleracea

B. rapa

B. oleracea

B. B. B. B. B. B.

B. rapa

B. oleracea

oleracea var. alboglabra oleracea var. alboglabra oleracea var. alboglabra oleracea

B. rapa

Background genome

B. oleracea

Genome dissected

Disomics Monosomics, disomics Disomics Monosomics Monosomics Monosomics Monosomics Monosomics

Monosomics Monosomics Monosomics Monosomics

Monosomics Monosomics, disomics Monosomics Monosomics

Monosomics

Monosomics, disomics Monosomics

Nature of additions

Table 2.18. Major articles on chromosome addition lines.

Isozymes, fatty acids, RFLPs Isozymes, RFLPs, RAPDs Black leg resistance Black leg resistance, isozymes Isozymes, RAPDs Isozymes, RAPDs

Cytology Cytology

Cytology, isozymes Chromosome morphology, Isozymes, RAPDs Chromosome morphology Cytology, RAPDs FISH Morphology, RAPDs

Phenotypes, isozymes, RFLPs Chromosome morphology, isozymes RFLPs Cytology

Isozymes, rDNA

Markers

Chevre et al. 1991 Struss et al. 1992 Zhu et al. 1993 Chevre et al. 1996 Struss et al. 1996 Chevre et al. 1997b

Jahier et al. 1989 Struss et al. 1991

(continued)

Cheng et al. 1994a,b, 1995a Heneen and Jrgensen 2001 Hasterok et al. 2005b Kaneko et al. 1987, 1991, 1996, 2000

Chen et al. 1992 Chen et al. 1997a,b

McGrath et al. 1990 McGrath and Quiros 1990; Hu and Quiros 1991 Hosaka et al. 1990 Lee and Namai 1992

Quiros et al. 1987

Reference

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Alloplasmic (Raphanus) B. napus Alloplasmic (Moricandia) Raphanus sativus B. napus B. napus

B. juncea

Raphanus sativus

Arabidopsis thaliana Crambe abyssinica

Orychophragmus violaceus

Moricandia arvensis

napus napus napus oleracea napus

B. B. B. B. B.

Diplotaxis muralis Sinapis arvensis Sinapis alba Sinapis alba Raphanus sativus

Brassica oxyrrhina

B. rapa

Diplotaxis erucoides

Background genome

Diplotaxis erucoides Raphanus sativus Alloplasmic (oxy) B. rapa B. napus

B. nigra

Genome dissected


Disomics Monosomics, disomics Monosomics

Monosomics

Monosomics, disomics

Monosomics, disomics Monosomics Monosomics Monosomics Monosomics Monosomics

Monosomics

Monosomics

Monosomics

Nature of additions

GISH

GISH, RFLPs GISH

Morphology, cytology, RAPDs

RAPDs, FISH

Flower color, morphology, RAPDs Male sterility GISH, black leg resistance GISH GISH FISH, RFLPs

Morphology, RAPDs

RAPDs, leaf shape

Isozymes, RFLPs

Markers

Xu et al. 2007

Leino et al. 2004 Wang et al. 2006a

Bang et al. 2002

Fan et al. 1985 Snowdon et al. 2000b Wang et al. 2005b Wei et al. 2007 Peterka et al. 2004; Budahn et al. 2006 Budhan et al. 2008

Chevre et al. 1994b

Srinivasan et al. 1998

Kaneko et al. 2001, 2003

This et al. 1990

Reference


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univalent at metaphase 1 of meiosis. However, it underwent pairing also and formed a trivalent as in R. sativus–B. oleracea (Kaneko et al. 1987), B. rapa–B. oleracea (Chen et al. 1992; Heneen and Jrgensen 2001; Hasterok et al. 2005b), B. rapa–B. oxyrrhina (Srinivasan et al.1998), B. napus–S. alba (Wang et al. 2005b) and B. napus–Crambe abyssinica (Wang et al. 2006a). These associations reflected intergenomic homoeology between the added chromosome and recipient genome chromosomes. Using GISH, Wang et al. (2005b) observed homoeologous associations between S. alba and B. napus chromosomes, and in some cases recombinant chromosomes could clearly be identified. Hasterok et al. (2005b) identified B. oleracea chromosomes undergoing pairing with B. rapa chromosomes including chromosome C5 with an intercalary 5s rDNA locus and chromosomes C8 and C9 involving the regions occupied by 18S-5.8S–25S rRNA genes. On the contrary, in B. nigra additions on B. napus, only occasional chromosome pairing was observed (Jahier et al. 1989; Struss et al. 1991), reflecting the genetic distance between B. nigra and B. napus (AC) genomes as proposed earlier by several investigators. Transmission frequency of added chromosomes through male and female gametes does not follow a Mendelian pattern. Many factors, such as meiotic behavior of added chromosomes and their integrity (intact or recombined), genotype, and ploidy level of the donor and recipient species, affect transmission. Transmission frequency is generally far higher through the ovules than the pollen. Reduction in transmission frequency of added chromosomes due to competition with normal gametes was a common feature, leading to production of normal euploid type. Transmission of B. nigra additions was assessed using isozyme markers carried by different chromosomes. It was on an average 14% to 23% through ovules while the male transmission values ranged from 27% to 39% (Chevre et al. 1997b) and 8% to 30% (This et al. 1990). However, in Oxyrrhina addition lines, there was a decrease in ovule transmission frequency (Srinivasan et al. 1998). Addition lines Raphanus sativus–B. rapa, R. sativus–B. oleracea and R. sativus–Moricandia arvensis were generally stable, and predominant formation of gametes with added chromosomes might explain these observations (Kaneko et al. 1991; Bang et al. 2002; Kaneko et al. 2003). The male and female fertility of B. nigra additions were highly variable. Pollen fertility in general was high. B. nigra chromosome 2 in B. oleracea induced complete male sterility but not in B. napus (Chevre et al. 1997b). A similar behavior was also observed by This et al. (1990), where two B. nigra addition lines were totally male sterile

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in D. erucoides background. Monosomic R. sativus additions to alloplasmic (R. sativus) B. napus showed disturbed stamen development with very poor pollen production (Budhan et al. 2008). Some of the monosomic additions have been observed to restore fertility to alloplasmics, and four such examples are reported. Synteny group 6 of B. oxyrrhina to (B. oxyrrhina) B. rapa (16% pollen fertility, Srinivasan et al. 1998), an unspecified chromosome of Moricandia arvensis to (M. arvensis) B. juncea (53% pollen fertility, Prakash et al. 1998), chromosome c of Moricandia arvensis to (M. arvensis) R. sativus (85.6 pollen fertility, Bang et al. 2002), chromosome III of Arabidopsis thaliana to (A. thaliana) B. napus (Leino et al. 2004), and Raphanus chromosome f to (R. sativus) B. napus (Budhan et al. 2008). Due to small size of chromosomes and nonavailability of precise cytological landmarks in the earlier years, addition lines were characterized either through rare association with specific morphological characters, such as flower color, male sterility, or disease resistance; in recent years, isozyme and DNA markers are widely employed to characterize them. Markers employed include RFLP, RAPD, SSR, and GISH and FISH. By making use of these techniques, substantial information has been accumulated. Isozymes were initially used to characterize addition lines. B. rapa-oleracea was identified using such enzyme systems, such as 6PGD, PGI, LAP, and PGM (Quiros et al 1987); 6 PGD-1, PGM-1, and GOT-5 (McGrath and Quiros 1990); and PGM-2, PGDH-1, and PGDH-2 (Hu and Quiros 1991). B. nigra chromosome additions in the background of B. napus genome were characterized extensively using a large number of isozymes, such as MDH, IDH, LAP, 6-PGDH, ACO, PGI, TPI, GOT, PGM, and ADH (Chevre et al. 1991; Struss et al. 1996). Monosomic additions of Diplotaxis erucoides–B. nigra revealed synteny associations for loci coding for isozyme markers GOT-2, 6PGD-2, MDH-2, LAP-2, and TPI-1 (Quiros et al. 1987). This et al. (1990) located these synteny associations on four different B. nigra chromosomes using six isozyme loci and confirmed the observations of Quiros et al. (1987). Also a Diplotaxis erucoides chromosome was observed to carry three isozyme alleles (Chevre et al. 1994b). RAPD analysis have been carried out extensively in brassicas. Use of this system has been demonstrated in B. nigra (Quiros et al 1991; Struss et al. 1992, 1996; Chevre et al. 1994a), B. oxyrrhina (Srinivasan et al. 1998), Diplotaxis erucoides (Chevre et al. 1994b), and Moricandia arvensis (Bang et al. 2002) additions. Similarly, addition lines in several instances have been characterized through RFLPs. Using these markers, many chromosomes of the different genomes could precisely


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be identified. The markers revealed extensive intergenomic recombination, presence of duplicated loci, and synteny rearrangements of chromosomes. GISH has been one of the major tools to identify alien chromosomes and was employed in addition lines for Sinapis arvensis (Snowdon et al. 2000b), Arabidopsis thaliana (Leino et al. 2004), S. alba, Crambe abyssinica (Wang et al. 2005b, 2006a), and Orychophragmus violaceus chromosomes (Li and Ge 2007) in the background of the B. napus genome. Recently FISH has been used to identify the addion lines. For example, Peterka et al. (2004) identified chromosome d of Raphanus sativus carrying a gene imparting resistance to beet cyst nematode. Hasterok et al. (2005b) characterized three of the nine B. oleracea var. alboglabra chromosome additions using double target FISH. Chromosome addition lines as such are commercially unacceptable because of their unstable nature, reduced fertility, and expression of undesirable traits due to alien chromosomes. However, these lines are of academic interest and important genetic stocks for introgressing alien genetic material that might ultimately confer agronomic or horticultural advantages. For achieving gene introgression, homoeologous recombination between the alien chromosome and its homologous counterpart of the recipient genome should occur. Location of genes for horticultural traits such as curd in cauliflower, heading in cabbage, root enlargement in turnip, and axillary bud enlargement in Brussels sprouts are envisaged (Quiros et al. 1987; This et al. 1990). Chromosomal location of various genes controlling important agronomic traits have been accomplished by dissecting the respective genomes in various nuclear background; these include determining black-seeded phenotype and erucic acid content on a B. oleracea var. alboglabra chromosome (Chen et al. 1997b); a male sterility factor on a Diplotaxis muralis chromosome (Fan et al. 1985); self-incompatibility locus in B. nigra on chromosome 3 (Chevre et al. 1997b); resistance genes to black leg (Phoma lingam) on B. nigra (Zhu et al. 1993; Chevre et al. 1996; Struss et al. 1996); and Sinapis arvensis chromosomes (Snowdon et al. 2000b); B. oleracea chromosome possessing genes conferring resistance to turnip mosaic virus (TuMV) (Kaneko et al. 1996); a fertility-restoring gene on chromosome III of Arabidopsis (Leino et al. 2004); resistance to beet cyst nematode on chromosome d of Raphanus sativus (Peterka et al. 2004); and high erucic acid content on a Crambe abyssinica chromosome (Wang et al. 2006a). A dominant fertility restoration gene was introgressed to CMS (Moricandia) B. juncea following generation of Moricandia chromosome addition on CMS (Prakash et al. 1998), as also from

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Enarthrocarpus lyratus to CMS (Lyratus) B. rapa (Deol et al. 2003) and B. juncea (Banga et al. 2003a). RAPD markers linked with the genes for erucic acid and seed color on B. oleracea var. alboglabra chromosomes have been established (Jrgensen et al. 1996; Chen et al. 1997b). It was obsereved that chromosome 4 carries the gene for seed color and exerts its control embryonically. Chromosome 1 carries a gene that controls seed color maternally (Heneen and Brismar 2001). Hasterok et al. (2005b) are of the view that precise identification of extra chromosome in addition lines could be accomplished by using chromosome-specific or even arm-specific sets of BAC clone-based probes, as has been demonstrated by Howell et al. (2002), Ziolkowski and Sadowski (2002), and Koo et al. (2004).

VII. MITOCHONDRIAL GENOME A. Organization Plant mitochondrial genomes are relatively large as compared with other eukaryotes and range in size from 200 kb to 2000 kb (Fauron et al. 2004). Among the angiosperm plants examined so far, members of Brassica and related genera have the smallest mitochondrial genome. For example, the mt-genome of Sinapis alba (syn. B. hirta) is 208 kb while the values for B. rapa, B. oleracea, and B. napus are 218, 219, and 222 kb, respectively (reviewed in Palmer 1985; Pring and Lonsdale 1985). Arabidopsis thaliana, the model eudicot plant with the smallest nuclear genome, has a mitochondrial genome of 367 kb (Unseld et al. 1997). The circular nature of the mitochondrial genome of plants was first deduced from physical mapping studies of B. rapa and B. oleracea (Chetrit et al. 1984; Palmer and Shields 1984). This is now confirmed from complete mitochondrial genome sequencing exercises in several organisms including fungi, plants, and animals. However, such single circular molecules have not been physically visualized from electron microscopic examinations. Instead, small circular molecules of varying sizes have been observed in plant mitochondrial preparations. Based on RFLP analysis, Palmer and Shields (1984) demonstrated a tripartite structure for the mitochondrial genome of B. rapa. They identified two 2 kb direct repeats in the master circular genome, which were found to undergo recombination, giving rise to subgenomic circles of 83 and 135 kB. Similar organization of mt-genome has now been found in several other plants. The repeats ranging in size from 50–100 bp to 1–10 kb have been recorded in mitochondria of several species. For


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example, the B. napus mt genome has two large repeats of 1 to 10 kb and 37 of 0.1 to 1.0 kb. A. thaliana has four repeats of 1 to 10 kb and 90 repeats of 0.1 to 1.0 kb. These repeats are involved in homologous recombination. Intra- and intermolecular recombination in the repeat region is believed to generate multipartite subgenomic circular molecules. Such recombination events have been implicated in substoichometric shift in mitochondrial genome in different tissues and accessions, and creation of novel open reading frames (orfs). S. alba carries only a single copy of the repeat found in B. rapa and thus is the only species known to lack any large direct repeats (Palmer and Herbon 1987). The repeat sequences contain protein coding sequences; hence such genes are duplicated. The repeat sequences including the protein coding genes are different in different species. For example, in B. napus, a part of the cox2 gene is found in the repeat region whereas in A. thaliana atp6 gene is duplicated (Handa 2003). Detailed restriction profiles of mitochondrial genomes of Brassica species have revealed very limited intraspecific variation within species. Intraspecific variations in the form of two short deletions (100 and 700 bp in B. nigra) and one inversion (in S. alba) were detected (Palmer 1988). Considerable variation is found among species in both mt-DNA restriction and RFLP patterns (Palmer and Herbon 1987; Palmer 1988; Pradhan et al. 1992). However, most of the variation appears to be restricted to noncoding regions (Palmer and Herbon 1986, 1987). Based on comparative restriction analysis of different mt-genomes, it was found that inversions and small deletions are mainly responsible for the observed variation in mt-genomes among species. For example, mt-genome restriction profiles of S. alba and B. rapa differ significantly. However, most of the mt-genome can be divided into 11 regions; sequences within each region have the same arrangement in the two genomes, but the relative orientation and order of these regions differ between the species (Palmer and Herbon 1987). Similarly, B. rapa and B. oleracea differ by three large inversions whereas B. rapa and Raphanus differ by 14 inversions (Palmer 1988). B. Gene Content Despite large variation in size, gene content in mitochondrial genomes of angiosperms appears to be comparable and limited. Thus much of the observed difference in mt-genome size among different species is attributable to the insertion of foreign DNA and the presence of large repeated regions. This conclusion is further strengthened

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following the availability of complete mitochondrial genome sequences of A. thaliana and B. napus (Handa 2003). Plant mitochondrial genomes contain about 50 genes coding for various functions such as transcription, protein synthesis and transport, oxidative phosphorylation, and so on. In addition, dozens of orfs of unknown function are also found in sequenced mitochondrial genomes of plants. The overall G þ C content of the B. napus genome is 45.2%, which is comparable to other plant mitochondrial genomes. The gene content of mitochondrial genomes of B. napus and A. thaliana is summarized in Table 2.19. The only major difference in gene content between mitochondrial genomes of B. napus and A. thaliana is with respect to rps14 gene, which is a nuclear gene in A. thaliana (Figueroa et al. 1999). Although A. thaliana contains 22 tRNA species (five more than B. napus), both the species can specify only 15 amino acids. Thus a complete set of t-RNA genes is lacking in Brassica and Arabidopsis mitochondrial genomes. Some of the sequences (about 3.6%) present in the B. napus mt-genome appears to be of plastid origin, including some tRNA species. Mitochondrial genes of B. napus share many features, such as the presence of introns and RNA editing with mt-genes of other species. Despite wide evolutionary divergence between A. thaliana and B. napus, there is a high degree of conservation at the functional level. The size and number of introns are identical between the two species. Similarly, the RNA editing sites (441 in Arabidopsis versus 427 in B. napus) are highly conserved (Handa 2003).

Table 2.19. Number of genes in mitochondrial genomes of B. napus and A. thaliana. Genes Respiratory Complex I Complex II Complex III Complex IV Complex V Cytochrome biogenesis Transcription Translation Transport t-RNA r-RNA orfs ( > 100 amino acids) Total (excluding orfs)

B. napus

A. thaliana

9 — 1 3 5 4 1 9 1 17 3 45 54

9 — 1 3 5 4 1 7 1 22 3 85 59


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It is now clear that during the course of evolution, much of the mitochondrial genome has been transferred to the nucleus. The complete genome sequencing of A. thaliana and rice have shown this more clearly. A 620-kb segment of mt genome is found on chromosome 2 of A. thaliana (Stupar et al. 2001). Similarly, a 190-kb sequence of rice mitochondrial genome is present on chromosome 12 (Ueda 2005). Therefore, it is not unexpected that other large segments of mt DNA will be found in nuclear genomes of Brassica species. C. Mitochondrial Plasmids Small autonomously replicating linear plasmids are also found in some accessions of Brassica. Palmer et al. (1983b) observed a 11.3-kb plasmid in B. rapa whose copy number varied 100-fold among accessions containing the plasmid. Its nucleotide sequence was found to differ from other known sequences. Further, the presence of plasmid was associated with cytoplasmic male sterility. Since this plasmid was absent in the cytoplasm donor species (R. sativus), its transmission from the male side was suspected. Handa et al. (2002) also reported a 11.6-kb linear plasmid in B. napus, which was capable of transmission through both maternal and paternal route. This plasmid contains six orfs (two coding for phage-type DNA polymerase and one coding for phage-type RNA polymerase). All six orfs were found to be transcribed, and proteins of at least three orfs are found at high levels in flower buds of B. napus.

VIII. PLASTID GENOME To date, complete plastid genome sequences are available for 55 species including A. thaliana. These studies have shown that cp-genomes of angiosperm plant species are highly conserved for both gene order and sequence. In general, plant cp-genomes are circular and contain two large (22–25 kb) inverted repeats. These features are found to be shared by members of genus Brassica. For example, Link et al. (1981) observed uniform 158-kb circular cp-DNA molecules of Sinapis alba by electron microscopic examination and later confirmed it through restriction analysis. Further, two inverted repeats accounting for nearly 30% of the cp-genome were identified. Most of the studies on cp-genomes in Brassica are focused on variation for restriction enzyme sites (Erickson et al. 1983; Palmer et al. 1983a; Vedel and Mathieu 1983; Warwick and Black 1991, 1993, 1994, 1997; Warwick et al. 1992).

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Palmer et al. (1983a) compared restriction patterns of six U-triangle species along with S. alba and R. sativus. Small insertions or deletions (indels, 50–400 bp) seem to be the cause of most of the variations observed among species. Total sequence variation among Brassica species was estimated to be about 2.4%. Low level (0–0.01%) of intraspecific variation was also reported by Warwick’s lab based on cp-DNA RFLP and restriction analyses. A majority (53–80%) of restriction site mutations recorded were found between species. These studies have been extremely useful in identifying the maternal parents of the allotetraploid species. Availability of the complete cpDNA sequence of A. thaliana (Sato et al. 1999) may provide further opportunity for more incisive investigation of cp-genome evolution in Brassiceae.

IX. POTENTIAL ROLE OF ARABIDOPSIS THALIANA IN BRASSICA IMPROVEMENT A. A. thaliana as a Model Crucifer The fact that Arabidopsis and Brassica are in the same family is of great advantage to Brassica researchers who are benefiting from the information generated by the completed Arabidopsis thaliana genome sequence. Although the taxonomic distance between the two genera is large, with approximate divergence of 15 to 20 million years (Yang et al. 1999; Wroblewski et al. 2000), there is a great deal of conservation. The genomes of diploid brassicas are three to four times larger than that of Arabidopsis (157 Mb, Bennett et al. 2003), ranging from 468 Mb for B. nigra to 662 Mb for B. oleracea (Arumuganathan and Earle 1991). In spite of these differences, sequence conservation and synteny are large enough in most cases to use the genome of A. thaliana as a guide to find genes of interest in Brassica species. B. Cytology and Possible Origin of the A. thaliana Genome Similar to Brassica, mitotic chromosomes of A. thaliana are too small and lack distinctive landmarks; thus developing a physical cytogenetical map is not possible. Its chromosome number (2n ¼ 10) was determined by Laibach in 1907. Since 1960s, several researchers reported mitotic and meiotic and a giemsa C-banded karyotypes. However, a major breakthrough in its cytogenetics was achieved with the introduction of FISH technology using repeat DNAs such as


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ribosomal (18S, 26S, and 5S rDNAs), pericentromeric, centromeric, and telomere repeats (reviewed in Koornneef et al. 2003; Lysak et al. 2003). Both mitotic and meiotic chromosomes have been investigated, but better resolution was achieved with meiotic prophase complements, and heterochromatic and centromeric regions could be clearly differentiated. Using BAC contigs as probes in FISH, Fransz et al. (1998) presented a comprehensive pachytene bivalents karyotype. Accordingly, the mean total length of pachytene bivalents is 331 mm. The major part is euchromatin, with heterochromatin regions comprising of only 7.1 %, confined mostly in pericentromeric regions and NOR. Chromosomes 1 and 5 are the longest and metacentric with average length of 80.76 and 76.32 mm respectively. Chromosome 5, the second largest, carries a major and a minor 5S rDNA loci. The major locus is in the pericentromeric heterochromatin region of the upper arm and the minor locus is in the opposite arm. Chromosomes 2 and 4 are acrocentric and carry NOR. Their average length is 52.12 and 52.65 mm respectively. Chromosome 4 contains a 5S rDNA locus in the pericentromeric heterochromatin region of the short arm. Chromosome 3, a submetacentric with an average length of 69.34 mm, contains a major 5S rDNA in the middle of the long arm. Polymorphism for 5S rDNA loci was also observed in different ecotypes. However, all of them possess chromosomes 4 and 5 in the short arms. Earlier investigations documented 45S rDNA on NOR of chromosomes 2 and 4 and 5S rDNA on chromosomes 4 and 5 and polymorphic sites on chromosome 3 (Murata et al. 1997). Chromosome painting has been fairly successful due to the high resolution of FISH on pachytene chromosomes using BAC contig pools as probes. Chromosomes 1, 2, and 4 were entirely painted (Fransz et al. 2000; Lysak et al. 2003). Of these, chromosome 4 became the first entirely painted chromosome of a euploid plant karyotype. Earlier, identifying Arabidopsis individual chromosomes was difficult; now FISH technique has made it possible to unambiguously identify chromosomes or specific chromosome regions. Sequencing of the A. thaliana genome has disclosed extensive duplication, indicating a paleotetraploid origin possibly resulting from three rounds of genome duplications (Henry et al. 2006). The first two rounds took place very early on in the evolution of the angisoperms, between 90 and 135 million years ago. According to this tentative hypothesis, the most recent polyplodization event took place 24 to 40 million years ago, before the divergence of the Arabidopsis and Brassica lineages, when an ancestral species of n ¼ 4 originated a tetraploid n ¼ 8 species. Therefore, the basal chromosome number of the family is considered to

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be n ¼ 4. A. thaliana evolved from the hypothetical tetraploid species approximately 5 million years ago by reduction in chromosome number caused mostly by chromosome fusions and also by translocations and inversions (Henry et al. 2006; Schranz et al. 2006). These chromosomal rearrangements were accompanied by substantial DNA losses in A. thaliana (Town et al. 2006), when compared to A. lyrata and other related species (Schranz et al. 2006). C. Synteny Conservation The first attempt to compare synteny between Arabidopsis and Brassica based on RFLP markers was reported by Kowalski et al. (1994), who found ‘‘islands of conserved gene organization.’’ This report was confirmed by subsequent studies using techniques of increasing levels of sophistication, such as construction of maps using Arabidopsis ESTs (Lan et al. 2000; Babula et al. 2003) and a transcriptome map based on cDNA polymorphisms (Li et al. 2003). The EST maps revealed colinear regions of 20 cM between B. oleracea and A. thaliana (Babula et al. 2003). Transcriptome mapping allowed a global gene for gene alignment of the genomes of B. oleracea and A. thaliana by construction of a map based on mRNA from leaf tissue. The resulting map consisted of 247 cDNA markers obtained by the cDNA-SRAP technique. After sequencing 190 of the polymorphic cDNA fragments, 169 of these sequences had similarity to genes reported in Arabidopsis, which allowed alignment to the physical map of this species. Extensive colinearity was found between the two genomes for large chromosomal segments (but not whole chromosomes), albeit often inversions and deletions/insertions were shown. This work has been expanded by adding genomic markers and sequences corresponding to known genes to construct a high-density map of B. oleracea (Gao et al. 2007). Lukens et al. (2003) compared positions of Arabidopsis BAC sequences with B. oleracea sequences from RFLP probes previously located on a genetic map. Complex level of duplications prevented identification of orthologous segments between the two species. Hence, Lukens et al. (2003) developed an algorithm to facilitate identification of collinear loci. Using this statistical tool, the authors found 34 regions in Arabidopsis that were colinear with over 28% of the Brassica map, a result very similar to the result of empirical genetic mapping by Kowalski et al. (1994). Various approaches have discerned at least 20 to 30 chromosome structural rearrangements between Brassica and Arabidopsis, and a smaller number among the diploid Brassica spp. (Kowalski et al. 1994;


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Truco et al. 1996; Lan et al. 2000; Schranz et al. 2002; Lukens et al. 2003). Some of these rearrangements may have been in association with large-scale chromatin duplication. However, it remains controversial how many such events have occurred, how much chromatin they involved, and whether they occurred before or after the divergence of the various branches of the Brassica genus. Parkin et al. (2005) expanded the EST-RFLP comparative genomic study by mapping over 1,000 of these markers in B. napus. They found more than 21 conserved genomic units in Arabidopsis of approximately 9 Mb that could generate the B. napus genome through rearrangements and duplications. Centromeric regions are also conserved. These authors were able to construct alignments with each of the A and C genome chromosomes of B. napus and the five chromosomes of A. thaliana. As progress is made sequencing the Brassica genomes (Ayele et al. 2006), this new information enables researchers to compare genome organization of B. napus with A. thaliana, whose genome is now completely sequenced (Arabidopsis Genome Initiative 2000). This will add to the previous studies on comparative genomics for these species and reveal general conservation of gene content and colinearity. However, this conservation is incomplete, due to extensive chromosomal rearrangements (Kowalski et al. 1994; Cavell et al. 1998; Lagercrantz 1998; Lan et al. 2000; O’Neill and Bancroft 2000; Bancroft 2001; Quiros et al. 2001; Ryder et al. 2001; Babula et al. 2003; Li et al. 2003; Lukens et al. 2003). Town et al. (2006) sequenced BAC contigs covering a 2.2-MB region of the B. oleracea genome, which is partially triplicated in this species but duplicated in A. thaliana. They found 177 conserved colinear genes between the two species, and to explain absence of perfect triplication they inferred that 35% of the genes were lost in B. oleracea, most likely by deletions. Other factors breaking colinearity were gene duplication by tandem arrays in B. oleracea of single-copy A. thaliana genes, insertions, and dispersal of gene fragments. Additionally, the sequencing of three complete B. oleracea BAC clones corresponding to two different chromosomes in A. thaliana revealed less gene density in the former species and frequent accumulation of transposable elements (Gao et al. 2004, 2005, 2006). Genome enlargement by TE has occurred mostly by accumulation of tandem repeats and retrotransposons largely centered in centromeric and peri-centromeric heterochromatic regions (Lim et al. 2007). Therefore, during the evolution of these two Brassicaceae species, changes have occurred not only in chromosome number but also in chromosomal structure, causing frequent breaks in synteny (Gao et al. 2005, 2006).

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D. Synteny-Based Gene Discovery and Cloning Based on genomic shotgun sequences covering close to half of the B. oleracea genome, Ayele et al. (2006) estimated that 84% of the A. thaliana genes have a match in B. oleracea. They called these regions CAGs (conserved Arabidopsis genome sequences) and found that the highest sequence alignments occur near the centromeres of the Arabidopsis chromosomes. Sequence conservation is high in exons, ranging from 70% to 90% with the majority having similarities higher than 80%, whereas for introns it is < 70%. Protein similarity or orthologs is often above 95% (Gao et al. 2006). These high similarity values along with synteny conservation make it possible, in most cases, to find Brassica orthologs based on A. thaliana gene models with ease. Sadowski et al. (1996) exploited the genetic map of A. thaliana (Hauge et al. 1993) to probe the Brassica genomes with an A. thaliana gene complex carrying five genes within a 20-kb span (Gaubier et al. 1993). This complex comprises a well-characterized Em-like protein coding gene and other four flanking genes on chromosome 3. Although the fivegene complex array from A. thaliana was conserved on a single chromosome of each Brassica genome, additional copies for most of the genes were found in one or two other chromosomes. A similar situation was observed for a six-gene complex on A. thaliana chromosome 4, including the disease resistance gene RPS2 (Sadowski and Quiros 1998). In this case, besides the conserved array in one Brassica chromosome, four other chromosomes contained copies for some of the genes. The benefit of synteny conservation for gene discovery in Brassica is well demonstrated in studies on genes coding for glucosinolates (GSL), which are secondary metabolites synthesized by many species of the order Capparales, including Brassica and Arabidopsis. Breakdown products of GSLs, particularly isothiocynates, have been found to be anticarcinogenic (Talalay and Zhang 1996). Therefore, consumption of some of the brassica crops, such as broccoli, has been reported to exert cancer-protecting effects due to the formation of sulforaphane, an aliphatic glucosinolate-derived ITC (Fahey et al. 1997). The pattern of GSL accumulation and variability has been well described during the life cycle of A. thaliana (Brown et al. 2003). Furthermore, the genetic control GSL content is best understood in this model species, which provides a starting point to genetically improve GSL content and profiles in related Brassica crops. At least 10 genes related to the GSL pathway have been identified in A. thaliana


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(Mikkelsen et al. 2002; Wittstock and Halkier 2002), and knockout mutants for most of these genes are available. The colinearity between A. thaliana and B. oleracea has been explored for three chromosomal regions carrying three glucosinolate genes. Two of them are involved in side-chain elongation and belong to a gene family of major genes encoding methylthioalkylmalate synthase enzymes (MAM). In A. thaliana, three loci are duplicated in tandem (MAM1, MAM2 and MAM-L) on chromosome 5, and their presence depends on the ecotype; MAM-L is always present, but MAM1 and MAM2 are dispensable. A functional allele of MAM1 results in the presence of GSL with side chains containing four carbons (4C-GSL), whereas the presence a MAM2 in the absence of MAM1 results in the presence GSL with side chains containing three carbons (3C-GSL). The function of MAM1 is dominant to that of MAM2, because when both are present, the plants produce 4C-GSL (Kryomann et al. 2003). It was found that in B. oleracea, the BoGSL-ELONG gene corresponds to MAM1 in A. thaliana, which results in plants with 4CGSL (Li and Quiros 2002). Comparing the sequence of a 96.7-kb-long BAC clone (B19N3) from Brassica oleracea (broccoli) harboring the BoGSL-ELONG gene with its equivalent regions in A. thaliana disclosed these breaks in synteny: B19N3 contains eight genes and six TEs. The first two genes in this clone, Bo1 and Bo2, have its corresponding region at the end of chromosome 5 of Arabidopsis (24 Mb). The third gene, Bo3, corresponds to an ortholog at the opposite end (2.6 Mb) of the same chromosome. The other five genes, Bo4 to Bo8, also have a equivalent region on the same chromosome but at 7.7 Mb. Bo5 is a tandem duplicate of BoGSL-ELONG (Bo4) and was named BoGSL-ELONG-L, which is equivalent to MAM-L in A. thaliana. These five genes are colinear with those found in the corresponding region of Arabidopsis, which contains, however, 15 genes. Therefore, a cluster of 10 genes is missing in B. oleracea clone B19N3. All five genes have the same order and orientation in the genomes of both species. Their 36 exons constituting the eight homologous genes have high conservation in size and sequence identity in both species. The presence of duplicate member MAM2 has not been surveyed in B. oleracea, although it is known that it is not present in the broccoli variety used to study the segment harboring the BoGSL-ELONG gene.

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The region was further expanded by constructing a contig primer walking and BAC-end sequencing, revealing general gene colinearity beyond the segment harboring the BoGSL-ELONG gene (Gao et al. 2005). Two other B. oleracea BAC clones were surveyed for colinearity. The second BAC clone contained gene BoGSL-PRO, which is also a homolog of the MAM A. thaliana gene family. This gene has its homolog at the top of chromosome I in A. thaliana (At1g18500, MAM4). A duplicate member of this gene is located in the opposite arm of the same chromosome (At1g74040, MAM3). This gene is likely orthologous to BoGSLPRO-L, another member in the family also at a different location in B. oleracea. Twelve protein-coding genes and 10 TEs were found in this clone. The corresponding region in A. thaliana chromosome I has 14 genes and no TEs. No synteny breaks was observed for this clone, except for the two missing genes absent in B. oleracea (Gao et al. 2006). Compared to its homologous A. thaliana sequence, the third B. oleracea BAC clone harbors the side-chain modification gene BoGSL-ALK and has 101 kb. It is involved in desaturation, addition of a double bond, and removal of the methylsulfinyl residue in both 3C- and 4C-GSL. It corresponds to the gene AOP2 in A. thaliana, which is a member of another gene family. These genes code for 2-oxoglutarate-dependent dioxygenases, forming part of the AOP gene family located on chromosome 4. It is found that the sequenced B. oleracea BAC clone harbors 23 genes, including BoGSL-ALK, while the equivalent region in Arabidopsis contains 37 genes. All 23 common genes have the same order and orientation in both Brassica and Arabidopsis. The 16 missing genes in the broccoli BAC clone constitute two major blocks of five and seven contiguous genes, two singletons and a twosome. The 118 exons corresponding to these 23 genes have high conservation between the two species. The arrangement of the AOP gene family in A. thaliana is: AOP3 (GS–OHP) AOP2 (GS– ALK) pseudogene AOP1. In contrast, in B. oleracea (broccoli and collard), two of the genes are duplicated and the third gene, AOP3, is missing, and genes are arranged as follows: Bo–AOP2.1 (BoGSL–ALKa) pseudogene—AOP2.2 (BoGSL–ALKb)—AOP1.1—AOP1.2 (Gao et al. 2004). The general conclusion from these studies is very much the same that Kowaltski et al. (1994) reached earlier. There is general gene colinearity between A. thaliana and Brassica, but this is incomplete, and a break in synteny is common due to chromosomal rearrangements. Furthermore, these differences are accentuated by the presence of tandem duplicates for some genes in Brassica. Another difference that can be inferred but not generalized, because of the small portion of the


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genome surveyed so far, is the lower gene density found in the three BAC B. oleracea clones. This is due mostly to the insertion of TE in intergenic spacers and introns. As a consequence of these changes and breaks in colinearity, especially the frequent absence of genes in corresponding segments of A. thaliana, using this species as a guide to find a corresponding Brassica gene is not a trivial task. The tandem duplicates often found in the latter species require further experimentation to determine the correct gene based on its functionality and expression. E. Arabidopsis Knowledge-Based Gene Discovery and Brassica Improvement Brassica and Arabidopsis genomes share a high degree of homology ( > 80%), particularly in the exon regions, and most of the genes present in Brassica are represented in Arabidopsis. Hence knowledge gained from Arabidopsis is highly transferable to Brassica, and is providing valuable insights into various aspects of Brassica, including domestication and speciation, growth and development, and metabolism. Various approaches and resources currently are being employed to accomplish the goal of assigning functions to all the genes in Arabidopsis by 2010. Brassica improvement is expected to get a boost from the availability of complete functional genomic information of Arabidopsis. Once the key genes responsible for expression of a given trait are identified in Arabidopsis, they can be used to engineer the trait in Brassica. The examples discussed next highlight the significance of Arabidopsis functional genomics to Brassica. 1. Understanding Domestication. Kempin et al. (1995) identified a gene mutation in Arabidopsis, which together with a mutation in another gene, AP1 conferred cauliflower phenotype. Further, the same gene was found mutated in cultivated cauliflower suggesting its role in cauliflower phenotype. Subsequent studies have demonstrated that mutations in the cauliflower orthologue gene (BoCAL) are, indeed, responsible for cauliflower phenotype and have been selected during domestication of cauliflower (Lowman and Purugganan 1999; Purugganan et al. 2000). Similalry, nonshattering siliques is a characteristic feature of crop domestication. Liljegren et al. (2000) identified that mutations in two redundant genes SHP1 and SHP2 lead to non-shattering siliques in Arabidopsis. SHP1 and SHP2 orthologues are present in cultivated Brassica species and are expected to play similar function. Additional genes FRUITFUL and REPLUMLESS have also been identified in

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Arabidopsis, which negatively regulate SHATTERPROOF (Roeder et al. 2003). Based on this information, stergaard et al. (2006) developed nonshattering B. napus lines. Genes governing vernalization response and flowering time have also been well characterized in Arabidopsis. Robert et al. (1998) isolated four orthologues of Arabidopsis CONSTANS gene from B. napus lines differeing in flowering time and showed that their function is conserved between Arabidopsis and Brassica. FLC is a major gene responsible for suppression of flowering in Arabidopsis and is downregulated upon exposure to cold temperature. Kole et al. (2001) found that the major QTL, VFR2 responsible for winter type B. rapa cosegregated with FLC orthologues. These studies illustrate how Arabidopsis could serve as a reference for Brassica improvement. 2. Understanding Metabolism. Fatty acid metabolism has been extensively studied in Arabidopsis, and genes encoding key enzymes involved in fatty acid synthesis, elongation, and modification have been cloned and characterized. Analysis of QTLs for oil quality in Brassica crops have revealed that, in a majority of cases, these QTLs correspond to the known Arabidopsis genes involved in fatty acid metabolism. For example, FAE1 gene encodes the enzyme responsible for erucic acid biosynthesis in Arabidopsis. Mutations in the othrologues of the FAE1 gene have been found to be responsible for low-erucic acid in seed oils of B. rapa and B. oleracea (Das et al. 2002). Similarly, in B. juncea, FAE1.1 and FAE1.3 genes have been shown to cosegregate with QTLs, which account for 60% and 38% varaince for erucic acid content (Mahmood et al. 2003). Vitamin E (a-tocopherol) synthesis is restricted to photosynthetic organisms. Molecular analysis of Arabidopsis mutants has helped unravel the genes involved in tocopherol biosynthesis. Shintani and Della Penna (1998) cloned the gene encoding the enzyme g-tocopherol methyltransferase, which catalyzes the final step of vit E biosynthesis. Seed-specific overexpression of this gene resulted in elevated accumulation of vitamin E in seeds of Arabidopsis. Transgenic B. juncea lines accumulating vitamin E have been generated through ectopic expression of A. thaliana gene (Yusuf and Sarin 2007). Engineering tolerance to abiotic stress is an important objective of Brassica improvement program. Apse et al. (1999) identified a gene encoding vacuolar Naþ =Hþ antiporter in A. thaliana. This gene conferred salt tolerance in transgenic B. napus (Zhang et al. 2001). Similarly, Meur et al. (2006) have cloned the NPR1 (nonexpressor of pathogenesis related proteins1) of B. juncea based on the information on the cDNA sequence in Arabidopsis. Availability of sequence information of transparent testa genes of Arabidopsis has opened


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opportunities for developing yellow-seeded Brassica varieties (Debeaujon et al. 2003; Gruber et al. 2007; Lu et al. 2007; Wei et al. 2007). These examples amply demnonstrate the usability of genetic information from Arabidopsis in Brassica molecular biology and improvement. 3. Testing for Gene Function by Complementary Transformation. The most common and straightforward method to demonstrate that a cloned candidate gene is in effect the correct gene searched for a specific function is by in planta complementary transformation. Unfortunately, transformation is not always an easy task in Brassica species, which is largely genotype dependent. However, A. thaliana is easily and efficiently transformed (Clough and Bent 1998). Furthermore, a series of knockout stocks are available in these species covering many of the major genes of interest. Therefore, a routine approach to test for Brassica gene function is to introduce these genes by Agrobacterium transformation to various A. thaliana ecotypes and knockout mutants, depending on the gene under scrutiny. Following phenotypic changes predicted by the introduced gene by gain in function often demonstrates that the candidate gene is indeed the right gene. An example of this approach is illustrated by Li and Quiros (2003) who tested the function of the BoGSL-ALK genes described in the previous section. In this study, they introduced a functional allele of BoGSL-ALK into A. thaliana ecotype Columbia, which has a nonfuctional allele for this gene. By doing so, they were able to change the GSL profile of the Arabidopsis ecotype, which normally produces 4-methylsulfinylbutyl and 3-methylsulfinylpropyl GSL. The transformants had a profile including three new additional compounds, 2-hydroxy-3-butenyl, 2-propenyl glucosinolate, and 3-butenyl glucosinolate, resulting from the conversion by desaturation of 4-methylsulfinylbutyl GSL precursor into 3-butenyl glucosinolate and the 3-methylsulfinylpropyl GSL precursor into 2-propenyl glucosinolate. The third compound resulted from hydroxylation of 3-butenyl glucosinolate, which is the next step on the side chain modification pathway and mediated by another gene in the AOP family.

X. CHLOROPLAST GENOMES AND THEIR PHYLOGENETIC IMPLICATIONS The tribe Brassiceae represents a natural grouping in the Brassicaceae characterized by conduplicate cotyledons, median nectaries, and/or two segmented fruits (Schulz 1919, 1936). The tribe has further been dissected

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into seven subtribes chiefly on fruit characters (Go´mez-Campo 1980). However, the morphology-based taxonomy is considered highly artificial by many taxonomists as the chromosome homology across the subtribes is often higher than within the subtribe. Possibilities of genetic exchange have been demonstrated. Molecular markers, particularly the chloroplast DNA restriction site variation, have been employed to infer phylogeny of subtribe Brassicinae and related subtribes, Raphaninae and Moricandiinae, and also to clarify the status and relationships among various species and genera. Such investigations were initiated by Warwick and Black (1991) and Pradhan et al. (1992) who studied chloroplast DNA RFLPs in a number of taxa. These studies were subsequently extended to other related subtribes encompassing a wider spectrum by Warwick and her colleagues in a series of articles (Warwick and Black 1991, 1993, 1994, 1997a; Warwick et al. 1992; Warwick and Sauder 2005). Phylogenetic analysis clearly revealed a vertical division of these subtribes into two lineages referred to as Rapa/Oleracea and Nigra lineages (Warwick and Black 1991; Pradhan et al. 1992). Earlier investigations on species relationships involving morphology and cytology had not suggested such dichotomy. However, the separation of the three cultivated diploid Brassica species into two lineages had earlier been suggested from cp DNA studies (Palmer et al. 1983; Erickson et al. 1983; Yanagino et al. 1987) and molecular DNA RFLP data (Song et al. 1988a,b, 1990). The smaller genera are monophyletic, while polyphyly is evident in large genera—Brassica, Diplotaxis, Erucastrum, and Sinapis, as these have taxa in both the lineages (Table 2.20). Recent investigations using ITS, trnL and combined ITS/trnL sequence data also supported it (Warwick and Sauder 2005). Interestingly, a high congruence is observed between genetically estabilished cytodemes and the clusters defined by cp DNA. Chloroplast genome information may form the basis for future taxonomic realignment and generic and specific delimitation along with morphological, cytogenetical, geographical and other molecular data for a more natural classification of the coenospecies. We will discuss the status of different genera separately.

A. Subtribe Brassicinae 1. Brassica. Schulz (1919, 1936) recognized 36 species in the genus Brassica and classified them into three sections: Melanosinapis, Brassicaria and Brassica. In recent years many of the taxa to which Schulz assigned species ranks have been identified only as varieties or


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Table 2.20. Genera and species of Brassica coenospecies in Nigra and Rapa/Oleracea lineage. Nigra lineage

n

GROUP I Brassica nigra Sinapis arvensis Diplotaxis ibicensis Diplotaxis siettiana Sinapis alba Brassica fruticulosa Erucastrum littoreum Trachystoma balii

8 9 8 8 12 8 16 8

GROUP II Brassica tournefortii Sinapis pubescens Brassica procumbens Diplotaxis brachycarpa Erucastrum varium Erucastrum virgatum Hirschfeldia incana

10 9 9 9 7 7 7

GROUP III Erucastrum canariense Diplotaxis assurgens Diplotaxis siifolia Sinapidendron spp. Diplotaxis berthautii Diplotaxis virgata Diplotaxis catholica Erucatrum brevirostre

9 9 10 10 10 9 9 9

GROUP IV Coincya spp.

12

Rapa/Oleracea lineage

n

GROUP I Brassica rapa Brassica oleracea Diplotaxis cossoneana Diplotaxis erucoides Erucastrum abyssinicum Erucastrum strigosum Erucastrum nasturtifolium Brassica deflexa Sinapis aucheri Enarthrocarpus lyratus Raphanus spp. Brassica barrelieri Brassica oxyrrhina

10 9 9 7 16 8 8 7 7 10 9 10 9

GROUP II Diplotaxis harra Eruca spp. Diplotaxis tenuifolia Rytidocarpus moricandiodes

13 11 11 14

GROUP III Moricandia arvensis Moricandia moricandiodes Moricandia suffruticosa

14 14 28

GROUP IV Brassica gravinae Brassica repanda Diplotaxis viminea

10 10 10

GROUP V B. elongata

11

Source: Arranged in groups defined by Warwick and Black 1991, 1997.

subspecies. Harberd (1972) established 10 cytodemes to which two more were added by Takahata and Hinata (1983). Cp DNA-based phylogenetic analysis and phenetic clustering separates the genus into two lineages (Warwick and Black 1991; Pradhan et al. 1992). Earlier cp DNA studies by Erickson et al. (1983), Palmer et al. (1983a), and Yanagino et al. (1987), and nuclear RFLP investigations by Song et al. (1988a,b, 1990) also suggested a vertical division. Based on cp DNA variations, B. rapa, B. oleracea, B. deflexa, B. oxyrrhina, B. repanda, B. gravinae, B. elongate, and B. barrelieri belong to Rapa/Oleracea lineage. The Nigra lineage includes B. nigra, B. fruticulosa, and

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B. tournefortii (Warwick and Black 1991; Pradhan et al. 1992). There are subgroups in both the lineages: three in Rapa/Oleracea and two in Nigra. A high level of congruence was found between cytodemes and the groups defined by chloroplast DNA restriction site variations. Rapa/Oleracea Lineage. There are three subgroups in the Rapa/ Oleracea lineage: 1. B. elongata (n ¼ 11) constitutes a very distinct group, which is reflected in its characteristic morphological traits: torulose pods with an inconspicuous seedless beak. It is endemic to southeastern Europe, western Russia, and the Near East. 2. Another group comprises three species: B. repanda, B. gravinae, and B. desnotesii (all n ¼ 10). Of these, B. repanda and B. desnotesii have very similar cp and are placed in the same cytodeme (Takahata and Hinata 1983). B. desnotesii is endemic to Morocco, and B. gravinae and B. repanda overlap in their distribution in northwestern Africa. All these species were ascribed to subgenus Brassicaria and have recently been transferred to a separate genus, Guenthera, based on a set of distinctive characters including seedless beak (Go´mez-Campo 2003). 3. Five species—B. rapa, B. oleracea, B. oxyrrhina (n ¼ 9), B. barrelieri (n ¼ 10), and B. deflexa (n ¼ 7) constitute the third group. B. rapa and B. oleracea form one subgroup, B. oxyrrhina and B. barrelieri another, and B. deflexa forms the third subgroup. Within B. oleracea, various wild taxa of the complex, including cretica, montana, insularis, incana, drapenensis, macrocarpa, and villosa, show a high degree of chloroplast genome similarity with cultivated forms, thus substantiating the proposals that these belong to B. oleracea (Snogerup 1980; La´zaro and Aguinagalde 1998a,b). A close relationship between B. rapa and B. oleracea is reflected in both possessing very similar chloroplast genomes, a fact supported from serological analysis of seed proteins (Vaughan 1977), isozyme patterns (Takahata and Hinata 1986), a high degree of chromosome affinities between their genomes (Olsson 1960b), and considerable similarities in size and morphology of their chromosomes and nuclear RFLPs (Song et al. 1988a,b, 1990; Hosaka et al. 1990). Brassica oxyrrhina (n ¼ 9) and B. barrelieri have close homologies in chloroplast genomes and ITS/trnL sequence (Warwick and Sauder 2005). Earlier, B. oxyrrihina was treated as a subspecies of B. barrelieri


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in Flora Europea (Tutin et al. 1964), but it is now recognized as a separate cytodeme by Harberd (1972). This separate status is confirmed by cp DNA studies (Warwick and Black 1991; Pradhan et al. 1992). B. oxyrrhina is proposed to have evolved from a loss of one pair of chromosomes from B. barrelieri (Harberd 1976). Both are identical in the vegetative stage, forming a rosette of leaves. B. deflexa shows strong homology with Sinapis aucheri. They have many similarities— for example, cp DNA, ITS/trnL sequence data, an overlap in distribution in the eastern Mediterranean, and pendant, torulose pods—but they form separate cytodemes (Warwick and Sauder 2005). Interestingly, the three species—B. oxyrrhina, B. barrelieri, and B. deflexa—close cp DNA homologies with Raphanus and S. aucheri and represent a unique trend in the evolution of pod morphology in the tribe. Although Raphanus with strong heteroarthrocarpic fruits (where the valvar portion is represented by vestigial scales and is formed entirely by the beak) represents an extreme, Brassica has a welldeveloped unsegmented portion. B. oxyrrhina and B. barrelieri represent an intermediate condition having disproportionally developed beaks. Nigra Lineage. Three taxa—B. tournefortii, B. nigra, and B. fruticulosa—form part of this lineage: B. tournefortii, which is cultivated as an oil crop in a limited area in India, has a distinct chloroplast genome and is quite distant from the other two taxa, B. nigra and B. fruticulosa. B. nigra is closely aligned with S. arvensis. Chloroplast genome homology confirms the earlier observation of close nuclear genome homology (Mizushima 1950a, 1968). It has been suggested that B. nigra be shifted to Sinapis (Warwick and Black 1991; Pradhan et al. 1992). Earlier, Linnaeus (1753) and de Candolle (1821) had also classified B. nigra as Sinapis. Three species—B. fruticulosa, B. maurorum, and B. spinescens—have very similar chloroplast genome. In spite of morphological differences, the hybridization data and chromosome pairing in hybrids and the isozyme profiles placed them into one cytodeme: diploid B. fruticulosa (Harberd 1972; Takahata and Hinata 1983). cp and mt DNA analysis are congruent with this grouping (Warwick and Black 1991; Pradhan et al. 1992). B. fruticulosa has a wide distribution in the Mediteranean basin with one diploid subspecies in southern Europe and several tetraploid subspecies in North Africa. However, B. maurorum and B. spinescens are confined to Algeria and Morocco, the former in stony pastures and fields in semiarid regions and the latter on coastal calcareous or siliceous cliffs (Tsunoda 1980). It was also suggested that the genus Sinapis could be redefined to include three

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species of Brassica: B. nigra, B. fruticulosa, and B. tournefortii (Warwick and Black 1991; Pradhan et al. 1992). 2. Diplotaxis. This genus contains about 27 species (Martıńez-Laborde 1993; Go´mez-Campo 1999c), which are mainly distributed in Central Europe and the Mediterranean region, particularly northwest Africa. It has been separated from other members of subtribe Brassicinae primarily in having biseriate, small, generally ovoid or ellipsoidal seeds (Schulz 1919; Tutin et al. 1964; Al-Shehbaz 1985). Interestingly, many primitive morphological characters for the tribe Brassiceae are present in Diplotaxis (Go´mez-Campo 1980). The leaves are generally pinnatifid or pinnatisect. Schulz (1936) recognized 22 species and grouped them into four sections: Rhynchocarpum, Catocarpum, Anocarpum, and Hesperidium. The different species have a continuous series of chromosome numbers from n ¼ 7 to n ¼ 13, also highchromosome allopolyploids with n ¼ 21, and have been grouped into 13 cytodemes (Harberd 1976; Takahata and Hinata 1983). Chloroplast DNA investigations clearly indicated a division into two lineages and the suggested level of divergence and taxon groupings are highly congruous with the cytodeme status (Warwick et al. 1992; Pradhan et al. 1992). However, the morphologically based delimitation of the species is not always consistent with these studies. All the species are separated into six groups, three each in both the lineages (Table 2.21). Interestingly, the boundaries of the sections established by Schulz (1919, 1936) correspond closely to the group defined by cp DNA. For example, groups B and C in Rapa/Oleracea and group F in Nigra lineages corresponds to sections Catocarpum, Anocarpum, and Rhyncocarpum, respectively. Rapa/Oleracea Lineage. The different species in the lineage do not form a single group but are separated into three major groups (Warwick et al. 1992). Diplotaxis erucoides (n ¼ 7) with two subspecies (subsp. erucoides and subsp. longisiliqua) form a distinct cp DNA entity in group A. The distinction between both subspecies is based on petal color, nervation patterns on petals, and fruit size (Schulz 1919; Maire 1965; Go´mez-Campo 1981; Martıńez-Laborde 1988). Both are also separated by strong breeding barriers. In areas of sympatric distribution, hybrids between the two are rare and completely sterile. Cp DNA data also substantiate this fact and might justify a specific rank (subsp. longisiliqua ! Diplotaxis cossoniana) and separate cytodeme status. The three species, D. tenuifolia, D. cretacea and D. simplex (all n ¼ 11), form a subgroup in group B and are placed by Harberd (1976)


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Table 2.21. Species of the genus Diplotaxis in Rapa/Oleracea and Nigra lineages. Rapa/Oleracea lineage GROUP A D. erucoides, n ¼ 7 D. cossoneana, n ¼ 7 GROUP B D. tenuifolia, n ¼ 11 D. cretacea, n ¼ 11 D. simplex, n ¼ 11 D. harra, n ¼ 13 GROUP C D. viminea, n ¼ 10 D. muralis, n ¼ 21

Nigra lineage GROUP D D. siettiana, n ¼ 8 D. brevisilique, n ¼ 8 D. Gomez-campoi, n ¼ 8 D. ibicensis, n ¼ 8 GROUP E D. brachycarpa, n ¼ 9 GROUP F D. assurgens, n ¼ 9 D. tenuisiliqua, n ¼ 9 D. virgata, n ¼ 9 D. siifolia, n ¼ 10 D. berthautii, n ¼ 9 D. catholica, n ¼ 9

Source: According to Warwick et al. 1992.

and Takahata and Hinata (1983) in one cytodeme. D tenuifolia and D. cretacea are morphologically very similar (Martıńez-Laborde 1988). While D. tenuifolia has a very wide distribution in Europe, D. cretacea is a narrow endemic in Eastern Europe and adjacent Russia (Tutin et al. 1964). Diplotaxis simplex has more similarities than differences in the other species; however, its distribution is different, as it occurs in Algeria, Tunisia, Libya, and Egypt. These facts coupled with low levels of chloroplast divergence do not warrant a separate specific status for these species and constitute a single cytodeme. Diplotaxis harra (n ¼ 13) has a wide distribution across northern Africa and the Middle East. It has several subspecies: harra, crassifolia, and lagascana. Two species— D. viminea (n ¼ 10) and D. muralis (n ¼ 21)—constitute group C. Diplotaxis viminea is assigned a separate cytodeme status while D. muralis is a naturally evolved allopolyploid between D. viminea D. tenuifolia (Harberd and McArthur 1980). Close similarities of cp and mitochondrial DNA between D. muralis and D. viminea suggest the latter as maternal parent and also indicate that D. muralis is of recent origin (Pradhan et al. 1992). D. simplex—a part of D. tenuifolia cytodeme—is morphologically very similar to D. muralis (Schulz 1936; MartıńezLaborde 1988) and is more likely the other parent (Warwick et al. 1992). Nigra Lineage. Three major groups have been recognized by cp DNA data in this lineage (Warwick et al. 1992) Four species, all n ¼ 8—D. siettiana, D. ibicensis, D. brevisiliqua, and D. ilorcitana—are included

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in one group (D). Each occupies a narrow region in the western Mediterranean. Genetically and morphologically all four taxa are very close (Martıńez-Laborde 1988). This closeness is also reflected in their cp DNA (Warwick et al. 1992). In fact, all four species constitute one cytodeme: D. siettiana. Diplotaxis brachycarpa (n ¼ 9) possesses a chloroplast genome very different from other species of Diplotaxis, and no information is available on its cytodeme status. It is placed in group E. Group F includes three subgroups: (1) comprising D. assurgens (n ¼ 9), D. tenuisiliqua (n ¼ 9), and D. siifolia (n ¼ 10); (2) comprising D. virgata, D. berthautii (n ¼ 9); and (3) D. catholica (n ¼ 9). Separate cytodeme status to D. assurgens, D. tenuisiliqua, D. virgata, D. berthautii, and D. catholica have been recognized (Prakash et al. 1999). The three species in subgroup 1 occur along the coast of Portugal and Morocco. The cp DNA data strongly supports the separate species and cytodeme status for D. virgata and D. berthautii in subgroup 2. Using intersimple sequence repeat nuclear DNA markers, Martin and Sańchez-Ye´lamo (2000) investigated 10 Diplotaxis species and observed that five species—D. tenuifolia, D. cretacea, D. simplex, D. viminea, and D. muralis—constitute one group. Morphologically, Prantl (1891) grouped them in section Anocarpum. Crossability and chromosome pairing in their hybrids also reflect high homologies among these five species (Harberd 1972; Takahata and Hinata 1983). One of the common shared characteristics is presence of glucosinolates giving a strong odor. Biochemical markers such as flavonoid (SańchezYe´lamo and Martıńez-Laborde 1991; Sańchez-Ye´lamo 1994), seed proteins and isozymes (Sańchez-Ye´lamo and Martıńez Laborde 1991), and cp and mt DNA analysis (Pradhan et al. 1992) also suggested such a close relationships. D. virgata, D. catholica, D. siettiana, D. harra, and D. erucoides constitute the second group. These are all odorless because of very low amount of glucosinolates (Sańchez-Ye´lamo 1994). The cp and mt DNA analysis shows close relationships among D. virgata, D. catholica, and D. siettiana (Pradhan et al. 1992). The status of D. siifolia (n ¼ 10, group F) has been a point of contention. It has variously been described as D. torulosa and D. catholica ssp. siifolia (Maire 1965). This taxon is regarded to have characters intermediate between Brassica and Diplotaxis. Its seeds are arranged in two rows in the locules (biseriate), which is characteristic of the genus Diplotaxis. However, this character is rather variable and the term ‘‘sub-biseriate’’ has been used for it. The sub-biserate character is also shared by two other species—B. maurorum and D. assurgens—in the subtribe. Delimitation of generic boundaries based on cotyledon shape by Go´mez-Campo and Tortosa (1974) strongly suggested


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D. siifolia’s placement in Brassica and of D. assurgens in Diplotaxis, although the two species have very similar cp DNA (Pradhan et al. 1992). D. siifolia shares cp DNA homologies with D. tenuisiliqua, D. catholica, D. virgata, E. cardaminoides, and Hirshfeldia (Pradhan et al. 1992). As no Brassica species is placed in this group indicating the remoteness between the taxa of this group and Brassica. D. siifolia has been reported to possess strong isolation barriers with Brassica species, which are mostly postfertilization. Although intergenomic homoeology between chromosomes of D. siifolia and B. rapa and B. nigra has been observed (Batra et al. 1990), placement of D. siifolia in the genus Diplotaxis rather than in Brassica seems appropriate. This genus is morphologically unique, having both types of taxa: some with seedless beaks and others with seeded beaks. Species in two of the subgenera—Diplotaxis and Hesperidium—always show seedless beak. Seeded beak (heteroarthrocarpic fruits) is also present in subgenera Rhynchocarpum and Heterocarpum. Go´mez-Campo (1999b) believed that much of the molecular heterogeneity is associated with beak duality. 3. Erucastrum. The genus Erucastrum comprises 21 species and is traditionally considered close to Brassica and Diplotaxis (Go´mezCampo 1999c). It has a distribution in the western Mediterranean and eastern and southern Africa. Polyphyly is evident in this genus, as indicated by placement of its species in both the lineages. Rapa/Oleracea Lineage. Five species form three subgroups in this lineage: 1. E. leucanthum and E. nasturtiifolium (both n ¼ 8) have close affinities and both belong to the same cytodeme. Morphologically they are similar. E. leucanthum has white flowers while E. nasturtiifolium is characterized by the retrorse lower segments of its leaves. 2. E. abyssinicum and E. strigosum (both n ¼ 8) are aligned together. They form a small group and both represent separate cytodemes. 3. E. gallicum (n ¼ 15) forms a group with Diplotaxis erucoides (both subspecies, n ¼ 7). E. gallicum is believed to be a natural alloploid between E. nasturtiifolium (n ¼ 8) and an unknown species (Harberd 1976). Chloroplast similarities suggest that D. erucoides could be the maternal parent.

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Nigra Lineage. Erucastrum species form three subgroups in this lineage: 1. E. canariense and E. cardaminoides (both n ¼ 9), endemic to Canary islands, have very similar cp genome and constitute one cytodeme. Both are morphologically very similar. 2. E. virgatum (n ¼ 7) and E. elatum (n ¼ 15) show close affinities in cp DNA and morphological attributes. The latter is an allopolyploid between E. virgatum (n ¼ 8) and Hirschfeldia incana (n ¼ 7) (Go´mez-Campo 1983; Sanchez-Yelamo 1992; Warwick and Black 1993). 3. E. brevirostre (n ¼ 9) forms a small group with Diplotaxis catholica. It is endemic to central and western Morocco. However, its cytodeme status is unknown. Go´mez-Campo (1982) suggested a close affinity with the Canarian species of group 1, supported by cp DNA analysis (Warwick and Black 1993).

4. Sinapis. Schulz (1919) recognized eight species under Sinapis and grouped them into four sections: Eriosinapis, Sinapis, Hebesinapis, and Chondrosinapis. Of these, five cytodemes were recognized by Harberd (1976) and Takahata and Hinata (1983), which include S. aucheri, S. arvensis, S. pubescens, S. alba, and S. flexuosa. All the species are heteroarthrocarpic (i.e. with seeded beaks). Chloroplast DNA studies recognized four groups corresponding to Schulz’s sections (Warwick and Black 1991; Pradhan et al. 1992) and placed three genera (S. arvensis, S. pubescens, and S. alba/flexuosa) in Nigra and one, S. aucheri, in Rapa/Oleracea lineage, thus suggesting the biphyletic origin of the genus. S. alba and S. flexuosa—both n ¼ 12—have been treated as separate cytodemes, although no divergence in their cp DNA was observed (Warwick and Black 1991; Pradhan et al. 1992), suggesting a recent reproductive isolation between the two species. Geographically they overlap in their distribution. While S. alba has a wide distribution—Europe and the Mediterranean—and is cultivated as an oil crop, S. flexuosa is restricted to Spain, Morocco, and Algeria and is a weed. S. arvensis has traditionally been considered very close to B. nigra. This is reflected in high levels of chromosome pairing in their hybrids (2n ¼ 17, up to 8 II) (Mizushima 1950a). Other investigations—including nuclear RFLPs (Song et al. 1988a), cp DNA analysis (Yanagino et al. 1987; Warwick and Black 1991; Pradhan et al. 1992), 5S rDNA spacer (Bhatia et al. 1993; Capesius 1993), repetitive DNA (Gupta et al. 1990, 1992; Kapila et al. 1996), chemotaxonomic markers


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(Tsukamoto et al. 1993; Simonsen and Heneen 1995), karyotypes (Yuan et al. 1995), RAPD patterns (Wu et al. 1996), nuclear sequence of Slocus related gene SLR1 (Inaba and Nishio 2002), and ITS/trnL sequence data (Warwick and Sauder 2005)—substantiate this closeness. S. pubescens (n ¼ 9) deserves a specific rank and separate cytodeme status. However, the close cp DNA affinities between S. pubescens and Hirschfeldia incana is intriguing, which is reflected morphologically also where only the degree of sepal erectness separates them (Schulz, 1919; Tutin et al. 1964). Sinapis aucheri has been placed in the annual section Chondrosinapis by Schulz (1936) Unlike other Sinapis species, which have multilocular pods and typical beak, S. aucheri has highly heterocarpic pods with long torulose, corky, and 6- to 10-seeded beak. Its distribution is confined to western Iran and eastern Iraq; all other Sinapis species are distributed in the Mediterranean region (Schulz 1936; Al-Shehbaz 1985). Chloroplast DNA analysis (Warwick and Black 1991; Pradhan et al. 1992) indicates close relationship between S. aucheri and Raphanus sativus. S. aucheri is often confused with Raphanus aucheri of section Hesperidopsis in taxonomy and nomenclature (Schulz 1936). It has strong heterocarpy like R. aucheri and has narrow endemism in western Iran. Considering its distribution and pod morphology, it would be justified to transfer S. aucheri to Raphanus. 5. Trachystoma. Trachystoma includes three species—labasi, ballii, and aphanoneurum—and all have similar chloroplast genomes in Nigra lineage and have been placed into one cytodeme (Harberd 1976). It has variably been treated under subtribes Brassicinae and Raphaninae (Go´mez-Campo 1980). One of its characteristic features is strongly heteroarthrocarpic silique. Chloroplast DNA studies supports its inclusion in subtribe Brassicinae and also suggest the close affinities with B. nigra and S. arvensis (Warwick and Black 1997). All the three taxa are endemic to Morocco. Its spontaneous hybridization with Ceratocnemum challenges the presently defined limits of coenospecies (Al-Shehbaz 1985). 6. Hirshfeldia incana. Hirshfeldia incana belongs to Nigra lineage (Warwick and Black 1991; Pradhan et al. 1992) and occurs in the entire Mediterranean. Schulz (1919) gave it status of a separate genus on the basis of multinerve character of the pod as against uninerve Brassica and Erucastrum. It has variably been named Erucastrum incanum, Sinapis incana, and B. adpressa (Mizushima and Tsunoda 1967). Its cp

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DNA has close homology with an allotetraploid species Erucastrum elatum (n ¼ 15) and is one of the components of it (Go´mez-Campo 1983). It also bears close genetic relationship with Sinapis pubescens (Warwick and Black 1991). In fact, Hirshfeldia is an Erucastrum with specialized fruits (Go´mez-Campo 1999c). 7. Sinapidendron. Three species—S. angustifolium, S. frutescens and S. rupestre—are endemic to Atlantic islands (Madeira, Canarias, and Cabo Verde) and regarded as Miocenic relic. The cotyledons exhibited by this genus (broad lamina and shallow notch) represent an ancestral type (Go´mez-Campo and Tortosa 1974). All three species constitute a single cytodeme, which is reflected in close cp DNA affinities and placed in Nigra lineage (Warwick and Black 1993). 8. Coincya. This is a highly heteroarthrocarpic genus with maximum variability in the Iberian peninsula and is placed in Nigra lineage. It was variously been described under different genera, such as Brassicella, Coincya, Hutera, and Rhynchosinapis (Go´mez-Campo 1980). Earlier, six species were recognized by Leadlay and Heywood (1980). However, cytological (Harberd and McArthur 1972) and molecular studies indicate a homogenous group (Warwick and Black 1991). 9. Eruca. This is a monotypic genus placed in the Rapa/Oleracea lineage. All the three species—vesicaria, sativa, and pinnatifida—are now treated as subspecies of sativa and constitute one cytodeme and possess similar cp DNA. Although partial sterility was observed in ssp. sativa vesicaria hybrids (Sobrino-Vesperinas 1995). E. sativa ssp. vesicaria is unique with nonheteroarthrocarpic silique and is widely distributed in the Mediterraneanregion; subspecies pinnatifida is endemic to southern Spain, Algeria, Morocco, and Tunisia. It has a very short life cycle and is well adapted to harsh drought conditions. Subspecies. sativa is cultivated in many parts of the world, particularly in drier habitats, for its oil (Tsunoda 1980; Go´mez-Campo 1999c). Its seeds are a common source of industrial oil in India. Ibn al-Awam, a Spanish Moor in the 12th century, mentioned its cultivation in Spain in his book Kitab-al-Falaha (Gomez-Campo and Prakash 1996). It is very popular as a pungent salad in Italy while nonpungent ones are grown in Turkey and Egypt. B. Subtribe Raphaninae Subtribe Raphaninae is very heterogenous, characterized by strongly segmented fruits and believed to be of polyphyletic origin. Raphanus is


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the core genus of this subtribe. Harberd (1972) placed two genera from this subtribe—Raphanus (n ¼ 9) and Enarthrocarpus (n ¼ 10)—in coenospecies. Nuclear DNA RFLP investigations by Song et al. (1990) also strongly supported the inclusion of Raphanus in subtribe Brassicinae. Both the genera are placed in Rapa/Oleracea lineage by Warwick and Black (1991, 1997) and Pradhan et al. (1992). Go´mez-Campo (1980) believed that Raphanus and Enarthrocarpus are intermediate between subtribes Raphaninae and Brassicinae, but are more closely related to Brassicinae. This closeness is also reflected in hybridization and chromosome pairing in hybrids. In fact, the intersubtribal hybrid Raphanus B. oleracea was obtained as early as 1927 by Karpechenko, and it exhibits high chromosome homologies (1 III þ 6 II, 2n ¼ 18, RC, McNaughton 1973). Similar high chromosome affinities were observed in hybrids E. lyratus B. oleracea, (2n ¼ 19, 1 III þ 4 II) and E. lyratus B. rapa (2n ¼ 20, 2 III þ 4 II, Gundimeda et al. 1992). Warwick and Black (1997) were of the view that five more genera of the subtribe— Cordylocarpus, Otocarpus, Guiraoa, Kremeriella, and Ceratocnemum—all North African endemics that fall in Nigra lineage, might also be considered for their inclusion into Brassica coenospecies. Ceratocnemum (n ¼ 8) shows close cp DNA and ITS/trnL sequence homology with Trachystoma (Warwick and Black 1991; Warwick and Sauder 2005). Both are also genetically close, as supported by the observation that an intergeneric hybrid Trachycnemum mirabile Maire and Samuels (Trachystoma ballii Ceratocnemum rapistroides) occurs in nature (Maire and Samuelsson 1937; Maire 1965; Al-Shehbaz 1985). C. Subtribe Moricandiinae Subtribe Moricandiinae is considered to be a heterogenous assemblage (Go´mez-Campo 1980). Taxonomic separation of this subtribe from subtribe Brassicinae has always been considered to be artificial as both are characterized by elongated siliquose fruits (Al-Shehbaz 1985). The only point of separation is absence of seed in the rostrum and median nectaries (Go´mez-Campo 1980). Chloroplast DNA and ITS/trnL data also do not support a separate subtribal ranking of Moricandiinae (Warwick and Black 1994; Warwick and Sauder 2005). Three genera of this subtribe—Moricandia, Rytidocarpus, and Pseuderucaria—show close cp DNA homologies with taxa of Brassicinae in Rapa/Oleracea lineage, thus strongly suggesting their inclusion in Brassica coenospecies (Warwick and Black 1994). Genus Moricandia is comprises of five species. Of these, four—arvensis, nitens, suffruticosa, and spinosa

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(all n ¼ 14)—are easily hybridized (Sobrino-Vesperinas 1997), have very similar cp DNA profiles, and are included into one cytodeme, M. arvensis. Their seed protein profiles also show large similarities (Sańchez-Ye´lamo et al. 2004). Earlier, Maire (1967) also treated these species as subspecies of M. arvensis. The other species, M. moricandioides (also n ¼ 14), has cp genome and seed protein profile distinct from the M. arvensis complex and is also included in Rapa/Oleracea lineage. Taxa of M. arvensis complex are widely distributed in the Mediteranean region and appear to be exclusively polyploids (AlShehbaz 1984). Close genetic affinities between M. arvensis and Brassica species is evidenced by the fact that sexual and somatic hybrids between M. arvensis / nitens and several Brassica species show a high degree of chromosome pairing (Takahata 1990; Takahata and Takeda 1990; Kirti et al. 1992b; Takahata et al. 1993; Meng et al. 1997, 1999; Meng 1998). The monotypic Moroccan genus Rytidocarpus is very close to Moricandia (Go´mez-Campo 1980) in morphology as it has Moricandia-like cotyledon with an almost absent notch, succulent and entire leaves, purple flower, and the same chromosome number n ¼ 14. Another genus, Pseuderucaria (n ¼ 14), earlier assigned to Moricandiinae (Schulz 1936; Go´mez-Campo 1980), has a weak relationship with Moricandia and Rytidocarpus but deserves a place in the coenospecies (Warwick and Black 1994). D. General Considerations The interesting taxonomic history of the genus Brassica has been reviewed by Go´mez-Campo (2003). It was a large genus in the 18th century from which some species were later withdrawn to create new genera, such as Eruca, Hirschfeldia, Moricandia, and Conringia. Others were transferred to existing genera, such as Coincya, Diplotaxis, Erucastrum, and so on. Pomel (1860) believed it was still highly polymorphic and split it into five genera: Brassica, Erucastrum, Brassicaria, Nasturtiops, and Melanosinapis which were used to define sections, subgenera, or new genera (Gren and Godron 1848; Schulz 1919; Go´mez-Campo 1999c). Recently Go´mez-Campo (2003) grouped species corresponding to Pomel’s Brassicaria and Nasturtiops and also B. elongata (Brassica subgenus Brassicaria) into a single entity with the generic denomination Guenthera. It is characterized by seedless stylar portion and the presence of a caudex. The cotyledon notch is shallower than in Brassica with a shorter petiole. Go´mezCampo described nine species: G. elongata (B. elongata), G. dimorpha (B. dimorpha), G. souliei (B. souliei, B. amplexicaulis), G. desnottessi


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(B. desnottessi), G. repanda (B. gravinae, B. repanda), G. nivalis (B. jordanoffii, B. nivalis), G. setulosa (Eruca setulosa), and G. loncholoma (B. loncholoma syn. Eruca loncholoma). Although ITS data does not provide support for such status because Guenthera itself, as defined, appears to be polyphyletic (Warwick and Sauder 2005), it is also certain that polyphyletism is still present in the remaining taxa of Brassica. The separate status of three subtribes—Brassicinae, Raphaninae, and Moricandiinae— has been questioned as morphological distinctiveness does not provide sufficient basis for it (Al-Shahbaz 1985; Warwick and Black 1994). Brassicinae and Moricandiinae have elongated dehiscent fruits while Raphaninae has reduced indehiscent fruits. Recent hybridization studies and phylogenetic analysis based on S-locus related gene SLR1 (Inaba and Nishio 2002) and cp DNA and ITS, trnL and ITS/trnL data also do not support separate recognition of subtribes (Warwick and Sauder 2005). The genus Orychophragmus was placed in the tribe Brassiceae subtribe Moricandiinae by Schulz (1936), but its position is not very clear (Go´mez-Campo 1980; Al-Shehbaz 1985). It has been excluded by Go´mez-Campo (1980) because it lacks the key tribal morphological features. However, several studies that include isozymes (Anderson and Warwick 1998), easy hybridization with cultivated Brassica species, and exchange of genetic material (Li et al. 2003; Li and Ge 2007), and ITS sequences and cp trnL intron information (Warwick and Sauder 2005) strongly support its inclusion and also of two more genera, Calepina and Conringia, in the tribe Brassiceae. However, Beilstein et al. (2006) placed Conringia and Calchanthus in a separate well-supported clade.

XI. EVOLUTION OF MORPHOLOGICAL CHARACTERS It has been suggested that the Himalayan region is the prime center of variation for several Crucifer tribes, where the area of dispersion extends from the region up to the Atlantic Ocean across vast regions of the Mediterranean, Irano-Turanian, and Saharo-Sindian phytochoreas (Hedge 1976). However, the maximum variability in Brassiceae occurs in the southwest Mediterranean area comprising chiefly Morocco, Algeria, and Spain. This can be regarded at least the secondary center of origin if not the primary one from which vigorous evolutionary radiations occurred. During speciation, a number of morphological characters evolved. Surprisingly these are genera specific and often incongruent with their

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position in the cp lineage. It appears that these morphological characters evolved much after lineage differentiation. We consider here three such characters: cotyledon, adult leaf, and fruit shape. We exclude flowers as these are rather homogeneous in the coenospecies and members of the tribe Brassiceae. Those interested in floral characters are referred to Clemente-Munõz. and Hernańdez-Bermejo (1980). A. Cotyledons An extensive investigation on cotyledonary characters has been carried out by Go´mez-Campo and Tortosa (1974, Fig. 2.5). In general, expanded

Fig. 2.5 Evolution of cotyledons in Brassica coenospecies. (Source: Adapted from Go´mez-Camp 1980.)


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cotyledons in the coenospecies are wide to oblong and variably notched. Diplotaxis species have small, slightly longer than wide and slightly notched cotyledons. These together with those present in Guenthera and Sinapidendron (wider but with shallow notch) probably represent the primitive type. Then heteroarthrocarpic genera (Brassica, Raphanus, Coincya, and Sinapis) undergo a progressive tendency toward wider cotyledons with deeper notches. Erucastrum, Eruca, Hirschfeldia, Enarthrocarpus, and Trachystoma represent intermediate steps between Diplotaxis and Brassica. However, there are some exceptions: D. siifolia, and Erucastrum cardaminoides show cotyledons that are very similar to Brassica. Conversely, cotyledons of Moricandia and Rytidocarpus have an almost absent notch and a short petiole, succulent appearance, and glaucous color representing xerophytic features. The only deviation from such types within Brassica coenospecies is in Pseuderucaria, which, like other psammophylls, have thick notchless cotyledons.

B. Adult Leaves Adult leaves in the coenospecies are of four types, as observed by Go´mez-Campo (1980). The names of leaf silouettes are here adapted to a more correct and updated nomenclature. These are: 1. 2. 3. 4.

Simple, entire to shallowly lobed Lobed to pinnatifid (sinuses not reaching the midnerve) Pinnatisect (divided with sinuses reaching the midnerve) Pinnatisect with reduced number (vestigial to two pairs) of lateral segments

Further evolutionary reduction in the number of lateral segments leads again to simple leaves of type 1. These basic four types constitute a so-called repeating polymorphic set and form a ring pattern (Fig. 2.6). A fifth type is pinnatipartite (deeply divided with narrow lobes), which is present in the group in a scattered way and seems to have evolved from any of the preceding types following mutations at one or two loci. In general, Diplotaxis and Eruca leaves are lobed to pinnatifid; while Erucastrum, Raphanus, Hirschfeldia, Trachystoma, Coincya, and Sinapis are pinnatisect, most often lyrate-pinnatisect (with a large terminal lobe). Diplotaxis has few exceptions: D. catholica and D. siettiana have pinnatipartite leaves; D. berthautii has both types: pinnatifid and partite in natural populations. Surprisingly, Brassica is

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Fig. 2.6 Suggested phenetic relations between different types of leaves in the tribe Brassiceae. (Source: Go´mez-Campo 1980.)

the most heterogenous and has leaves of every kind; for example, pinnatifid in B. repanda, B. elongate (Guenthera); pinnatisect in B. barrelieri, B. oxyrrhina, and B. tournefortii; lyrate-pinnatisect with variable reduction in segments in all the cultivated species. Xeromorphous species such as Moricandia and Rytidocarpus have simple entire leaves. Simple entire leaves might be a basic type from which others evolved, but the habit of the species (annual, biennial, perennial, etc.) has probably been determinant for a rapid evolution of the different types.

C. Fruits Many authors have studied the fruit characters in a wide range of taxa of the coenospecies. The siliqua consists of two separate cavities.


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The distal cavity is formed by the substylar region and is empty in most crucifers. Only in a part of the tribe Brassiceae it can have seeds—a phenomenon referred to as heterocarpy or, more correctly, heteroarthrocarpy. The valvar portion is, in general, the seed-bearing cavity, dehiscent by separation of the valves. In Raphanus and some species of Enarthrocarpus, the beak is highly developed and is dehiscent by fragmentation. Raphanus is an extreme case where the valvar portion is only vestigial. Trachystoma, Enarthrocarpus, Sinapis aucheri, and Coincya may also exhibit strong heteroarthrocarpy. A moderate reduction in fruit size may also occur in some cases (such as some Diplotaxis, Erucastrum, or Brassica species), but it is much stronger in some Raphaninae. Most of the genera have pods that are erecto-patent. However, adpressed pods also occur in Hirschfeldia, B. nigra, and some Erucastrum species while Coincya longirostra and Diplotaxis harra have reflexed or pendulous fruits. Heteroarthrocarpy and fruit reduction plus some additional characters, such as ribs, rugosities and wings, have resulted into a diversity of pods and have been assigned a major importance in taxonomy. D. Isthmus Concept The isthmus concept was introduced by Go´mez-Campo (1999c) and emphasizes the importance of the presence of seeds within the stylar cavity as a singular morphogenetic achievement in the evolution of the tribe Brassiceae. This character is present only in this tribe; half of the genera exhibit this trend—heteroarthrocarpy—with several forms of seeded beaks. The other half do not show such development and thus resemble all other tribes in the family. Two successive evolutionary radiations are envisaged: The first involves genera possessing the primitive character—seedless beak—and is present in Sinapidendron, Eruca, Moricandia, Rytidocarpus, Pseuderucaria, subgenera Diplotaxis (e.g., D. tenuifolia, D. muralis, D. viminea, etc.), and Hesperidium (e.g., D. harra, D. acris) of Diplotaxis and subgenus Brassicaria of Brassica (e.g., B. repanda, B. gravinae, B. elongata, B. desnotesii). The last subgenus has been recently moved to Guenthera (Go´mez-Campo 2003) due not only to its lack of heteroarthrocarpic fruits but also by the presence of a set of primitive characters. The archetypic background of the tribe might today be represented by an ancient disjunction between fruticose nonheteroarthrocarpic Sinapidendron spp. (Madeira Island) on the west side and Guenthera somaliensis (Somalia) with similar

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characters on the east side. Evolution of heteroarthrocarpic fruits might have occurred with the origin of the subgenus Rhynchocarpum of Diplotaxis (D. assurgens, D. virgata, D. tennuisiliqua, D. catholica, D. berthautii, and D. siifolia). Thus, Diplotaxis occurs on both sides of the ‘‘isthmus’’ or ‘‘bridge’’ between both radiations. The second radiation involves Erucastrum, Hirschfeldia, Sinapis, Coincya, Erucaria, Trachystoma, Raphanus, Enarthrocarpus, and Brassica (excluding Guenthera), which have heteroarthrocarpic fruits with varying degrees of beak development sometimes accompanied with fruit reduction. A set of predominantly west Mediterranean genera with reduced fruits such as Rapistrum, Ceratocnemum, Otocarpus, Guiraoa, and so on might be not far, phylogenetically, from Brassica coenospecies. Other genera, such as Crambe, Crambella, Kremeriella, and so on, are more distant and represent extreme situations of globose beaks with null or vestigial valvar portions. The heteroarthrocarpic radiation may not be completely monophyletic as both cp lineages seem to occur at both sides of the isthmus.

XII. CONCLUDING REMARKS The genus Brassica with its vast diversity of forms and uses has been subjected to intensive investigations by researchers and has served as a model for studies on cytogenetics, speciation, and domestication. The choice of Arabidopsis as a model eudicot plant for genomics investigations has given new impetus to Brassica research. Brassica and allied genera constitute a potential germplasm pool possessing many desirable horticultural traits. The last few decades have witnessed a spectecular progress in cytological, in vitro, and molecular techniques. Thus, classical cytogenetics has given way to molecular cytogenetics. As Brassica chromosomes are relatively small and lacking distinctive physical landmarks, their precise identification and generating reliable karyotypes is difficult. In situ hybridization techniques (GISH, FISH) and a spectrum of molecular markers allow identification of individual chromosomes through direct localization of DNA probes on chromosomes and are very helpful for structural and functional chromosome analysis. Brassica researchers encounter several major constraints in improvement programs. These include a lack of useful variability in allopolyploid species and the nonavailability of genes conferring resistance to diseases in crop germplasm. Although many synthetic


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lines of B. napus, B. juncea, and B. carinata have become available during the last 60 years, this added variability is still inadequate. More synthetic genetic variants are yet to be obtained by utilizing the enormous morphological, physiological, and geographical variability of the diploid constituent parents. As many of these current diploid variants have evolved after the natural syntheses of the allopolyploids, they are likely to produce new useful variability. Hybridization in nature was always unidirectional. Synthetics with new cytoplasms as compared to the natural ones and also new combinations of cytoplasmic organelles following protoplast fusion can be obtained easily at present, generating further variability. As in any crop improvement program, wild germplasm always plays a pivotal role. Nuclear genes conferring desirable traits as well as cytoplasmically controlled characters, such as male sterility, herbicide resistance, and photosynthetic activity, are frequently distributed in the related wild germplasm in the tribe Brassiceae. Enriching conventional germplasm with alien genetic diversity is a much-desired goal. Introgression of traits can be achieved successfully in view of the advances made via in vitro protoplast fusion methodology. In recent years, a large number of wild species have been combined with crop species, overcoming even intertribal barriers. However, introgression of traits across generic boundaries has not been very successful in a majority of instances due to a general lack of intergenomic chromosome homoeology. It is necessary to devise ways to induce such homoeologous pairing to facilitate alien gene transfer. One such approach might be a chromosome-5B-like manipulative system used in wheat. Although the occurrence of a pairing regulator gene has been proposed in B. napus and B. juncea based on indirect evidence, it remains to be clearly demonstrated. A large number of accessions of wild species are stored in gene banks across the world. However, these have not been evaluated scientifically and systematically for desirable traits. Their evaluation could uncover new genes and QTLs for breeding pest and diseases resistant varieties, and for breaking present yield barriers. At present, cytogenetic stocks, such as monosomics, trisomics, and nullisomics, are limited in number, but these are integral for cytogenetical analysis. Efforts to develop and characterize such lines should be encouraged. Although several genomes have been dissected; monosomic and disomic addition lines have been obtained; and genes of agronomic and horticultural importance have been located, comprehensive are needed on well-defined cytogenetical stocks, which are unavailable at

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present. Molecular investigations indicate enormous variability for chloroplast and mitochondrial genomes. This offers opportunity for generating novel cytoplasmic male sterile lines for use in hybrid seed production. As discussed in earlier sections, mitochondrial genome rearrangement/recombination are a rule rather than an exception in somatic hybrids, particularly in Brassicaceae. It has also been demonstrated that mitochondrial genome organization and its expression in synchrony with the nuclear gene expression control flower morphology. Different flower types could be produced by developing cybrid lines for correcting the defects in flower morphology. It has been demonstrated time and again that some CMS systems in Brassicaceae were associated with defects in floral morphology. Although intensive efforts in the past three decades have made available an array of CMS and restorer lines through convetional methods, the challenge is to develop better ones using the in vitro biotechnological methods. These include rectifying developmental and floral abnormalities in the traditionally developed CMS lines following protoplast fusion. Protoplast fusion techniques can also remove excessive alien mitochondrial DNA through intergenomic mitochondrial recombination, which makes restoration easier. With the availability of genetic systems for controlled pollination, hybrids are likely to become popular in most countries in the near future. Given the current status of Brassica genomics and recombinant technology, it is worth exploring the possibility of fixing heterozygosity and hybrid vigour through apomixis. Some species of the genera Boechera and Draba, both crucifers, reproduce through diplosporous apomixis (Sharbel and Mitchell-Olds 2001; Richards 2003). Investigations are under way to unravel the genetic and molecular mechanisms that cause apomixies expression. Introgression of this trait will have significant impact on Brassica production. Extensive information on genome structure and mapping have been generated during the past 17 years since construction of the first linkage map of B. oleracea (Song et al. 1990). At present, high-density maps are available for the crop species. Many useful genes for various traits have been tagged, including disease resistance, abiotic stress, fertility restoration, oil quality, and morphological traits (Snowdon et al. 2005). Intensified research to develop more SSR markers for generating saturated genetic maps is needed as only a limited number of such markers are currently available for marker-assisted breeding and map-based cloning of genes for agronomically desirable traits. These studies will be of immense value in Brassica breeding in coming years. Genome sequencing of B. rapa is advancing rapidly. Information


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emerging from these investigations will contribute to unraveling the structure and origin of Brassica genomes. Traditional classifications of the Brassicaceae are mostly based on flower and fruit characters and also geographical distribution. However, the subdivision of the family into tribes and subtribes and also generic delimitation have been contentious issues. Molecular phylogeny, in recent years using molecular markers, specifically the maternally inherited cpDNA and biparently inherited ITS sequences (internal transcribed spacers of nuclear ribosomal DNA and 5.8S rRNA gene), strongly suggest massive incongruities in the generic and specific delineations. Chloroplast DNA, ITS, and cp trnL intron information do not support the separate recognition of subtribes Brassicinae, Moricandiinae, and Raphaninae. Surprisingly, the information from ITS and ITS/trnL data does not provide evidence of cp lineages in Nigra and Rapa/Oleracea as suggested and discussed earlier, but clearly indicates the polyphyletc origins for the larger genera: Brassica, Diplotaxis, and Erucastrum. However, as Go´mez-Campo (1999b) suggests, it is premature to disturb their current. Rapid-cycling Brassica (RcBr) developed by Paul Williams of Wisconsin University (Williams and Hill 1986) have become model organisms for basic and applied research primarily because of their short life span, small size, and absence of seed dormancy. Rapidcycling plants of all the six crop species are available with life spans ranging from 35 days for B. rapa to 60 days for B. oleracea. These stocks have been used in protoplast fusion for resynthesis of alloploid B. napus, developing cytoplasmic male sterility systems, and transferring cp genome encoded characters. Another major application of Rc is in undergraduate research and education related to plant breeding, genomics, and ecology where one of the goal is to have undergraduates do independent research projects. Results emanating from Arabidopsis structural and functional genomics are proving a great boon to both basic and applied researches with Brassica. High-throughput functional genome analyses using microarray platform has also become available to Brassica researchers. This will accelerate gene discovery and allele mining. Hence Brassica improvement through a transgenic approach, especially for speciality traits and stress tolerance, using genes and promotors from other species is also expected. Oilseed Brassica spp. are considered as a crop for producing biodiesel, particularly in developed countries. Considering the enormous advanages that this crop enjoys in terms of technology and knowledge base, and also natural adaptability, it is expected to gain further importance in future.

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ACKNOWLEDGMENTS We thank Robert Hasterok, Silician University, Poland; Xiaoming Wu, Oil Crop Research Institute, Wuhan, China; and Y. P.Wang, Yangzhou University, China, for providing publications. Special thanks are due to Professor C. Go´mez-Campo, University Polytechnica, Madrid, Spain, Professor K. Hinata, Tohoku University, Sendai, Japan, and Dr. R.K. Downey, AAFC-Saskatoon Research Centre, Saskatoon, Canada for their valuable comments and suggestions on this chapter. Financial assistance from the Indian National Science Academy, New Delhi, to Shyam Prakash in the form of a senior scientist position is gratefully acknowledged.

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3 Genetic Enhancement for Drought Tolerance in Sorghum Belum V. S. Reddy, S. Ramesh, P. Sanjana Reddy, and A. Ashok Kumar International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) Patancheru 502 324, Andhra Pradesh, India

I. INTRODUCTION II. BREEDING FOR DROUGHT TOLERANCE III. SELECTION AMONG CULTIVARS AND LANDRACES A. Cultivar Options B. Selection among Landraces and Breeding Material IV. BREEDING FOR DROUGHT ESCAPE V. GROWTH STAGE–SPECIFIC SCREENING TECHNIQUES A. Germination and Seedling Emergence B. Postemergence and Early-Seedling Stage C. Midseason and Preflowering Stage D. Terminal and Postflowering Stage 1. Screening Techniques 2. Breeding VI. PHYSIOLOGICAL RESPONSE TRAITS FOR DROUGHT TOLERANCE A. Leaf Water Potential B. Osmotic Adjustment VII. MARKER-ASSISTED BREEDING FOR DROUGHT TOLERANCE VIII. OUTLOOK LITERATURE CITED

I. INTRODUCTION Sorghum (Sorghum bicolor (L.) Moench) is a predominant food and fodder crop grown in the semiarid tropical (SAT) regions of south Asia and the Sahelian-Sudanian zone of Africa that are characterized by Plant Breeding Reviews, Volume 31 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 189

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high temperatures, high radiation, high evaporative demand, unreliable and irregular rainfall, and soils of low fertility and low waterholding capacity. Sorghum production is affected by a range of biotic and abiotic yield constraints (drought, temperature, and nutritional stresses). Among these, drought will likely be the primary yield constraint throughout the SAT in the coming years (Ryan and Spencer 2001). Most of the reduction in yield due to abiotic stresses is attributed primarily to drought (Kramer and Boyer 1995). Sorghum, being C4 plant species, is better adapted to stress environments, especially soil moisture stress, compared to maize (Nagy et al. 1995). As such, it is the logical crop to support the poor of the world, 25% of whom are expected to experience severe water scarcity by 2025 (Ryan and Spencer 2001). Genetic enhancement of sorghum for drought tolerance is a cost-effective approach to further increase its productivity, stabilize production, and contribute to food security. This chapter reviews and discusss various mechanisms, screening techniques, inheritance and efforts to breed sorghum for drought tolerance and output.

II. BREEDING FOR DROUGHT TOLERANCE Drought is recognized as a condition where the water requirement of the plants, at different crop growth stages, exceeds the available water by more than 50% in the root zone because of inadequate precipitation (excluding other soil problems, such as excessive concentration of aluminum (Al), sodium (Naþ ), chloride (Cl ), or clay), leading to perceptible reduction in crop growth and economic yield. Drought tolerance refers to physiological or biochemical adaptations that enable plant tissues to withstand water deficits (Clarke and Durley 1981) or the ability of plant tissues to function under stress or adapt to low tissue water potential (i.e., osmotic adjustment) (Blum 1979a). From a crop production point of view, drought tolerance is defined as stability of crop yield under a specific target drought stress environment, resulting from the operation of drought tolerance mechanisms. Turner (1979) defines crop drought tolerance as the ability of a genotype to yield satisfactorily in areas subjected to periodic water deficits. However, to be agronomically useful, a drought-tolerant cultivar should also have a good yield potential under favorable moisture conditions, since it is diffiult to predict drought in time and space. The effective exploitation of genetic diversity in drought tolerance requires unraveling the mechanisms that ameliorate internal stresses and those that minimize drought injury (Steponkus et al. 1980). The

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mechanisms of drought tolerance in sorghum can be described as escape, avoidance, and tolerance (Levitt 1972; Blum 1979b; Ludlow 1993). Early maturity is a well-known ‘‘drought-escape’’ mechanism through which the crop completes its life cycle before the onset of severe moisture deficits and is often associated with reduced yield potential; other two mechanisms are physiological responses to water stress (Blum et al. 1992). Short-duration sorghums have lower evapotranspiration rates due to smaller leaf area and smaller root density compared to those of long-duration types (Blum 1979b) as well as reduced seasonal transpiration due to a shorter life cycle. Avoidance is defined as the plant’s ability to maintain a relatively higher level of hydration (i.e., maintenance of higher turgor or leaf-water potential [LWP] under conditions of soil or atmospheric moisture stress). Given sufficient time, plants subjected to moisture stress may avoid dehydration by maintaining higher LWP or adapt to low tissue water potential (osmotic adjustment). Sorghum avoids low LWP by one or more mechanisms, such as a change in rooting pattern, an increased root growth, or an adjustment in leaf area (Seetharama et al. 1982). Leaf area adjustment has been suggested as one of the most powerful mechanisms of drought avoidance in sorghum (Passioura 1976). Apart from these physiological adaptations, certain biochemical compounds and micronutrients are known to confer drought tolerance in sorghum. Increased levels of glycine betaine and proline levels are reported to contribute to drought tolerance in sorghum (Wood et al. 1996). Abu et al. (2002) have identified significant association of grain micronutrients (potassium and iron) with drought tolerance in sorghum. Ability of the genotype to yield reasonably high in specified drought stress environments is considered drought tolerance. As already mentioned, it is desirable that drought-tolerant lines have the ability to manifest high yield potential, if drought stress is relieved or a better environment is provided. Strategies for genetic enhancement of crop plants for drought tolerance have been widely discussed (Hurd 1976; Blum 1979b; Sharma and Saxena 1979; Townley-Smith and Hurd 1979; Reddy et al. 1980; Saxena and O’Toole 2002; Luigi et al. 2008; Blum et al. 1992; Wang et al. 2005). It has been postulated that drought tolerance can be improved without sacrificing substantial yield (Muchow et al 1996). Four basic approaches to the breeding for drought tolerance have been proposed. The first is to breed for high yields under optimal conditions (i.e., to breed for yield potential and then to assume that this will provide a yield advantage under suboptimal conditions). The second is to breed for maximum yield by empirical selection in the field in the target drought-prone environment. The success of this

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approach depends entirely on how variable the target environment is. It works well in the Indian post–rainy season environment, which is very predictable, but not in the rainy season environment, which is highly unpredictable. The third approach is to incorporate the selected physiological and morphological mechanisms conferring drought tolerance into traditional breeding programs. For example, Blum (1979b, 1983) has recommended selection in the F5 and F6 generations for yield and yield components under optimal production conditions and simultaneous selection of duplicate samples under moisture-stress conditions. The fourth breeding approach involves identifying a key trait that confers drought tolerance at specific growth stages and its introgression into the high-yielding background. This method was established and followed at the International Crops Research Institute for Semi-Arid Tropics (ICRISAT, Patancheru, India (17 300 N, 78 160 E, altitude 545 m); Reddy 1986). This method involves pedigree selection of breeding materials for specific traits, such as (1) longer mesocotyl length for emergence under crust, and grain yield under drought-prone and yield potential areas for early seedling stage drought; (2) for grain yield under drought-prone and yield potential areas alternatively for midseason drought; and (3) for stay-green and nonlodging and grain yield under drought-prone and yield potential areas alternatively for terminal drought. Crosses were made between high-yielding adapted lines, and lines were selected for high yields under drought or with one or more drought-related traits. Selections from F2 onward were made by evaluating the segregating material in alternate generations under specified drought (early, midseason, and terminal stage) and in yield potential environments. The F5/F6 pure lines were evaluated for drought yield, potential yield, and for specific drought-related traits. Testing for yield under mild stress was adequate, as the rankings of genotypes for potential and drought yields were similar, since the drought-tolerant lines selected under mild stress had high yield potential in nonstress environments. These practical investigations agree well with those of Rosielle and Hamblin (1981), who indicated general increase in mean yield in both stress and stress-free environments if selection is practiced for mean productivity (i.e., average yield in stress and stress-free environments). Fischer and Maurer (1978) proposed an empirical drought susceptibility index (DSI) to screen the genotypes for drought tolerance under field conditions. This index is calculated as shown: DSI ¼ Y ½1 Y =YP=D


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where Y ¼ Yield in stress conditions YP ¼ Yield in irrigated conditions Y ¼ Mean yield of all genotypes under stress conditions YP ¼ Mean yield of all genotypes in irrigated conditions D ¼ Drought-stress intensity ðD ¼ 1 ½Y =YPÞ The DSI estimates indicate reduction in yield of a genotype under drought stress conditions relative to mean reduction in yield of all genotypes under drought stress. Also, the sum of ratios of yield of a particular genotype in stress (rain-fed) and the ratio of yield in relieved stress (irrigated) provides information on mean relative performance (MRP), which is calculated as MRP ¼ ½Y =Y þ YP=YP: The higher the MRP, better the performance under stress. Osmanzai (1994b) demonstrated the usefulness of DSI and MRP indices for screening and evaluating the sorghum cultivars for drought tolerance (Table 3.1). This index, however, has a weakness in that it identifies both drought-escaping and drought-tolerant genotypes as ‘‘tolerant.’’

Table 3.1. Agronomic performance of sorghum hybrids and cultivars under two soil moisture regimes at Matapos and Kadoma (Zimbabwe), and Kasinthula (Malawi) during 1991/1992.

Hybrids/cultivars Hybrids Improved cultivars Local landraces z

Number

Yield reduction (%)

Drought susceptibility Y indexz

Mean relative performance (MRP)y

12 11 5

62 63 69

0.98 1.00 1.09

2.47 1.83 1.76

Estimation of the reduction in yield by drought stress for genotypes relative to the mean reduction in yield by drought stress y Mean relative performance (MRP) ¼ the sum of the ratios of yield of genotypes in stress (rainfed) and yield in relieved stress (irrigated). Source: Osmanzai (1994b).

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III. SELECTION AMONG CULTIVARS AND LANDRACES A. Cultivar Options The greater grain yield of hybrids, compared to the improved and landrace cultivars in drought-prone environments, has been demonstrated on a number of occasions. For example, sorghum hybrids showed superior performance across variable environments, as compared to cultivars in both rainy (Table 3.2) and post–rainy seasons (Table 3.3) in India (Patil 2007; Reddy et al. 2007). As growing conditions become stressed, the yields of both hybrids and cultivars decline, but the yield difference between hybrids and cultivars becomes larger by about 30%, favoring the hybrids (House et al. 1997). Blum et al. (1992) and Osmanzai (1994a) showed that hybrids performed better than cultivars under moisture-stress conditions and recover faster when moisture stress was relaxed. Evaluation of 12 Table 3.2. Comparative performance of sorghum hybrids over improved varieties/ landrace cultivars during rainy season in India.

1

Grain yield (T HA )

Year of testing

Hybrid

Cultivar

1985-90 1993 1994 1995 1996

3.7 3.4 3.6 3.9 3.6

3.2 3.1 3.0 3.0 3.3

(07) (09) (10) (17) (06)

(04) (09) (10) (12) (05)

Increase over cultivar or local check (%) 14.9 8.3 18.5 28.9 8.8

Reference Murty (1992) — — — Rana et al. (1997)

Figures in parentheses denote the number of hybrids or cultivars tested. Table 3.3. Comparative performance of sorghum hybrids over improved varieties/ landrace cultivars during post–rainy season in India.

1

Grain yield (T HA )

Year of testing

Hybrid

Cultivar

1996 2000 2001 2002

2.8 4.9 4.8 4.2

2.2 4.1 4.7 3.9

z

z

(4) (16) (13) (10)

(04) (03) (02) (01)

Increase over cultivar or local check (%) 28.4 18.6 1.8 7.4

Reference Rana et al. (1997) SPSHTy 2000 SPSHT 2001 SPSHT 2002

Figures in parentheses denote the number of hybrids/cultivars tested. Seed Producers Sorghum Hybrid Trial conducted at several seed producers’ experimental plots and ICRISAT, Patancheru, India. y


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single-cross sorghum hybrids along with their parents and two local cultivars in Kenya under varying levels of drought stress, ranging from nonstress to extreme stress (preflowering and terminal), revealed mean hybrid superiority over midparent values by 54% and by 12% over local cultivars for grain yield (Haussmann et al. 1998). The single-cross hybrids were consistently superior to their parents with an average heterosis of 54% across eight frequently drought-prone environments in the semiarid Makueni district of Kenya (Haussmann et al. 2000). Rao and Khanna (1999) have also reported superiority of sorghum hybrids over their parents for leaf area and dry-matter production under both preflowering and postflowering drought stress. Greater performance of hybrids than their parents or cultivars under drought stress or related abiotic stresses was also reported. Field research based on a limited number of genotypes (Peng et al. 1994; Azhar et al. 1998) indicated that hybrids have better salinity-stress (which mimics drought stress) tolerance than their parental lines or pure-line cultivars. It has been established in many studies that F1 hybrids had superior stability (or buffering capacity) across variable environments as compared to homozygotes in sorghum (Blum 1988). For a given growth duration and biochemical photosynthetic efficiency, the total photosynthetic product of a crop species is finite. Hence increased performance of hybrids from their parents is due to greater growth rates and greater total biomass production and higher harvest index (Blum 1966; Blum et al. 1977b; Gibson and Schertz 1977) with or without an apparent increase in leaf photosynthetic rates (Sinha and Khanna 1975). The advantages of the hybrids are often associated with reduced crop growth duration (Quinby 1974). As the heterozygote may contain more than one gene product than the homozygote, it becomes biochemically diversified. This biochemical diversification allows better adaptation to diverse environments (Srivastava 1981). Bhale et al. (1982) found that some sorghum hybrids showed heterosis for proline accumulation (known to confer drought tolerance) under moisture stress. All these evidences suggest that the wider adaptability of hybrids is due to their relative tolerance to a wide range of abiotic stresses including soil moisture stress and related factors. Therefore, breeding for hybrid cultivars is a better option than open-pollinated (OP) cultivars for improving sorghum grain yield in water-scarce environments. Furthermore, to increase the grain yield within the limits of the available water supply, female parents for hybrid production should be chosen based on both leaf area and photosynthetic rate. Pollinators should be selected for maximum seed number per panicle (Krieg 1988). The improvement of performance per

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se and combining ability of parents for agronomic traits and grain yield under drought stress should be given strategic importance, considering that parental performance per se and general combining ability (GCA) in sorghum is strongly correlated with hybrid performance (Quinby and Karper 1946; Murty 1991; Murty 1992; Bhavsar and Borikar 2002). It is well established in sorghum breeding that good OP cultivars make good parents for hybrids, because heterosis results primarily from additive gene action (Kambal and Webster 1965; Miller and Kebede 1981). Doggett (1988) showed the importance of the parental improvement to increase the hybrid performance and claimed that about 50% of the sorghum grain yield increase could be ascribed to better parents. B. Selection among Landraces and Breeding Material Landraces are valuable genetic resources for environmental stress resistance as they have evolved under natural selection pressure over several years. Drought tolerance of landrace sorghum selection M 35-1, which is a highly popular post–rainy season adapted cultivar in India, has been repeatedly demonstrated (Seetharama et al. 1982; Shackel and Hall 1983). Blum and Sullivan (1986) evaluated of 13 sorghum landraces (six from Mali and seven from Sudan) that evolved along geographical gradients of rainfall for drought tolerance under hydroponics conditions. The experiment was carried out in a growth chamber and water stress was induced by adding polyethylene glycol-800 to the nutrient solution with a solute water potential of 0.5 MPa compared to a control solution at 0.03 MPa. The result indicated that drought tolerance in terms of relatively less growth inhibition under stress was higher in races from dry regions than in those from humid regions. Germplasm lines and breeding lines tolerant to drought at specific growth stages have been identified at ICRISAT (Table 3.4). Table 3.4. Sorghum germplasm and breeding lines tolerant to drought at specific growth stages, ICRISAT, Patancheru, India. Growth stage Seedling emergence

Early seedling Midseason Terminal drought

Tolerant sources/ improved lines IS 4405, IS 4663, IS 17595 and IS 1037, VZM1-B and 2077 B, IS 2877, IS 1045, D 38061, D 38093, D 38060, ICSV 88050, ICSV 88065 and SPV 354 ICSB 3, ICSB 6, ICSB 11 and ICSB 37, ICSB 54 and ICSB 88001 DKV 1, DKV 3, DKV 7, DJ 1195, ICSV 272, ICSV 273, ICSV 295, ICSV 378, ICSV 572, ICSB 58 and ICSB 196 E 36-1, DJ 1195, DKV 3, DKV 4, DKV 17, DKV 18, ICSB 17

Source: ICRISAT (1982), Reddy et al. (2004).


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IV. BREEDING FOR DROUGHT ESCAPE In regions where end-of-season drought stress is common, such as those in Indian peninsula in rainy and post–rainy season environments, the most effective way to reduce losses due to drought is through the development of early-maturing genotypes to enable them escape endof-season drought (Blum 1979b; Turner 1979). The relative yield advantage of early genotypes, especially under late-season moisture stress, has been reported by Saeed and Francis (1983) and Saeed et al. (1984). In the Indian peninsula, the replacement of traditional longduration OP cultivars (130 to 180 days) with early hybrids and OP cultivars of 100- to 110-day duration, which mature before the end of rains or before soil moisture is depleted, has resulted in a remarkable increase in rainy season sorghum production (Rao et al. 1979). However, early-maturing cultivars have become highly prone to grain molds, as the grain-filling and maturation periods normally coincide with continuous rains. Breeding sorghum for grain mold resistance for rainy season adaptation is therefore one of the major objectives of most sorghum improvement programs globally. Under terminal drought typically experienced by post–rainy season sorghums in India, earlymaturing improved sorghum cultivars, such as CSH 1 (100 days and 4 t ha1), CSH 6 (95 days and 3.2 t ha1), and NK 300 (88 days and 4 t ha1), produced better grain yields than long-duration cultivars, such as M 351 (105 days 1.9 t ha1) and SPV 86 (108 days and 3 t ha1) (Seetharama et al. 1982). Sorghum production in the Great Plains of the United States is based on the development of early-maturing genotypes that escaped late-season stress by maturing before soil moisture reserves are exhausted (Smith and Frederiksen 2001). Sorghum improvement programs at ICRISAT and elsewhere are most successful in exploiting a ‘‘drought-escape’’ mechanism and have bred specific maturity cultivars that match the available soil moisture. Selection for enhanced grain yield under conditions of moisture stress resulted in a genetic shift towards early flowering (Blum 1980). Most of these studies have also confirmed the positive association between the long growth duration and yield potential in high-potential environments. It is therefore evident that while exploiting drought escape as a solution, some of the potential grain yield must be sacrificed in return for improved stability under moisture stress (Blum, pers. comm.). The reduced yield potential in early genotypes may be compensated for to some extent by increasing plant density (Blum 1970). Under terminal water stress during the post–rainy season, short-duration sorghum genotypes produce equal grain but less dry matter than late cultivars.

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Some early-maturing A/B lines that facilitate development of early-maturing hybrids, which can escape drought, have been identified at ICRISAT: for example, ICSA/B-615, 628, 629, 630, 634, and 635. Drought escape is, however, not drought tolerance per se. Therefore, breeding for early maturity may not be always associated with higher yield in regions with erratic rainfall pattern. Concerted breeding for tolerance to a given pattern of drought in a target region is the best way to improve grain yield under moisture-limited conditions. Successful breeding for tolerance to a particular drought requires the availability of large-scale, cost-effective screening techniques, which can facilitate efficient discrimination of genotypes for drought tolerance. However, responses of genotypes to drought stress are complex. Moisture stress is known to cause differing degrees of reduction in grain yield, depending on the stage of the crop and frequency, duration, and severity of moisture stress. Nevertheless, four growth stages are recognized as most vulnerable to moisture stress (Reddy 1985; Rooney 2004): 1. Germination and seedling emergence 2. Early seedling stage (from seedling emergence to panicle initiation) 3. Midseason (from panicle differentiation to flowering [preflowering]) 4. Postflowering (from flowering to grain-filling stage) The responses of sorghum to moisture stress at these four growth stages have been well characterized. Genetic variation for drought tolerance at these growth stages has been observed and found to be heritable (Reddy 1985; Mkhabela 1996). Repeatable genotypic differences in drought response are observed, if the moisture stress is confined to one stage, but differences are masked if it occurs at more than one stage (Garrity et al. 1982). It is therefore suggested that screening techniques aim to discriminate genotypes for drought tolerance at each of the growth stages separately (Reddy 1985). Sandy soil or shallow soil sites are best suited for preflowering field evaluation of stress response; heavier and deeper soils are best for evaluating postflowering drought stress (Rosenow et al. 1997c). Several effective and reliable screening techniques were developed during the late 1970s and early 1980s at ICRISAT, India and in the United States and Australia, and drought-tolerant sources at different growth stages, from the germplasm and breeding lines, were identified. This approach led to several attempts to breed sorghum for


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drought tolerance based on either the plant responses at these growth stages or physiological response traits conferring drought tolerance. These techniques are discussed in the next two sections.

V. GROWTH STAGE–SPECIFIC SCREENING TECHNIQUES A. Germination and Seedling Emergence The condition ideal for seedling survival is hardly present in the SAT, especially those of sub-Saharan Africa. A hot dry seedbed environment with soil surface temperature often greater than 55 C during crop establishment due to absence of subsequent rain after initial planting rain is a common occurrence in most regions of SAT (Gupta 1986; Hoogmoed and Klaij 1990; Peacock et al. 1993). Seedling death can occur at one of three defined stages—germination, emergence, and postemergence—during crop establishment. Under such situations, longer mesocotyl and ability of seedling emergence from deep planting (where soil moisture is greater) and from soils with high surface temperatures and tolerance to, or recovery from, drought at the seedling stage are considered important for crop establishment. Experience at ICRISAT showed that initial selection for coleoptile length (the trait associated with germination under deep sowing, which is desirable to capitalize on initial rains) of test lines 5 days after planting in germination boxes followed by selection in raised brick tanks using charcoal and heavy kaoline (to simulate higher or less than normal temperatures, respectively) in 12 cm planting depth based on plant counts (as a reflection of mesocotyl length) is highly effective in identifying lines with long mesocotyl length that is necessary for emergence in deep sowing. Useful genetic variation was noted for seedling emergence (10–50%) at 5 days after sowing among 166 sorghum genotypes grown in alfisols with limited soil moisture during a hot dry summer season and under differential irrigation (5–30 mm) using a line-source sprinkler system (Seetharama et al. 1982). Diminishing soil water availability after germination due to dry spells after initial rains during sowing greatly affects seedling growth and survival. Selection of breeding lines in the sandy soil-filled brick tanks spread uniformly with charcoal powder at 125 gm2 (which induces high soil surface temperatures) based on the seedling emergence counts 6 days after planting was effective in identification of lines with high seedling emergence under the high surface soil

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temperature that is prevalent in Africa during the sowing season (Reddy 1985). Significant cultivar differences for seedling emergence at low soil moisture conditions (Soman 1990) and subsequent growing ability have been reported in sorghum (O’Neill and Diaby 1987). B. Postemergence and Early-Seedling Stage Sustained seedling growth following emergence depends on the capacity of the seedling to elongate, produce leaves, and become autotrophic. Postemergence seedling death due to moisture stress under field conditions is primarily caused by the prevalent high soil surface temperatures, at least in the first 10 days following sowing, and only after that does water deficit start to take effect (Stomph 1990; Peacock et al. 1993). Selection of sorghum breeding lines for recovery from severe seedling drought, induced after germination for 24 days followed by termination of drought in the 29th day after planting in polyvinyl chloride (PVC) vases based on recovery scales, number of plants recovered/vase, and number of green leaves/vase, was effective to screen for recovery from seedling drought (Reddy 1985). Wenzel (1991) reported that additive effects controlled variation for seedling drought tolerance and that the trait was highly heritable. However, the relative magnitude of genotypic variation is far lower than that of soil temperature variation. C. Midseason and Preflowering Stage The preflowering response is expressed when plants are stressed during panicle differentiation prior to flowering. Symptoms of midseason or preflowering drought stress susceptibility include leaf rolling, uncharacteristic leaf erectness, leaf bleaching, leaf tip and margin burn (leaf firing), delayed flowering, poor panicle exertion, panicle blasting and floret abortion, and reduced panicle size (Seetharama et al. 1982; Rosenow et al. 1997a,b,c). Tolerance to preflowering drought stress is indicated by the alternative condition in each instance. Since the panicle is directly affected, severe preflowering stress can result in drastic reductions in grain yield. For screening for midseason stress, which represents the midseason drought pattern of the rainy season in many parts SAT, mild moisturestress is not sufficient for the expression of the genotypic differences for responses to moisture stress. Curtailing irrigation 3 weeks after sowing for over 45 days in a rain-free season was found to provide the required


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level of drought stress for effective screening for preflowering drought stress (Reddy 1985). Specific nurseries were used in dry environments in Sudan (Ejeta 1987) and in Mexico (Rosenow et al. 1997a) to screen sorghum genotypes for preflowering drought stress. Excellent sources of tolerance to preflowering drought have been identified (Rosenow et al. 1997a,b,c). These sources of tolerance have been utilized by researchers to develop inbred lines, hybrids, and cultivars that have good levels of preflowering drought tolerance (Rooney 2004). At ICRISAT, India a large number of progenies were screened for tolerance to preflowering drought stress. Those with better tolerance were selected further for grain yield alternatively under midseason drought and yield potential environments in India. Some of the most promising lines are DJ 1195, ICSV 213, ICSV 221, and ICSV 210. When these lines were tested in drought-prone environments in sub-Saharan Africa, they showed greater stability for grain and biomass yields than other cultivars that were developed for favorable environments (ICRISAT 1982, 1986, 1987). Selection of sorghum breeding lines for grain yield in field conditions under midseason drought (induced for 35 days), after initial nonstress growth period of 21 days after planting, was effective to identify promising lines for midseason drought stress tolerance. In replicated trials at Sangareddy (20 km west of ICRISAT, Patancheru, India), 364 advanced selections from the drought tolerance breeding project were evaluated during the hot summer (April–May) of 1980, when the maximum daily temperature varied between 35 C and 43 C. Many entries showing tolerance to leaf firing were also agronomically good. Good correlations between scores for leaf firing and ability to recover were reported (Seetharama et al. 1982). Little effort was made to breed sorghum specifically for drought tolerance at specific growth stages, particularly at germination and emergence, and early-seedling stages and relate it to whole-plant tolerance to drought. The earlier efforts for genetic enhancement of sorghum for preflowering drought tolerance in the United States and ICRISAT, Patancheru helped in identification of improved sorghum lines for drought tolerance (Reddy et al. 2004). D. Terminal and Postflowering Stage 1. Screening Techniques. Postflowering stress is due to inadequate soil moisture during the grain-filling stage, especially during the later portion of grain filling. Postrainy season sorghum in India typically experiences such postflowering stress. It is similar to that experienced

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by crops grown under receding soil moisture conditions in the West Asian region (Israel and Yemen) as well as parts of West Africa (Lake Chad area in Nigeria and in Mali). Symptoms of postflowering drought stress susceptibility include premature plant (leaf and stem) death or plant senescence, stalk collapse and lodging, and charcoal rot (caused by Macrophomina phaseolina), along with a significant reduction in grain size, particularly at the base of the panicle. Tolerance is indicated when plants remain green and fill grain normally. Such green stalks also have good tolerance to stalk lodging and to charcoal rot (Rosenow and Clark 1995; Garud et al. 2002). The cultivars that remain green under postflowering drought are referred to as having the ‘‘stay green’’ trait. Selection of breeding lines for the stay-green trait was useful for screening for terminal drought tolerance. The stay-green trait is expressed only when the materials at postflowering stage are exposed to severe moisture stress. Comparison of yield on shallow vertisols or on partially saturated deep vertisols with an irrigated control has been advocated to screen genotypes for terminal drought tolerance in receding moisture conditions. Effective screening for terminal drought tolerance can be carried out under field conditions by choosing the appropriate time of sowing to ensure that the crop experiences terminal drought stress. The line source (LS) sprinkler irrigation technique developed at Utah State University was followed at ICRISAT for screening sorghum genotypes for terminal drought tolerance. Each side of the LS formed one replication. The field was uniformly irrigated until the crop reached boot stage, and the LS were used at 50, 61, and 77 days after sowing to create a gradient of soil moisture (stress). The amount of water received across the plot was measured in catch cans placed at crop height. The LS was also used to study the effect of soil moisture stress on charcoal rot incidence (Seetharama et al. 1987). The rows of plants farthest from the LS showed disease earlier than those nearest. This was apparent for each of the three parameters of disease spread: percentage of soft stalks, number of nodes crossed, and the length of fungal spread (Seetharama et al. 1987). Specific nurseries were used in dry environments in Sudan (Ejeta 1987) and in Mexico (Rosenow et al. 1997c) to screen sorghum genotypes for postflowering drought tolerance. In the sorghum improvement program of University of Queensland (Australia), breeding progenies are routinely evaluated in regular field screening nurseries for premature leaf and plant senescence at or near physiological grain maturity (Borell et al. 2000a, 2000b).


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2. Breeding. Unlike the situation at other stages, considerable progress has been made for genetic enhancement of sorghum for drought tolerance at the postflowering stage. Stay-green trait is now considered as an important postflowering drought tolerance trait (Rosenow et al. 1997c). Genotypes possessing the stay-green trait maintain a greater green leaf area during grain filling and extend photosynthesis in upper canopy leaves after physiological grain maturity under postflowering drought compared to their senescent counterparts (Rosenow et al. 1977). Earlier reports indicated that the leaves remain green due to a lack of assimilate demand because the plants have small panicles under postflowering drought (Henzell and Gillerion 1973; Rosenow et al. 1983a). However, recent studies have shown that they are stay green not only because of their small sink demand but also due to their higher leaf nitrogen status (Borrell and Douglas 1997; Borrell et al. 1999; Borrell and Hammer 2000). Although small sink demand enables plants to maintain photoynthetic capacity and ultimately result in higher grain yield and lodging resistance (Borrell et al. 2000a), higher leaf nitrogen status retards the decline in protein content of the aging leaves (Humphreys 1994) and higher transpiration efficiency (Borrell et al. 2000b). The stay-green sorghums accumulate more soluble sugars in stems than do senescent counterparts, both during and after grain filling (Duncan et al. 1981; McBee et al. 1983). The higher concentration of stem sugars improves the digestible energy content of the stover (Vietor et al. 1989). Staygreen genotypes are less susceptible to lodging, more resistant to charcoal rot, and retain higher levels of stem carbohydrates than non– stay-green genotypes (Mahalakshmi and Bidinger 2002). Inheritance of Stay Green. Walulu et al. (1994) have concluded that the stay-green trait in B 35 is influenced by a major gene that exhibits varied levels of dominant gene action depending on the environment where evaluations are made. Its control in Q 141, which is derived from B 35, appeared to be however multigenic (Henzell et al. 1992). Greater green leaf-area duration during grain filling appears to be a product of different combinations of three distinct factors: green leaf area at flowering, time of onset of senescence, and subsequent rate of senescence (Van Oosterom et al. 1996; Borrell et al. 2000a). All the three factors appear to be inherited independently (Van Oosterom et al. 1996), and thus sources expressing these components can be combined in breeding programs (Borrell et al. 2000a). This is supported by the identification of multiple quantitative trait loci (QTL) affecting the trait (Harris et al. 2006).

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Stay Green, Grain Yield, and Size. Stay green and grain yield were positively associated in a range of studies conducted in both Australia (Borrell et al. 2000a) and India (Borrell et al. 1999), highlighting the value of retaining green leaf area under terminal drought. Grain yield is the product of grain number and grain size (completeness of grain filling). Grain number is generally the main determinant of differences in grain yield, and this has also been observed in sorghum grown under terminal drought stress in southern India (Borrell et al. 1999). Grain size is a secondary yield determinant and is often negatively associated with grain number under nonstress conditions. Grain size was correlated with relative rate of leaf senescence during grain filling such that reducing rate of leaf senescence from 3% to 1% loss of leaf area per day resulted in doubling grain size from about 15 to 30 mg (Borrell et al. 1999). Hence, the stay-green trait can potentially increase grain yield by improving grain number and grain filling ability. Sorghum hybrids containing the stay-green trait have been found to yield significantly more under water-limited conditions compared with hybrids not possessing this trait (Rosenow et al. 1983b; Henzell et al. 1992; Borrell and Douglas 1996). Stay-green hybrids have been shown to produce significantly greater total biomass after anthesis, retain greater stem carbohydrate reserves, maintain greater grain growth rates, and have significantly greater yields under terminal drought stress than related but senescent hybrids (Henzell et al. 1997; Borrell et al. 1999; Borrell et al. 2000b). Conventional breeding for the stay-green trait has been based primarily on two sources for this trait, B 35 and KS 19 in programs in Australia and the United States (Mahalakshmi and Bidinger, 2002). KS 19 is a selection from a cross of short Kaura, an improved landrace cultivar from northern Nigeria, with Combine Kafir 60 (Henzell et al. 1984). B 35 (PI 534133) was selected from a converted (dwarf, earlyflowering) version of IS 12555, an Ethiopian landrace (Rosenow et al. 1977c, 1983a). The stay-green trait from IS 12555 (as SC 35) has been successfully used by Queensland Department of Primary Industries (QDPI) program in Australia to develop postflowering drought stress tolerance and lodging resistance in parental lines and commercial hybrids (Henzell et al. 1992b; Henzell and Hare 1996). KS 19 has been used commercially primarily in the breeding program of QDPI, while B 35 is widely used in both public and private sector breeding programs in United States (Mahalakshmi and Bidinger 2002). The partially converted (B 35) and fully converted (SC 35C-14E) versions of IS 12555 (Rosenow et al. 1983b) have provided the major and best sources of the trait used in the QDPI program (Henzell et al. 1997).


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QL 41 was the key line developed, and was derived from the cross QL 33 B 35 (Henzell et al. 1992 a). It has a high level of expression of stay-green. Crosses of it with QL 38 and QL 39 (sorghum midge– resistant lines) formed the basis of the female stay-green and midgeresistant gene pool at QDPI), although less progress has been made in developing such germplasm in the restorer program (Henzell et al. 1997). Hybrids involving nonsenescent lines QL 40 and QL 41 showed the least stalk rot incidence and severity, particularly with natural exposure, and performed better than commercial hybrids Texas 671 and E 57 (Dodman et al. 1992). In most sorghum improvement programs globally, E 36-1 and B 35 have been extensively used for developing hybrid seed parents (B-lines) and pollen parents (R-lines) and cultivars. E 36-1 is a widely adapted zera-zera germplasm line from Ethiopia. Several stay-green hybrid seed parents were developed prior to 2000 following a traitbased breeding approach at ICRISAT (Reddy et al. 2005). These seed parents were evaluated for stay-green and grain yield potential during the 2003 post–rainy season at ICRISAT in Patancheru. Some of these seed parents are better than the stay-green source, E 36-1, for stay green and grain yield under terminal drought (Table 3.5). The grain size of

Table 3.5. Performance of selected ICRISAT-bred sorghum stay-green B-lines (ICRISAT, Patancheru, 2003 post–rainy season).

Stay-green lines ICSB ICSB ICSB ICSB ICSB

375 405 675 676 677

Controls 296By E 36-1 (Stay green control) Mean SE LSD (5%) CV (%)

Days to 50% flowering

Plant height (M)

Stay green scorez

Grain yield (T HA1)

100-grain weight (G)

71 72 72 73 72

1.7 1.2 1.0 0.9 1.1

2.0 1.5 2.5 1.5 2.5

3.5 3.0 3.1 3.4 3.3

3.14 3.27 3.28 3.00 3.28

76 65

1.1 1.5

2.5 2.0

2.6 2.8

3.01 3.71

73 0.87 2.68 1.68

1.4 0.05 0.15 5.31

2.6 0.43 1.30 23.57

3.5 0.36 1.10 14.26

3.12 0.17 0.53 7.58

z Stay green score taken on a 1 to 5 scale at harvest, where 1 ¼ > 75% green; 2 ¼ 51–75%; 3 ¼ 26–50%; 4 ¼ 10–25%; and 5 ¼ < 10% green. y Hybrid seed parent of several popular and released or marketed hybrids in India.

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these B-lines is significantly better than 296B, the hybrid seed parent (known for its large grain) of several popular and released/marketed sorghum hybrids in India. Since 2000, several improved stay-green R-lines and cultivars were developed. Some of these R-lines and cultivars are significantly better than E-36-1 for grain yield with comparable stay greenness and grain size under terminal drought (Table 3.6). These hybrid parents have good potential for developing stay-green hybrids. Although most U.S/ commercial sorghum hybrids possess good tolerance to preflowering drought stress, only a few have good postflowering drought tolerance (Nguyen et al. 1997). Despite the availability of simply inherited stay-green trait associated with terminal drought tolerance, progress in enhancing postflowering drought tolerance is slow (Rosenow et al. 1997c). This is because the expression of stay-green trait is dependent on the occurrence of a prolonged period of drought stress of sufficient severity during the grain-filling period to accelerate normal leaf senescence, but not of sufficient magnitude to

Table 3.6. Performance (mean of two years) of selected ICRISAT-bred sorghum stay-green cultivars and R-lines (ICRISAT, Patancheru, 2003 post–rainy season). Cultivars/ R-lines

Days to 50% flowering

Plant height (M)

Stay green scorez

Grain yield (T HA1)

100-grain weight (G)

ICSV 21001 ICSR 21004 ICSR 21005 ICSR 21006 ICSR 21009 ICSR 21010 ICSV 21012 ICSV 21013

77 77 76 77 71 73 73 71

1.6 1.5 1.5 1.5 1.6 1.5 1.7 1.5

1.5 2.5 2.5 2.5 2.0 2.5 2.5 2.0

3.4 4.1 4.9 5.4 3.6 3.8 4.4 3.5

3.45 3.67 3.69 3.63 3.61 3.34 3.26 3.48

Controls E 36-1 (stay green control) M 35-1y R 16y Mean SE LSD (%) CV (%)

65 69 67 73 0.87 2.68 1.68

1.5 2.2 1.8 1.4 0.05 0.15 5.31

2.0 3.0 2.5 2.6 0.43 1.30 23.57

2.8 3.6 3.3 3.5 0.36 1.10 14.26

3.71 3.45 3.08 3.12 0.17 0.53 7.58

z Stay green score taken on a scale 1 to 5 at harvest, where 1 ¼ > 75% green, 2 ¼ 51–75%, 3 ¼ 26–50%, 4 ¼ 10–25%, and 5 ¼ < 10% green. y Popular post–rainy season cultivars released in India


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cause premature death of the plants. Only a limited number of sources are currently in use in sorghum breeding programs (Mahalakshmi and Bidinger 2002). Because of this precise requirement for the trait expression, natural field environments do not offer ideal conditions for selection. QTL for stay-green trait and the molecular markers tightly linked to these QTL have been identified and are serving as powerful tools to enhance terminal drought tolerance in sorghum (details are discussed in the Section IX).

VI. PHYSIOLOGICAL RESPONSE TRAITS FOR DROUGHT TOLERANCE Several physiological traits such as leaf water potential (LWP), osmotic adjustment (OA), heat tolerance, desiccation tolerance, rooting depth, and epicular wax (Downes 1972; Levitt 1972; Sullivan 1972; Sullivan and Ross 1979; Turner 1979; Jordan and Monk 1980; Kramer 1980; Jordan and Sullivan 1982; Peacock and Sivakumar 1987; Krieg 1993; Ludlow 1993) are known to contribute to drought tolerance. Screening techniques and genetic variability based on some of these traits (LWP and OA) have been reported (Christiansen and Lewis 1982; Garrity et al. 1982; Seetharama et al. 1982; Blum 1983; Jordan et al. 1983; Blum 1987; Ejeta 1987) and are discussed in the next sections. A. Leaf Water Potential (LWP) The physiological adaptations effective in improving tolerance to moisture stress were found to vary with plant growth stage in sorghum (Ackerson et al. 1980). Before flowering, plants avoid dehydration largely by maintaining higher LWP; after flowering, plants avoid dehydration by maintaining higher turgor at a given level of moisture stress. This activity could be partly responsible for the different classification of drought tolerance before and after flowering in sorghum (Rosenow et al. 1983a). The most evident control of LWP is at the root system. Small root resistances and a large root-length density would contribute to the maintenance of a higher LWP. The root-length density increased with reduced soil moisture only at certain soil depths (Blum and Arkin 1984). Genotypic differences in sorghum root growth have been noted under moisture stress (Blum et al. 1977b,c). Blum (1979a) has shown that early-maturing sorghum genotypes not only escape drought but also avoid it because of reduced transpiration as a result of increased root length accompanied by reduced leaf area (high

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Table 3.7. Genotypic differences in expansion rates of culm (leaf þ stem; Ter) and of leaf (Ler) under control and moisture-stress during the panicle development stage in sorghum (ICRISAT, Patancheru, India). Terz (MM H1)

Reduction

Genotype

Control

Stress

(%)

CSV 15 V 302 CSH 8 M 35-1 IS 12611 CS 3541 CSH 1 CSH 5 Mean Se Range

2.42 2.37 2.09 2.45 4.05 3.08 2.55 2.87 2.61 0.22 2.08–4.06

1.42 1.53 1.79 2.28 2.67 1.49 1.72 2.2 1.89 0.16 1.42–2.67

41.3 35.4 14.4 6.9 34.1 29.5 32.6 23.3 27.19 4.10 6.9–41.3

Lerz (MM H1) Control 2.18 1.76 1.74 1.95 2.42 1.49 1.30 2.06 1.86 0.13 1.30–2.42

Reduction

Stress 1.23 1.11 1.47 1.70 1.72 1.16 0.88 1.65 1.37 0.11 0.88–1.72

(%) 43.6 36.9 15.5 12.8 28.9 22.2 32.3 19.9 26.51 3.80 12.8–43.6

z

Data for period 1600 hrs on 31 January 1981 to 1530 hrs on 2 February 1981. Source: Seetharama et al. (1982)

root length to leaf area ratio). Because of high sensitivity of leaf area expansion to changes in turgor, several researchers (Hsiao and Acevedo 1974; Boyer and McPherson 1975) suggested the use of leaf area expansion as the criterion for screening the genotypes for drought tolerance. Large differences for leaf expansion rates among sorghum cultivars and hybrids have been observed in Patancheru. The cultivars CSV 5 and V 302, which were sensitive to drought, showed more reduction in leaf expansion than M 35-1 or CSH 8 (known to be drought tolerant) (Table 3.7). At ICRISAT, leaf firing was found to be a simple phenotypic trait that allows large populations to be screened (Andrews et al. 1983). Leaf rolling is an established symptom of wilting in cereals (Jones 1979), and delayed leaf rolling under drought stress is being used as one component of a selection index for drought tolerance (avoidance) in sorghum (Rosenow et al. 1983a). Greater leaf rolling was indicative of reduced LWP in different sorghum genotypes (Blum et al. 1989). Gaosegelwe and Kirkham (1990) suggested that LWP could be used as an easy and fast way to screen sorghum genotypes for drought avoidance. Under relatively mild stress, delayed leaf rolling may be associated with sustained plant growth and production. However, under severe drought and heat stress conditions, greater leaf rolling may be associated with better chances for recovery when moisture stress is relieved (Blum et al. 1992). Stricevic and Mastrorilli (1992) and Stricevic and Caki (1997) showed a predawn LWP of 0.5 MPa as


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the threshold value for scheduling irrigation. Physiological processes were significantly decreased below this value, which suggests that those genotypes that maintain predawn LWP above this level can be considered as drought tolerant. However, breeding programs are slow to adopt this trait for selection. B. Osmotic Adjustment (OA) Prolonged periods of water stress, a characteristic of SAT, cause low tissue water potential and tolerant plants adapt to low tissue water potential (OA). The genotypes with high OA retain higher turgor at a given level of plant water deficit and produce higher leaf area and subsequently support carbon assimilation. Based on this, Tangpremsri et al. (1995) concluded that the adverse effect of water stress could be reduced by selecting sorghum genotypes with high OA. However, Flower et al. (1990) concluded that while drought tolerant sorghum cultivars had better OA and consequently less leaf rolling under stress compared with susceptible cultivars, these responses did not influence growth under very dry and hot conditions. Studies on OA have been accelerated by the use of pressure chamber method and analysis of pressure volume graphs to measure water, osmotic, and turgor potentials (Tyree and Hammel 1972). Thermocouple psychrometry has also aided in measurement of water and osmotic potentials (Parsons 1982). Variation in osmotic adjustment (OA) among sorghum genotypes was found to range from nil to 1.7 Mpa (Blum and Sullivan 1986). Landraces from dry habitats compared to those from humid regions have greater capacity for OA (Blum and Sullivan 1986). Diurnal and seasonal OA to water stress have been noted in sorghum (Jones and Turner 1979). OA has direct positive effect on yield under moisture stress (Ludlow et al. 1990; Santamaria et al. 1990) and is largely ascribed to increase in root size, root length density, and soil moisture extraction (Tangpremsri et al. 1991a,b). At ICRISAT, genotypic differences have been detected in predawn osmotic potentials even under mild stress. Post–rainy season cultivars, such as M 35–1 and CSH 8, have a greater capacity to decrease their osmotic potential under stress than the rainy season cultivar CSH 6 (Seetharama et al. 1982). Two independent major genes (OA I and OA 2), have been reported to control the inheritance of OA in sorghum (Basnayake et al. 1995). Little if any progress in breeding for drought tolerance using either OA or any of the other physiological traits has been documented in sorghum, partly because of poor understanding of the traits conferring drought tolerance (Bonhert et al. 1995), and lack of

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procedures to impose reproducible stresses and rapid methods to measure these traits (Santamaria et al. 1990). It appears that individual physiological traits identified to date are not sufficiently related to overall drought tolerance under field conditions to merit selection based on them (Rosenow et al. 1997c).

VII. MARKER-ASSISTED BREEDING FOR DROUGHT TOLERANCE The direct selection for drought tolerance components using conventional breeding approaches has been slow and difficult (Ejeta et al. 2000), largely due to unpredictable timing, duration, and severity of drought occurrence under natural conditions and difficulty of establishing screening environments. The use of molecular markers and QTL analysis based on carefully managed replicated tests therefore has the potential to alleviate the problems associated with inconsistent and unpredictable onset of moisture stress or the confounding effect of other related stresses, such as heat (Ejeta et al. 2000). Many researchers (Tuinsta et al. 1996, 1997, 1998; Crasta et al. 1999; Ejeta et al. 2000; Subudhi et al. 2000; Tao et al. 2000; Xu et al. 2000; Kebede et al. 2001; Coulibaly 2002; Sanchez et al. 2002) identified QTL associated with pre- and postflowering drought tolerance in sorghum. Tuinsta et al. (1996) found six distinct genomic regions that were associated with preflowering drought tolerance in sorghum in recombinant inbred lines (RIL) derived from the cross Tx7078 B 35. These loci accounted for approximately 40% of the total phenotypic variation for yield under preflowering drought and were detectable across a range of environments. Kebede et al. (2001) reported four QTL that confer preflowering drought tolerance in sorghum RIL derived from the cross SC 56 Tx7000. However, these QTL were not consistent across the environments. Kebede et al. (2001) also noted a strong association between preflowering drought tolerance and days to 50% flowering. The molecular genetic analysis of QTL influencing the stay-green trait, which is an important postflowering drought tolerance character (Tao et al. 2000; Xu et al. 2000; Haussmann et al. 2002), resulted in the identification of up to four QTL located on linkage groups B, G, and I. Subudhi et al. (2000) confirmed all four QTL (Stg –1, –2, –3, –4) that were identified earlier by Xu et al. (2000) by evaluating RIL populations derived from B35 Tx700 in two locations for two years. Similarly, comparisons of stay-green QTL across locations with earlier reports


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indicated that three of the four stay-green QTL showed consistency across different genetic backgrounds and environments. They concluded that Stg-2 is expected to increase the understanding of the staygreen trait, leading to either marker-assisted introgression of this QTL into elite agronomic background or map-based cloning to genetically engineer this locus into improved cultivars. The efficient map-based cloning requires the availability of a high-resolution integrated genetic and physical map, large segregating populations, and accurate phenotyping (Mullet et al. 2001). The construction of an integrated sorghum genome map is well under way (http://www.cropscience.org.au/ icsc2004/poster/3/2/1/965_borrellak.htm verified 15 October 2008). Significantly, one of the markers linked to stay-green QTL located on linkage group B is a PCR-based SSR marker. This type of marker is ‘‘user friendly’’ and therefore can readily be used in breeding programs (http://www.cimmyt.org/ english/docs/proceedings/molecApproaches/ pdfs/physiologBasis.pdf verified 15 October 2008). QTL for stay-green trait have been mapped by phenotyping RIL from two-Striga tolerance mapping populations having agronomically elite, Striga susceptible, stay-green parent E 36-1. Results have indicated that this source has several QTL for the stay-green trait that were not detected in previous research using as sources B 35 and SC 5. Across the three available stay-green sources (B 35, SC 56 and E 36-1) for which QTL have been mapped to date, QTL have been identified on all 10 sorghum linkage groups (Reddy et al. 2006). Although it has been possible to identify several regions of the sorghum genome that condition the expression of drought tolerance, little information is available on the expression of individual QTL. Therefore, analysis of near-isogenic lines (NIL) that differ at QTL of interest can be an effective approach for the detailed mapping and characterizing the individual QTL (Ejeta et al. 2000). Tuinsta et al. (1998) developed a procedure for developing NIL for any region of the genome that can be analyzed with molecular or other genetic factors to identify heterogeneous inbred families that are isogenic at most loci in the genome from NIL that differ in marker linked to QTL of interest. Tuinsta et al. (1996) used these NIL to test the phenotypic effects of three different genomic regions associated with various measures of agronomic performance in drought and/or nondrought environments. In most cases, NIL contrasting for a specific locus differed in phenotype, as predicted by QTL analysis. NIL contrasting at QTL marker tM5/75 indicated large differences in yield across a range of environments. Further analysis indicated that differences in agronomic performance might be associated with a drought-tolerance mechanism that also

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influences heat tolerance. NIL contrasting at QTL marker tH19/50 also differed in yield under drought and nondrought conditions. The analysis of these NIL indicated that these differences might be influenced by a drought-tolerance mechanism that conditions plant water stress and expression of stay green. NIL contrasting at QTL marker t329/232 differed in yield and seed weight. These differences appear to be the result of two QTL that are closely linked in repulsion phase. Molecular marker-assisted backcross (MABC) introgression of the stay-green QTL from B 35 and E 36-1 donors into R 16 (a popular postrainy season cultivar in India); ISIAP Dorado (a popular cultivar across Latin America); and IRAT 204, ICSV 111 and S 35— a selection from ICSV 111 (all popular cultivars in several African countries) is under way to develop improved sorghum lines for drought tolerance (ICRISAT 2005). At present, the derivatives are in advanced backcross generations. The identification of several (e.g., IS 22380, QL 27, QL 10, E 36 R 16 8/1) tropically adapted lines with stay-green expression equivalent to those of the best temperate lines B 35 and KS 19 (Mahalakshmi and Bidinger 2002) is further expected to hasten the process of mapping QTL and their subsequent introgression into agronomically elite lines. Among the other biotechnological methods, it was reported that increase of glycinebetamine (GB) synthesis improved drought tolerance in cotton (Sulian et al. 2007) and efforts were also made in sorghum (Yang et al. 2003) to study the variability for GB in 240 genotypes at post-flowering stage. The total quatenary ammonium compound (QAC) levels in the betain fraction of the flag leaves were found to range from as low as 0.1 mmol g1 fresh weight (FW) to as much as 33 mmol g1 FW indicating high variability. Transgenic sorghums for drought tolerance are at infancy.

VIII. OUTLOOK Sorghum is one of the most important food-fodder-feed crops in the SAT worldwide. Sorghum with greater water use efficiency is relatively more drought tolerant than maize, making it a logical cereal to support the tropics. Genetic enhancement of sorghum for drought tolerance would stabilize productivity trends and contribute to sustainable production systems in drought-prone environments. The extent of grain yield losses due to drought stress depends on the stage of the crop and the timing, duration, and severity of drought stress. However, four growth stages in sorghum have been considered as most


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vulnerable: germination and seedling emergence, postemergence or early seedling stage, midseason or preflowering, and terminal or postflowering stages. Sorghum responses to moisture stress at these four growth stages have been characterized. Variation in these responses has been observed and found to be heritable. Genotypic differences are observed, for drought tolerance through appropriate screening techniques for each of the growth stages, separately (Reddy 1986; Blum et al. 1989; Muchow et al. 1996; Hausman et al. 1998; Borrell et al. 200a,b; Harris et al. 2007). Of the several mechanisms to circumvent drought stress in sorghum, drought escape (related to maturity duration), drought avoidance (maintenance of higher LWP), and drought tolerance (related to OA) are considered as most important and have been characterized. However, LWP and OA do not relate to the field response well enough to merit selection based on them; in addition, the screening techniques developed based on them are not cost effective. Stay-green trait, which is known to confer postflowering drought tolerance, has been exploited to enhance postflowering drought tolerance in sorghum. In most sorghum improvement programs including that at ICRISAT, growth stage–specific breeding for drought tolerance (which involves screening in specific drought and yield potential environments) is used to breed sorghum that can yield well in high-yield-potential environments as well as in drought-prone environments at specified growth stages. Since hybrids exhibited relatively better performance than OP cultivars for grain yield under water-limited environments, hybrid cultivar development (including their parents) should be given strategic importance for enhancing sorghum production in water-scarce environments. The progress in enhancing drought tolerance in sorghum through conventional approaches is limited by the quantitative inheritance of drought tolerance and yield coupled with complexity of timing, and severity and duration of drought. Biotechnology appears to offer promising tools, such as marker-assisted selection, for genetic enhancement of drought tolerance in sorghum. Four stable and major QTL were identified for stay-green trait and are being introgressed through MAS into elite agronomic backgrounds at ICRISAT, QDPI, Purdue University, and Texas A&M University in the USA. The integration of sorghum genetic map developed from QTL information with the physical map will greatly facilitate the mapbased cloning and precise dissection of complex trait like drought in sorghum. Sorghum is a drought tolerant crop with compact genome size (2n ¼ 20) can be an excellent model for identifying genes involved in drought tolerance to facilitate their use in other crops.

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ACKNOWLEDGMENTS We gratefully acknowledge the grants from Sorghum Hybrid Parents Research Consortium in partial support of preparation of this chapter.

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4 Breeding for Resistance to Stenocarpella Ear Rot in Maize Johannes D. Rossouw Monsanto Singapore Co (PTE) Ltd. 151 Lorang Chuan 06-08 New Tech Park Singapore 556741 Z. A. Pretorius Department of Plant Sciences University of the Free State Bloemfontein, 9300 South Africa H. D. Silva Monsanto Brazil Rodovia Uberlaˆndia-Araxa´ Uberlandia, 38405232 MG, Brazil K. R. Lamkey 2101 Agronomy Hall Iowa State University Ames, Iowa, 50011-1010 USA

I. INTRODUCTION II. DISTRIBUTION AND IMPORTANCE A. United States B. South Africa C. Brazil D. Argentina E. Mexico F. Occurrence of Diplodiosis


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J. D. ROSSOUW, Z. A. PRETORIUS, H. D. SILVA, AND K. R. LAMKEY

III. PATHOGEN A. Taxonomy B. Symptoms C. Variability D. Host Range IV. EPIDEMIOLOGY V. DISEASE MANAGEMENT A. Crop Rotation B. Cultural Practices C. Stress D. Fungicides E. Genetic Resistance 1. Inheritance of Resistance 2. Breeding for Resistance VI. SUMMARY AND CONCLUSION LITERATURE CITED

I. INTRODUCTION Stenocarpella maydis (Berk.) Sutton is the most important ear rot pathogen in nearly all countries where maize is produced. Maize breeders have traditionally experienced difficulties in improving resistance to ear rot caused by S. maydis, also known as Diplodia ear rot, in public and private breeding programs. This is mainly due to the fact that yield is the breeder’s main objective, and, resistance to S. maydis is quantitatively inherited, screening techniques are expensive, labor intensive, and time consuming. Furthermore, known sources of resistance are mostly tropical and large genotype-byenvironment effects usually are involved. As for any quantitative trait, selection for resistance needs to be done in every generation to ensure maximum genetic gain. This means annual inoculation of field plots and assessment of expression of resistance levels. All of this will add to the development cost of inbreds and hybrids, and usually a yield penalty is paid in exchange for improved levels of resistance. If the S. maydis scenario is considered globally, the economic importance of the disease is increasing and of concern in maize production worldwide. The objective of this chapter is to emphasize the growing importance of S. maydis and to document what is known of the disease globally. We also address what needs to be done to improve resistance through breeding to ultimately prevent loss in global maize production.

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II. DISTRIBUTION AND IMPORTANCE A. United States S. maydis has been recognized as an important disease of maize in the United States for nearly a century. Burrill and Barrett (1909) estimated losses in the Illinois maize crop due to ear rots at 4.5% in 1906 and 2% in 1907. They attributed most of the damage, as much as 90%, to Diplodia ear rot. Later reports indicated that S. maydis was the most important ear rot pathogen of maize during the 1950s (Holbert et al. 1924, Koehler and Holbert 1930, Koehler 1959). Starting in the 1930s, an increased emphasis was placed on identifying resistance to Diplodia. A number of inoculation techniques were described for both Diplodia ear and stalk rot (Ullstrup 1949; Koehler 1959). Both public and private breeding programs used these techniques to enhance Diplodia resistance in the maize germplasm base. The resulting resistant hybrids contributed to the decline of Diplodia as a major pathogen of maize. In 1976, Hooker and White noted that Diplodia ear and stalk rots had been minor problems during the preceding 10 years. They attributed this to the development of Diplodia stalk and ear rot resistance and the deployment of resistant hybrids. During the 1970s and 1980s, Diplodia was of minor importance. During the 1990s, however, localized epidemics of the disease occurred in limited areas of southern Illinois, Indiana, Ohio, and central Missouri. The warm, humid conditions in these areas favored the development of Diplodia. Although the disease was often noted in individual fields, it was not widespread. More widely use of reduced tillage in these areas and a reduction to breed for resistance to Diplodia allowed the pathogen to become reestablished as a stalk and ear rot pathogen. The result was a widespread epidemic of Diplodia ear rot in the summer of 2000. The disease was present in much of the central to south-central U.S. corn belt and frequently observed in the states of Ohio, Indiana, Illinois, Missouri and Iowa. In these areas, fields containing more that 50% mummified ears were reported (J. M. Perkins pers. comm., Monsanto, USA). S. maydis remains a problem in the United States where climate and reduced tillage practices favor development of the disease. With breeding efforts focusing on other maize diseases, such as gray leaf spot (Cercospora zeae-maydis), anthracnose stalk rot (Colletotrichum graminicola), and Fusarium ear rot (Fusarium verticillioides), the S. maydis resistance level in current hybrids will continue to erode.

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Consequently, potential exists for S. maydis ear rot to become a devastating disease over a large geographical area of the United States (J. M. Perkins pers. comm., Monsanto, USA). B. South Africa In South Africa, the unpredictability of epidemics is a major problem. Control measures are primarily preventive, and it is almost impossible to predict epidemics. These epidemics usually occur during earlyseason drought and late-season rains. Epidemics were noted in the 1980–1981, 1985–1986, 1986–1987, 1999–2000, and 2003–2004 seasons (B. C. Flett pers. comm., ARC, Grain Crop Institute, Potchefstroom, South Africa). Although favorable climatic conditions are the primary reason for occurrence of epidemics, the introduction and increased use of minimum and reduced tillage for monoculture maize production ensures the presence of inoculum. In addition, the extensive use of certain susceptible lines, mainly U.S. corn belt material (e.g., B73) increased susceptibility. Changes in pathogen virulence or aggressiveness were also considered to play a role. Over the last 15 years, there have been distinct improvements in the general resistance of maize hybrids to ear rot, particularly those bred and tested locally. More recently, the importing of maize hybrid seed and germplasm has resulted in an increased susceptibility particularly in ultra-short-season hybrids because of the susceptible heterotic backgrounds used. The production of these hybrids caused the buildup of inoculum and spread of the disease in the maize-growing areas. S. maydis caused an annual loss of R400 million to the South African Maize Board during the epidemic seasons of 1985–1986 and 1986–1987. These losses were due to reduced grain yield and quality and diplodiosis, an endemic neuromycotoxicosis of domestic ruminents grazing on infected maize stubble. During the 1997 to 2000 seasons, losses varied from R1.5 million to R40 million per annum (B. C. Flett pers. comm., ARC, Grain Crop Institute, Potchefstroom, South Africa). The ear rot epidemics of the 1980s exposed the genetic vulnerability of the breeding material used in South African maize hybrids at that time and resulted in considerable research. This research was focused on identifying inoculation techniques, sources of resistance, selection criteria, inheritance of resistance and genotype environment interaction. Different inoculation techniques were compared, and the technique of putting ground infected kernels in the leaf whorl was recommended (Bensch 1995a). Van Rensburg and Ferreira (1997) studied isolate differences and sources of resistance. They found that under South


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African conditions, inbreds B37, DO620Y, and E739 showed levels of resistance superior to that of the U.S. resistant standard H111, but that isolates provided inconsistent infection levels over years. All interactions were significant, with seasonal effects predominant over the genotype isolate interaction. Rossouw et al. (2002a,b) evaluated the different selection criteria to screen germplasm and studied the inheritance of resistance. The incidence of rotten ears was the most practical and reliable method to evaluate lines. In these studies, resistance was controlled by additive gene effects with low dominance and interaction effects. Sufficient general combining ability (GCA)) and specific combining ability (SCA) effects exist in the resistant inbreds to serve as donors of resistance genes. DO620Y and MON1 showed good GCA and B37 and E739 showed good SCA for resistance. Genotypes environment effects were pronounced, indicating the necessity to screen across environments. In South Africa, S. maydis remains problematic in commercial maize production because of maize monoculture, reduced tillage (in particular no-till), stressful environmental conditions, and the planting of susceptible hybrids due to the widespread use of susceptible U.S. germplasm. C. Brazil Over the last 30 years, Brazil has experienced significant losses in maize production in due to several diseases, in particular ear rots caused by S. maydis and S. macrospora. Increased use of no-till practices contributed to disease development. S. maydis is a problem in the southern and central (Cerrados Highlands, altitude above 700 m) regions of Brazil. It is problematic on 6 million ha of which 3.5 million ha show high levels of infection. Incidence of rotted ears could be as high as 45% but are, in general, about 12%. With a mean yield of 5.5 tons/ha, at an estimated price of US $100 per ton, the total losses per season are estimated to be US $231 million. S. maydis will increase in Brazil in future, with a strong impact on yields and grain quality (I. Resende pers. comm., Monsanto, Brazil). D. Argentina Although it is well known that S. maydis is present in maize fields in Argentina, little is known about the spread of the disease, susceptibility or resistance of commercial hybrids, and yield losses. The disease has been reported in all areas where maize is grown, but in the last two years, it has been observed more frequently in the Argentine maize belt area, from the area south of Santa Fe to north of Buenos Aires.

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Ear rot infection levels in the maize belt ranged from 2% to 8% across hybrids and seasons. S. maydis stalk rot data from the 2003 season suggested that the levels would be very similar to those reported for S. maydis ear rot. A case of diplodiosis, where cattle grazed on a maize field infected with S. maydis, resulted in the death of some 10 steers of the 27 that ate the infected grain in 2003 (Odriozola et al. 2005). Given the expansion of the no-till method of maize production, it is expected that problems associated with S. maydis will increase in frequency and intensity. Use of genetic resistance and cultural practices would be of primary importance to control the disease, since no cost-effective treatment is available to control the pathogen (J. Cerono, pers. comm., Monsanto, Argentina). E. Mexico Despite being prevalent in all maize-growing regions, S. maydis incidence appears to be higher in the tropical and central areas of Mexico. These areas are characterized by abundant rainfall, high humidity, reduced tillage, and monoculture production systems. Incidence of ear rots due to Gibberella zeae and/or Fusarium verticillioides in the tropical and central areas is considered to be higher and more destructive than S. maydis. In Mexico, ear rot varies considerably in severity in different years and in different locations, indicating unequal influence of environmental conditions on the disease. S. maydis is usually found close to sources of inoculum and rarely occurs where inoculum has been buried by tillage or reduced through rotation with other crops. Open husk cover and ears that are in an upright position until late in the season have been reported to enhance susceptibility, whereas tight husk coverage and hard endosperm kernels are associated with reduced susceptibility to S. maydis. However, S. maydis has not yet become of sufficient economic importance to stimulate research for resistance and incorporation of resistance into adapted hybrids (C. Leon-Ochoa pers. comm., Monsanto, Mexico). F. Occurrence of Diplodiosis In addition to yield loss and quality problems in grain, S. maydis also causes diplodiosis, which is an endemic neuromycotoxicosis of domestic ruminants grazing on infected maize stubble (Prozesky et al. 1994).


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Diplodiosis is not only of historical interest but is also currently one of the most common nervous disorders of livestock in southern Africa. Although S. maydis is present wherever maize is grown, authenticated outbreaks of diplodiosis have apparently not been reported outside southern Africa until a 2003 report from Argentina 2003 (Kellerman, Coetzer, and Naude 1988; Odriozola et al. 2005). According to Kellerman et al. (1985), the clinical signs of diplodiosis are ataxia, paresis, and paralysis. Although mortality may be high, the prognosis is good if livestock are removed from the toxic field as soon as the first signs appear. Recovery is usually complete. However, in one experiment, an animal became permanently paralyzed and another developed an irreversible change in gait after being dosed with cultures of the fungus. S. maydis cultures have been found to be extremely fetotoxic to ruminants. With the exception of diplodiosis, mycotoxicoses are not considered to be an important cause of reproductive failure in livestock (Prozesky et al. 1994). Dosing trials on ewes revealed that 66% of the offspring of dams exposed to cultures of S. maydis in the second trimester of pregnancy, and 87% of ewes exposed in the third trimester, were either stillborn or died soon after birth. Histopathological examination of the central nervous system of the affected lambs revealed a status spongiosus in the white matter, similar to that of stillborn lambs and calves in flocks and herds grazing on harvested lands naturally infected by ear rot. Lambs of control ewes and those of ewes in the first trimester of pregnancy showed no ill effects (Kellerman et al. 1991).

III. PATHOGEN A. Taxonomy The principal pathogen is the anamorphic ascomycetous fungus Stenocarpella maydis (Berk.) Sutton first described by Berkeley as Sphaeria maydis from an Ohio, United States, specimen in 1847, and then as Stenocarpella macrospora (Earle) Sutton 50 years later in Alabama (Latterell and Rossi 1983). Other synonyms for S. maydis are Diplodia maydicola Speq., Diplodia maydis (Berk.) Sacc., Diplodia zeae Le´v., Diplodia zeae-maydis Mekht., Hendersonia zeae (Le´v.) Hazsl., Macrodiplodia zeae (Le´v.) Petr. and Syd., Phaeostagonosporopsis zeae (Le´v.) Woron., Sphaeria zeae Curr., and Sphaeria zeae Schwein (www.indexfungorum.org, 16 January 2007).

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B. Symptoms S. maydis symptoms are expressed in different ways, which makes accurately assessment of levels of infection difficult. Initial symptoms are yellowing and drying of infected leaves. White fungal mycelium starts at the base of the ear, may cover the entire ear, and produces pycnidia at the kernel base. These symptoms are also called mummification. In South Africa, another symptom is discolored kernel embryos, also termed hidden diplodia, that can be seen only if the ear is broken (Flett 1999). This concealment of symptoms makes it difficult to determine the true state of S. maydis infection without opening the ear, and therefore requires labor-intensive and timeconsuming screening procedures. Primary infection occurs from the shank of the ear rather than the ear tip and is caused by a nonuniform mycelial ramification of S. maydis through the kernel endosperm. Sometimes rot appears to begin at the silk end of the ear, probably associated with ear worm feeding, or sometimes at the middle of ear, when a lesion is centered on the husk at this position. Due to a reduction in kernel moisture, the rate of mycelium ramification is slower in hybrids infected late in the season. Infection is more visible in kernels than in pith tissue, which is colonized only when the entire cob is heavily infected with S. maydis (Koehler 1942). Bensch (1995b) confirmed that S. maydis prefers to colonize the embryo and pedicel of the kernel. The pathogen grows from the sclerenchymatous tissues of the cob into the kernels during the early stages of ear development. Embryos are then colonized before the black layer is formed at the pedicel. The black closing layer of the seed-hilar orifice is impervious to fungi. When hilar layer formation is incomplete or delayed, hyphae could pass around the end of the testa and enter the embryo by way of the hilar orifice. The delayed or less effective closing of the hilar orifice and delayed maturation of the caryopsis may increase susceptibility to ear rot (Johann 1935). Enzymatic activities involved in the mycelial ramification in maize stalk tissue, which disrupt cell wall structures through cellulase production, have been reported for S. maydis (BeMiller, Tegtmeier, and Pappelis 1969). Bensch and Van Staden (1992) conducted a histological study on S. maydis and suggested that, given the lack of differences observed in S. maydis germination and growth between susceptible and resistant maize plant material, resistance to the pathogen is not regulated at the epidermal surfaces of the stalk, ear leaf sheath, or shank. Based on the degree of degradation of the cell walls of the plant tissue, it was suggested that enzymes are involved in the


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infection of maize by S. maydis. This also explains the occurrence of intra- and intercellular growth of S. maydis in maize plant tissue. Similar to many other fungi, S. maydis produces polygalacturonase at early stages of infection to degrade the pectin component (complex polysaccharide) of plant cell walls. Polygalacturonase-inhibiting proteins (PGIPs) have been identified in the cell walls of many plant species (Favaron, Castiglioni, and Di Lenna 1993). PGIPs contribute to the defense response by modulating the activity of fungal polygalacturonase to release active oligogalacturonides that delay the rapid degradation of pectin to inactive monomers (Hahn et al. 1989). Berger et al. (2000) indicated that an extract containing PGIP from Phaseolus vulgaris inhibited 66% of S. maydis polygalacturonase. Maize transformation with the pgip-1 gene needs to be done to address whether the in vitro evidence can be reflected by increased resistance to S. maydis in vivo. C. Variability Several reports of variability in S. maydis exist. Differences were found in mycelial growth temperature requirements and in pathogenicity, with isolates being more virulent at the locality of origin (Young et al. 1959). Aversions and inhibition of growth at the edge of different cultures have been observed. In addition, variation occurs among isolates for aggressiveness, phenotypic color, and pycnidiospore production. Pycnidiospore production is, however, not a stable phenotype due to the influence of environment (Hoppe 1936; Kappelman, Thompson, and Nelson 1965; Maxwell and Thompson 1974; Dorrance, Miller, and Warren 1999). Significant differences occurred for stalk rot severity between combinations of maize inbreds and testers inoculated with isolates differing in virulence (Maxwell and Thompson 1974). However, using 10 enzymes, a collection of S. maydis isolates from the United States and South Africa revealed no significant isozyme polymorphisms (Dorrance et al. 1999). In one study, the rating of maize resistance depended on environmental conditions and aggressiveness of the pathogen strain (Van Rensburg and Ferreira 1997). In contrast to this, no differences were found in the incidence of ear rot caused by five isolates collected from various maize production areas in the United States (Ullstrup 1949). Isolate differences should be considered when using artificial inoculation during screening for S. maydis. Maxwell and Thompson (1974) inoculated maize plants with a highly and a weakly virulent isolate of Diplodia zeae. Significant differences between lines were

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observed for two combinations, resistant lines by the highly virulent isolate and susceptible lines by the weakly virulent isolate. For proper discrimination between susceptible and resistant lines, a balance between host resistance and isolate aggressiveness should be maintained. If not balanced, phenotypes may become inadequately infected or overwhelmed with ear rot. D. Host Range The primary hosts of S. maydis are maize (Zea mays L.), an Arundinaria sp. (Sutton and Waterston 1966, Flett 1991), and teosinte (Zea diploperennis Iltis) (Morant, Warren, and Von Qualen 1993). The Arundinaria sp. is commonly known as river or giant cane and is native to North America. Maize is the only commercial host for the fungus, making crop rotation an excellent practice to reduce inoculum buildup (Sutton and Waterston 1966; Sutton 1980; Flett 1991).

IV. EPIDEMIOLOGY Given a susceptible host and presence of inoculum, the environment is a critical factor for disease and possibly epidemic development of S. maydis. Resistant reactions of hybrids over locations and seasons vary due to climatic conditions and inoculum potential (Flett and McLaren 1994). A complex relationship also exists in which the crop phenotype and the pathogen are independently and differentially affected by environmental conditions favorable to a particular isolate of the pathogen. The most consistent infection levels were recorded in seasons with extreme droughts after flowering, high summer temperatures, and late-season rains; the lowest average infection rates occurred in years with cool and humid conditions (Van Rensburg and Ferreira 1997). Under natural infection, hybrids may respond differently to environmental conditions, resulting in variation for resistance between years (Hooker 1956; Thompson, Villena, and Maxwell 1971). Similarly, hybrid response to S. maydis was not consistent over locations and/or seasons, and ‘‘resistant’’ hybrids showed severe ear rot symptoms from time to time (Nowell 1997; Van Rensburg and Ferreira 1997). Inconsistent results have been obtained across locations and seasons in both natural and controlled infections (B. C. Flett pers. comm., ARC, Grain Crop Institute, Potchefstroom, South Africa). This variation has prevented reliable discrimination between resistance and susceptibility over a long period of time and has made it difficult to determine


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if severity of S. maydis ear rot is genetically controlled or rather the result of climatic conditions or inoculum potential at a specific time. Locations in the drier western maize production areas of South Africa are less suitable for disease development than locations in the eastern areas, where more moisture is available at the critical plant growth stages. Environmental variation also compounds uncertainty about the inheritance of resistance (Rossouw et al. 2002b). V. DISEASE MANAGEMENT A. Crop Rotation Crop rotation has been used to control plant diseases effectively, primarily by reducing the amount of pathogen inoculum. The success of crop rotation depends on the inability of the pathogen to infect an alternative host and/or the time needed for inoculum reduction in the field (Curl 1963). S. maydis inoculum builds up and overwinters on maize stalk stubble retained on the soil surface or partially buried (Flett 1991). Since maize is the only crop host for the fungus, any other crop can be used successfully in crop rotation systems. Flett et al. (2001) found that wheat, soybean, and peanut were the most, and sunflower the least, effective rotation crops for reducing S. maydis. However, in countries such as South Africa, where maize is the commercial crop that yields the highest income, farmers are reluctant to plant alternative crops that yield a lower income. Even with substantial losses in yield and quality, farmers will still make more profit by planting monoculture maize rather than having a crop rotation system. B. Cultural Practices The utilization of cultural practices such as no-till and minimum tillage will result in an increase in inoculum due to more stubble being left on the soil surface where the pathogen can overwinter (Latterell and Rossi 1983). Significantly less S. maydis ear rot was observed in fields where the infected stubble was plowed under, in comparison with minimum and no-tillage practices. Furthermore, a linear relationship was found between severity of S. maydis ear rot and surface stubble mass. Therefore, moldboard plowing was recommended for long-term cultural control (Flett and Wehner 1991; Flett 1999). If disease management is to be achieved through cultural practices, it should be kept in mind that changes in tilling practices should go along with increased host resistance toward S. maydis and preferably other

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pathogens that overwinter on maize stubble. It is generally accepted that a shift toward conservation tillage will increase inoculum buildup of most foliar fungal pathogens as well as the ear rot pathogens. C. Stress Any form of stress endured by plants could predispose them to S. maydis infection. Precautionary measures include ensuring optimum nutrient levels in the soil, avoiding abnormally high plant populations that could increase water stress, and planting at optimum times to reduce cold and heat stress. Other conditions that the grower cannot control are hail, wind damage on sandy soils, and severe drought periods during the season, especially during anthesis, and late-season rains. D. Fungicides S. maydis can be controlled successfully with applications of benomyl, mancozeb, or a mixture of benomyl and carbendazim fungicides (Warren and Von Qualen 1984; Beukes and Flett 1992; Marley and Gbenga 2004). However, chemical control is often not economically justified due to low yields and the relatively low producer prices of maize. Chemical control is therefore regarded as the last option. In environments where high yields are achieved, farmers can justify the costs of spraying fungicides that can prevent and reduce both foliar disease and ear rots. However, worldwide, most maize is produced on marginal soils, and spraying is not economical. E. Genetic Resistance The only long-term and cost-effective solution to manage S. maydis is through genetic resistance and the use of resistant hybrids (Hooker and White 1976; Rossouw 2001). Resistant hybrids in combination with sanitation practices can provide effective control (Flett 1999). Quantitative resistance to S. maydis remains the main focus in breeding programs. Most germplasm used in breeding programs is susceptible to S. maydis, which makes it difficult to breed for resistance in elite inbreds (Dorrance et al. 1998; Rossouw et al. 2002b). 1. Inheritance of Resistance. The inheritance of resistance to ear rot is complex, and various mechanisms have been documented: Dominant inheritance (Koehler and Holbert 1930) Additive inheritance with genotype by environmental interaction having a large influence on the expression (Koehler 1953)


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Quantitative, partially dominant resistance (Wiser et al. 1960) Recessive resistance (Wiser et al. 1960) Quantitatively inherited resistance with additive effects important (Villena 1969; Das, Chattopadhyay, and Basak 1984; Dorrance et al. 1998) Resistance controlled by additive gene effects with low dominance and interaction effects (Rossouw et al. 2002b) Tight husk cover reduced susceptibility (Boewe 1936), and a relationship was found between ear declination and ear rot (Koehler 1959). Hanging ears had less ear rot than those erect in relation to the plant stalk. Closely associated with the effect of ear declination on ear rot was the protection of the ear by the husks. Ears with loose husks and/or those that were open at the tips had significantly more ear rot than tightly covered ears, especially in association with upright ears. Ears in which husks were opened by hand had more ear rot than the closed ones. It was also found that lodged plants with ears that touched the ground had significantly more ear rot (Boewe 1936; Koehler 1959). Ferreira (1994) indicated that ear declination and prolificacy (number of ears per plant) were correlated to ear rot resistance under South African conditions. No correlation was found between infection and shank length. Rossouw et al. (2002b) did not find any correlation between S. maydis and agronomic characteristics such as ear declination and husk cover or tightness. The upright orientation of ears was the result of infection, which made them lightweight. Thus, ear declination was a consequence, not a predisposing condition, of ear rot. No correlation has been found between inheritance of S. maydis ear rot and S. maydis stalk rot or any other ear rot pathogen (Hooker 1956; Thompson et al. 1971). A relationship exists between pith parenchyma cell death and infection of maize ears by S. maydis. A hybrid with a slow death rate of the parenchyma cells will reduce the infection of S. maydis (Pappelis et al. 1973). 2. Breeding for Resistance. Infection levels vary from year to year and from environment to environment, making it difficult to assess levels of resistance or susceptibility among lines on the basis of natural epidemics. Because natural infection is so unreliable, it was important to develop an artificial inoculation technique to induce disease and allow discrimination among lines. The biggest limitations of artificial inoculation techniques are their resource intensity and the time required to measure the expression of levels of resistance or susceptibility. This is the main reason

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why maize breeders have given less attention to S. maydis or any of the other ear rots compared to foliar diseases. Artificial Inoculation. According to Ullstrup (1949): The success and usefulness of a method of inoculating corn ears with Diplodia zeae depend on several criteria: (1) Clear differentiation between resistant and susceptible host materials (2) Disease reactions of inbred lines and hybrids comparable to those experienced under natural epidemics of the disease (3) Reproducibility of results from year to year, and no great effect of environment on the relative disease reaction of the host plants (4) Simplicity of execution and freedom from complicated and laborious procedures and (5) Simulation of those phenomena attending the natural mode of infection by the parasite Based on Ullstrup’s criteria, pathologists and breeders have achieved great successes in developing methods of artificial inoculation for S. maydis to simulate natural epidemics. However, environmental influences on the disease reaction of the host plant remain large, and inoculation and assessment are complicated and labor intensive. Several methods of direct introduction of S. maydis into the ear result in successful infection. Methods include: Inserting infested toothpicks into the middle of the ear (Villena 1969; Chambers 1988; Drepper and Renfro 1990) Inserting infested toothpicks into the butt of the ear (Villena 1969) Drilling holes in the ear followed by insertion of infected grain or spores (Kang et al. 1974) Injecting a conidial suspension into the middle, tip, or butt, or both the tip and butt of the ear (Villena 1969; Laterall and Rossi 1983). Other techniques used in the past include brushing the ear or silk with an agar-spore slurry and dropping a spore suspension among the silks with a medicine dropper (Villena 1969). White et al. (2002) compared different methods and found that infested sorghum resulted in good separation between resistant and susceptible hybrids under irrigated conditions. In South Africa, different methods of ear rot inoculation have been developed in recent years, such as the toothpick method, spraying the ears with a conidial suspension, application of conidial suspension either in the leaf whorl or behind the ear between the husk and stalk


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without wounding, and the placing of ground, infected kernels in the leaf whorl. Ground infected kernels in the leaf whorl were the most consistent in inducing ear rot (Nowell 1992; Bensch 1995a). A revised technique was later used in which ground infected kernels were placed in the whorl of each plant 2 weeks prior to tasseling (Flett and McLaren 1994). The whorl method has the advantage that the inoculum is easy to apply and does not injure the plant. Following inoculation, overhead irrigation or sufficient rainfall is needed to create a microclimate for the spores to germinate and the disease to develop (Klapproth and Hawk 1991). With the whorl inoculation technique, disease incidence (number of ears infected as a percentage of total ears) among lines can be as high as 50%, but an incidence between 17% and 20% allows differentiation among degrees of resistance. This can be achieved with artificial inoculation at a wide range of experimental sites (Flett 1999). Hybrids react nonlinearly to different disease levels. When the incidence exceeds 50.6%, S. maydis could not be controlled by resistance (Flett and McLaren 1994). The pathogen grows well on maize ears above 21.5% grain moisture, indicating that the time of artificial inoculation is important for successful differentiation among lines (Koehler 1938; Ullstrup 1949; Kerr 1965; Fajemisin et al. 1987). To achieve this, it was suggested that inoculations should be done prior to 20 days after midsilk, before the kernel moisture decreases too much (Chambers 1988). Inoculations done at silking resulted in the highest disease incidence (Latterell and Rossi 1983; Bensch, Van Staden, and Rijkenberg 1992). Inoculations up to 15 days after silking provided less disease incidence. This shows that the pathogen has a critical period for colonization and symptom development before the plant reaches physiological maturity ( 50 days after silking) (Bensch et al. 1992). Therefore V8 through V12 appears the most effective time for inoculations (White et al. 2002). Disease Measurement. Ullstrup (1949) described three methods of disease measurement: percentage rotted ears, percentage rotted kernels (by mass) in a representative 250 g sample of shelled maize, and a disease index, provided results that were closely correlated. Percentage rotted ears was the easiest method of measurment. Nowell (1997) evaluated four methods for ear rot assessment: a nonlinear scale method, percentage rotted ears, percentage rotted kernels in a representative sample, and a method in which infected ears were categorized according to their causal organism (Stenocarpella, Fusarium, Gibberella, stalk borer, and other). The percentage rotted ears method was the most practical, yet accurate, method for ear rot assessment. For

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hidden diplodia ear rot assessment, the grain should be removed from the base and tip of the ear to observe the symptoms. Both percentage rotted ears and disease severity were assessed in a diallel analysis (Dorrance, Hinkelmann, and Warren 1998). Only percentage rotted ears was used for data analysis because disease severity parameters that indicated the colonization of the ears by S. maydis were limited by grain moisture and not by host resistance. Percentage rotted ears, percentage rotted kernels by mass, and percentage rotted kernels by number are effective for disease measurement or as selection criteria when breeding for resistance to S. maydis. All three methods were highly correlated and heritable. Percentage rotted ears is the preferred methods because it is practical, simple, and less work than the other methods (Rossouw, Van Rensburg, and Van Deventer 2002a). Another important factor is the sample size. Due to poor growing conditions, Dorrance et al. (1998) sampled only 10 to 15 plants per plot. Van Rensburg and Ferreira (1997) sampled 20 plants per plot in their study. Rossouw et al. (2002a) and Rossouw et al. (2002b) used 44 plants per plot as their sample size. The preferred disease evaluation is based on a percentage; therefore, as many plants as possible need to be sampled. It is important to sample the primary ear on each plant because the measurement method of percentage infected ears per plot will be influenced by the larger sample size of prolific lines. Sources of Resistance. To successfully breed for resistance, breeders need to identify sources of resistance and continue to search for new sources in germplasm that can be adapted for local conditions or incorporated into locally adapted germplasm (Van der Plank 1984). For S. maydis, the donors are usually tropical, which makes introgression and improvement of resistance more difficult in temperate and subtropical areas. Variation was found for levels of resistance and susceptibility to S. maydis in a collection of inbreds (Wiser et al. 1960; Thompson et al. 1971). These results were interpreted as the ability of the resistant lines to resist initial infection rather than differences in the development rate of the fungus (Wiser et al. 1960). High lysine maize in the United States was shown to be particularly susceptible to S. maydis, depending on the background that the opaque2 gene was introduced into (Ullstrup 1971). In contrast, high lysine maize inbreds and hybrids have been developed in South Africa that have important levels of resistance to S. maydis (Gevers 1989). White-grained hybrids are generally more resistant to ear rot than yellow-grained hybrids (Rheeder 1988; Gevers 1989; Nowell 1992;


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Table 4.1. GCA effects for different S. maydis characteristics using Griffings Model 1, (parents, F1s and reciprocals) for Petit, Hillcrest and Lichtenburg (1999/2000). Genotypes

Locations

I137TN

Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg Petit Hillcrest Lichtenburg

B73

B37

F2834T

MON1

DO620Y

E739

H111

Mo17

ARC1

LSD(0.05)

REz

RMy

RKx

RSw

3.63 2.84 0.80 16.16 3.89 4.21 9.20 1.27 0.60 9.41 0.83 1.62 5.64 10.42 2.20 11.33 12.09 2.28 1.89 8.50 0.15 4.62 6.17 0.44 4.23 4.29 0.94 6.93 17.30 2.34 9.34 5.21 2.48

1.94 1.39 1.25 10.11 0.70 2.61 5.51 1.12 0.18 6.43 1.01 1.31 2.53 3.59 1.08 6.54 4.89 0.92 3.58 1.99 0.08 4.74 2.45 0.22 4.99 2.75 1.26 5.16 7.46 1.03 6.26 3.91 2.14

2.13 1.60 1.00 11.67 0.65 3.06 7.89 0.71 0.35 9.32 0.78 1.42 2.77 3.49 1.30 7.86 5.05 0.87 4.70 1.90 0.16 6.18 1.66 0.11 5.76 2.93 1.29 7.64 7.50 1.12 7.92 4.43 2.11

0.23 0.13 0.72 1.25 0.22 1.24 0.18 0.33 0.16 1.90 0.69 2.09 0.30 1.74 1.06 0.05 0.41 0.86 0.15 1.09 0.76 1.58 0.34 0.74 1.17 0.41 1.21 0.72 2.06 1.92 1.67 1.26 1.46

RE ¼ percentage rotted ears. RM ¼ percentage rotted kernel mass. x RK ¼ percentage rotted kernels. w RS ¼ infection severity index. Source: Rossouw et al. 2002b. z

y

Ferreira 1994; Flett and McLaren 1994; McLaren and Flett 1994). For yellow-grained inbreds, the heterotic groups F (F2834T) and M (M37) produced significantly more resistant inbreds than the other groups, with more resistance in the F group. Derivatives from Reid, B73, or I137TN were responsible for the susceptibility in South African

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Table 4.2. Comparison of general combining abilities (GCA) of maize inbreds tested in 1994 and 1995. 1994 Parent B68 H111 H99 B37 MS MB271 VA26 B73

1995 z

Parent

a a ab ab ab b b b

B68 B37 MS H111 VA26 H99 B73 MB271

GCA 4.27 3.20 2.04 1.51 0.68 3.62 3.98 4.09

GCA 5.48 3.82 3.52 2.03 0.44 1.29 5.42 7.69

a ab ab ab bc bc c c

z Numbers followed by the same letter do not differ significantly at P ¼ 0:01. Source: Dorrance et al. 1998.

germplasm, along with the direct introduction of U.S. germplasm in hybrids that were not selected and screened for S. maydis resistance. Opaque-2 genotypes provided the most inbreds with resistance in South Africa (Gevers 1989; Gevers, Lake, and McNab 1992). Sufficient SCA effects exist in certain inbred lines, in particular B37 (Iowa Stiff Stalk Synthetic) and E739 (Kroonstad Robyn), that they can serve as donor parents for the improvement of susceptible germplasm. Inbreds DO620Y and MON1 showed good GCA (Table 4.1) and Inbreds B37 (Iowa Stiff Stalk Synthetic) and H111 (Mayorbella/B37//B37) have large GCA effects (Table 4.2), whereas SCA effects are significant only in some years (Dorrance et al. 1998). The South African inbred D940Y showed high SCA for resistance whereas epistasis in ear rot resistance was present in a number of other inbreds (McLennan 1991). Das et al. (1984) found SCA to be more important than GCA in a diallel study on open-pollinated maize varieties. Van Rensburg et al. (2003) developed new S5/F7 inbreds with high levels of resistance through the use of donor parents in a normal breeding program. They stated that future improvement of resistance in germplasm can be even more effective by means of marker-assisted selection, provided that suitable markers are found.

VI. SUMMARY AND CONCLUSION Stenocarpella ear rot is apparently becoming a greater problem in countries where it was historically of minor economic importance. Use of crop rotation, cultural practices, and fungicides to control the disease


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will make maize production less profitable. The only real long-term solution is genetic resistance. In reviewing what global S. maydis research, it is clear that we have to focus on incorporating resistance into elite germplasm and create economic value and benefits for all maize farmers. Most research on Stenocarpella ear rot has been focused to better understand the economic impact, the pathogen, epidemiology, and the role the environment plays, and disease management, through crop rotation, cultural practices, and fungicides. However, little has been done to improve our understanding of inheritance and to improve resistance in elite germplasm. Due to the smaller economic impact of the pathogen on yield and quality on the maize production of the past few decades in the United States, most research has been conducted in countries such as South Africa, where economic losses have been experienced. The question is whether maize breeders have the resistance available to prevent epidemic outbreaks of the disease. Due to the complexity of the disease, inheritance of resistance, and the genotype-by-environment interaction, it will take too long to improve resistance incrementally through recurrent selection. The best and quickest approach to address the problem will be to use molecular breeding and to identify QTL for resistance. Plant breeders must: Find resistant lines. Identify QTL for resistance. Validate the expression of those QTL across environments and different heterotic backgrounds. Incorporate these QTL into elite, susceptible germplasm through a marker-assisted backcrossing program. The biggest challenge for breeders will be to implement an effective artificial screening method in environments where disease expression is good enough to discriminate among genotypes to identify significant differences and minimize the QTL-by-environment interaction. This should result in an economic benefit to both commercial and subsistence maize farmers worldwide. LITERATURE CITED Bemiller, J.N., D.O. Tegtmeier, and A.J. Pappelis. 1969. Effects of phenolics and indole-3acetic acid on production and activity of cellulolytic and pectolytic enzymes of Diplodia zeae. J. Phytopath, 59:674–676.

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Bensch, M.J. 1995a. An evaluation of inoculation techniques inducing Stenocarpella maydis ear rot on maize. S. Afr. J. Plant Soil 12:172–174. Bensch, M.J. 1995b. Stenocarpella maydis (Berk.) Sutton colonization of maize ears. J. Phytopath. 143:597–599. Bensch, M.J., and J. Van Staden. 1992. Ultrastructural histopathology of infection and colonization of maize by Stenocarpella maydis (¼ Diplodia maydis). J. Phytopath. 136:312–318. Bensch, M.J., J. Van Staden, and F.H.J. Rijkenberg. 1992. Time and site of inoculation of maize for optimum infection of ears by Stenocarpella maydis. J. Phytopath. 136:265–269. Berger, D.K., D. Oelofse, M.S. Arendse, E. Du Plessis, and I.A. Dubery. 2000. Bean polygalacturonase inhibitor protein-1 (PGIP-1) inhibits polygalacturonases from S. maydis. Physiol, Molec. Plant Pathol. 57:5–14. Beukes, H.M.B., and B.C. Flett. 1992. Efficacy of three fungicides in the control of S. maydis ear rot of maize. Proceedings of the 30th South African Society of Plant Pathology Congress, Cintsa, East London. Phytophylactica 24:106. Boewe, G.H. 1936. The relation of ear rot prevalence in Illinois corn fields to ear coverage by husks. State of Ill. Nat. Hist. Surv. Div. Contrib. Sec. Appl. Bot. Plant Pathol. Pub. 273. Burrill, T.J., and J.T. Barrett. 1909. Ear rots of corn. Univ. Illinois Agr. Expt. Sta. Bul. 133. Chambers, K.R. 1988. Effect of time of inoculation on Diplodia stalk and ear rot of maize in South Africa. Plant Dis. 72:529–531. Curl, E.A. 1963. Control of plant diseases by crop rotation. Bot. Rev. 29:413–479. Das, S.N., S.B. Chattopadhyay, and S.L. Basak. 1984. Inheritance of resistance to Diplodia ear rot of maize. Sabrao J. 16:149–152. Dorrance, A.E., K.H. Hinkelmann, and H.L. Warren. 1998. Diallel analysis of Diplodia ear rot resistance in maize. Plant Dis. 82:699–703. Dorrance, A.E., O.K. Miller, and H.L. Warren. 1999. Comparison of Stenocarpella maydis isolates for isozymes and cultural characteristics. Plant Dis. 83(7):675–680. Drepper, W.J., and B.L. Renfro. 1990. Comparison of methods for inoculation of ears and stalks of maize with Fusarium moniliforme. Plant Dis. 74:952–956. Fajemisin, J.M., J.A. Durojaiy, Y. Efron, and S.K. Kim. 1987. Inoculation studies of three ear rot diseases of tropical maize. Phytopathology 77:1747. (Abstr.) Favaron, F., C. Castiglioni, and P. Di Lenna. 1993. Inhibition of some rot fungi polygalacturonases by Allium cepa L. and Allium porrum L. extracts. J. Phyotpath. 139:201–206. Ferreira, M.J. 1994. Relationship between ear-rot incidence and morphological characteristics of maize. pp. 60–63, In: J.G. du Plessis et al. (eds.), Proc. 10th S. Afr. Maize Breeding Symp., Potchefstroom 1992 Tech. Comm. 238, Dept. Agr., Pretoria, South Africa. Flett, B.C. 1991. Crop plants as hosts and non-hosts of Stenocarpella maydis. Phytophylactica 23:237–238. Flett, B.C. 1999. Epidemiology and management of maize ear rot. Ph.D. diss. Faculty of Natural, Agricultural and Information Science, Department of Microbiology and Plant Pathology, Univ. Pretoria, Pretoria, South Africa. Flett, B.C., and N.W. Mclaren. 1994. Optimum disease potential for evaluating resistance to Stenocarpella maydis ear rot in maize hybrids. Plant Dis. 78:587–589. Flett, B.C., N.W. Mclaren, and F.C. Wehner. 2001. Incidence of Stenocapella maydis ear rot of corn under crop rotation systems. Plant Dis. 85:92–94. Flett, B.C., and F.C. Wehner. 1991. Incidence of Stenocarpella and Fusarium cob rots in monoculture maize under different tillage systems. J. Phytopath. 133:327–333. Gevers, H.O. 1989. The ear rot epidemic. pp. 77–80. In: J.G. du Plessis (ed.), Proc. 8th S. Afr. Maize Beeding Symp., Potchefstroom 1988, Tech. Comm. 222, Dept. Agr. and Water Supply, Pretoria, South Africa.


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5 Cassava Genetic Resources: Manipulation for Crop Improvement Nagib M.A. Nassar Departamento de Genetica e Morfologıá Universidade de Brasilia 70919 Brasilia, Brazil Rodomiro Ortiz Centro Internacional de Mejoramiento de Maı´z y Trigo (CIM-MYT) El Batań, Texcoco Apdo. Postal 6-641 06600 Mexico, D.F.

I. INTRODUCTION A. Origin B. Distribution of Manihot Species and Cassava II. WILD MANIHOT SPECIES: A BOTANICAL REVIEW III. INTERSPECIFIC HYBRIDS A. Polyploidization B. Chimeral Instability 1. Identification of Chimeras 2. Instability C. Production of Cassava Cultivars IV. CASSAVA DIVERSITY AS REVEALED BY DNA MARKERS AND GENETICS A. Diversity among and between Manihot Species B. Landraces and Bred Clones in the Agro-Ecosystems C. Trait Level D. Gene Expression

The authors dedicate this article to the late Dr. David Rogers, a leading botanist whose passion for research on Manihot species will be an example for the next generation of cassava scientists. Plant Breeding Reviews, Volume 31 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 247

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V. TRAIT TRANSFER A. Useful Traits B. Apomixis C. Carotenoids D. Amino Acids VI. OUTLOOK LITERATURE CITED

I. INTRODUCTION Farmers grow cassava (Manihot esculenta Crantz, Euphorbiaceae) throughout the lowland tropics. This crop is cultivated between 30 north and 30 south of the equator, in areas where the annual mean temperature is greater than 18 C (Nassar and Ortiz 2007). This tuberous root crop ranks among the two most important staples of sub-Saharan Africa, which today accounts for most of the cassava harvest worldwide, followed by Asia and South America—the main diversity center of Manihot species. In sub-Saharan Africa and Latin America, cassava is mostly used for human consumption; in Asia and parts of Latin America, it is also used commercially for the production of animal feed and starch-based products. Cassava has been regarded as a crop adapted to drought-prone environments, where cereals and other crops do not grow well (Cock 1985), and it also grows well in poor soil. The professional genetic enhancement of cassava started in the 20th century and was spurred by increasing population demands, which were affected by the limited supply of energy food crops, particularly in sub-Saharan Africa (Ortiz et al. 2006). The Consultative Group on International Agricultural Research (CGIAR) centers have been active in cassava breeding since the late 1960s: the Centro Internacional de Agricultura Tropical (CIAT, Cali, Colombia) in Latin America and the Caribbean, Asia, and the Pacific. In Africa, the major undertakings were under the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. Among the breeding goals of the two CGIAR centers were yield potential as well as stability-enhancing traits for marginal or pest-prone areas (Jennings and Iglesias 2002). Ceballos et al. (2004) provides an overview of the state of the art of cassava breeding as well as remaining challenges. Nassar and Ortiz (2007) assess the impacts on cassava breeding programs worldwide. Although significant gains are reported in Africa (Dixon et al. 2003) and Asia (Kawano 2003), pests and other constraints affect crop yield. More recently, breeding programs began using biotechnology tools that may assist on the genetic enhancement of cassava (Fregene and Puonti-Kaerlas 2002). For example, DNA markers are providing a

5. CASSAVA GENETIC RESOURCES

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better understanding the relationships among Manihot species (Olsen 2004) and insights into the cassava genome through gene mapping (Okogbenin et al. 2006), which can led further to marker-aided breeding (Fregene et al. 2006). Ongoing transgenic programs aim to engineer cassava with enhanced resistance to pests, improved nutritional content, modified and increased starch metabolism, and reduced cyanogenic content of processed roots (Taylor et al. 2004). However, transgenic cassava has not been yet grown by farmers in the main producing tropical areas Wild Manihot species are gene reservoirs for tackling abiotic and biotic stresses as well as for improving the root quality of cassava. Most species are perennial and vary in growth pattern from nearly acaulescent subshrubs to small trees. The Universidade de Brasilia (Brazil) maintains a living collection of Manihot species since the 1970s (Nassar 1981). The collection was because such genetic endowment was used before in cassava breeding, especially for host plant resistance to cassava mosaic disease (CMD) (Nichols 1947), and the species may be potential sources for other traits, such as high protein content (Jennings 1959; Nassar and Costa 1978), seed fertility (Jennings 1962), apomixis, resistance to mealybug, and tolerance to drought-prone environments (Nassar 2000a). Likewise, indigenous clones are potential sources of b-carotene and lycopene. This chapter updates previous reports (Byrne 1984; Nassar 1999, 2000a) on the use of Manihot genetic resources (including DNA markers) for cassava improvement. A. Origin A total of 98 Manihot species have been recognized (Rogers and Appan 1973), with one species (Manihotoides pauciflora) known in the closest related genus. Several of its attributes are not found in any Manihot species, including monoflower inflorescences and leaves borne at the apex of short and condensed stems arising from branchlets. These primitive characters suggest M. paciflora as a probable progenitor of all Manihot groups. Unfortunately, this species is on the verge of extinction (Nassar 1999). Cassava, a native crop of South America, was grown in Colombia and Venezuela between 3,000 and 7,000 years ago (Bonierbale et al. 1997). Ugent et al. (1986) cite evidence of domestication on the Peruvian coast earlier than 4000 BCE. Further research has shown that modern domesticated cassava roots produce significantly larger-size starch granules than those of its putative wild ancestor (Perry 2002). The coastal Peruvian and lowland neotropical cassava types differ, and appear to be separated by several millennia, which suggests that the

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crop may have been domesticated more than once. Nassar (1978a) emphasizes that M. esculenta species does not grow wild elsewhere. According to this author, domestication could occur from a natural hybrid between two species, with M. pilosa perhaps being one of the parents. Enlarged roots could happen as a result of this hybridization. The ability of cassava cultivars to set seed has been reduced since they evolved from wild Manihot species (Jennings 1962), indicating that cassava has long been propagated asexually likely by cuttings. Conscious and unconscious farmers’ selection for increasing the number and quality of cassava cuttings would act to favor plants with thicker stems (Elias et al. 2007). This selection for increased asexual propagules can have led to a reduction in the degree of branching, which is regarded as one of the most striking differences between the cultigen and its wild ancestors. Although the cultigen is vegetatively propagated, farmers still incorporate plants from seedlings as plantings stocks (Elias et al. 2001). The outcrossing rate ranged from 0.69 to 1 among eight cassava ethnovarieties, which indicates that they are preferentially allogamous (da Silva et al. 2003). Natural hybridization between cassava and wild Manihot relatives does occur; coupled to weak interspecific barriers, this hybridization has led to an extremely heterozygous gene pool that may begin a sequence of hybridization followed by speciation (Nassar 2003b). Comparative analysis between cassava and its wild relatives showed that epigeal germination was primitive in Manihot. Hypogeal germination was a feature of wild Manihot species because it confers better adaptation to the risky savanna environments; epigeal germination and photosynthetic cotyledons evolved through domestication in cassava, since both traits provide the cultigen with fast initial growth in the farming systems (Pujol et al. 2005). B. Distribution of Manihot Species and Cassava All Manihot species are native to tropical regions of the New World, particularly Brazil and Mexico. The only species found in other tropical regions of the world are those that were introduced after Columbus’s voyages to the American continent. The species of Manihot are sporadic in their distribution and rarely become dominant in the local vegetation. The majority of these species are in relatively dry regions, and only a few are found in the rain forest. Their typical habitats are openings in the forest, as the case of M. anomala. They are therefore heliophiles that grow only in the absence of shading. Many of these species (e.g., M. pohlii, M. zehntneri, and M. grahamii) are weedy types capable of invading new agitated areas and frequently are found on


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limestone-derived and well-drained soils. With a few exceptions, such as M. grahamii and M. neusana, whose native distribution includes areas with occasional frost, most species are damaged by frost. Rogers and Appan (1973) classified Manihot species into 19 sections, varying from trees in the section Glazioviannae to subshrubs, nearly acaulescent, in the section Stipularis. The species in this latter section are also characterized by being more dioecious than monoecious, a condition reversed in all other Manihot species. Other sections, such as Tripartitae and Graciles, are perennial subshrubs with large woody roots, and whose stems frequently die back to the root crown in response to dry periods or fires. There are four widely known centers of diversity for Manihot species: Mexico and northeast and central Brazil, plus southwest Brazil and Bolivia. Microcenters of diversity of these species exist within central Brazil where large numbers of species are concentrated in small areas; that is < 50 km in diameter (Nassar 1978a,c,d,e,f, 1979a,b, 1980b, 1982, 1985, 1986). These microcenters arose from the frequent hybridization between species and the heterogenic topography of their habitats, which help isolate fragmented gene pools that lead to speciation. For example, Goia´s Velho and Corumba´ de Goia´s are regarded as two microcenters of cassava diversity (Nassar 2003a), following Harlan’s concept of geographic pattern of variation of cultivated crops (Harlan 1951, 1971). Likewise, treelike species such as M. glaziovii and M. pseudoglaziovii are found in northeastern Brazil whereas short species and subshrubs are found in central Brazil. The species within each Manihot section, their growth form, and native distributions are provided by Rogers and Appan (1973) and tabulated by Byrne (1984, Table 3.1, pp. 84–87). Central Brazil (southern Goia´s and eastern Minas Gerais) bears the highest Manihot diversity (38 out of the 98 accepted species). Mexico, the second largest center of diversity, includes 17 Manihot species (Nassar 1978a, 2000a). The third largest center of diversity is northeast Brazil with 16 species. There are six species in south Matto Grosso and Bolivia that together are the fourth center of Manihot diversity. The M. esculenta cultigen appears to be a complex species with multiple sites of initial domestication (Rogers and Fleming 1973), although Allem (1994) proposed that M. esculenta derived from two primitive forms instead of being a cultigen, and having three subspecies. They are M. esculenta subsp. esculenta (in which all known cultivars and landraces should be included) and two wild types (M. esculenta subsp. peruviana (which occurs in eastern Peru and western Brazil), and M. esculenta subsp. flabellifolia (with a distribution range from Goia´s in

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Brazil northwards to Venezuelan Amazonia). Allem (1994) concluded that the large area of distribution of the two wild subspecies cannot allow assigning a place of initial domestication. Allem (1999) regarded M. esculenta ssp. flabellifolia as a wild ancestor of modern cassava cultivars, and therefore included it in the primary gene pool (GP1) of this root crop together with M. peruviana (Allem et al. 2001). Despite the fact that they did not do any experimental hybridization or carry out a cytogenetic exam of chromosome pairing, they indicated that M. pruinosa was the nearest species to the GP1 of cassava and can hardly be separated from the wild strain M. esculenta ssp. flabellifolia on morphological grounds. However, Nassar (1978a, 2001a) did not agree with Allem’s views that cassava arose from M. flabellifolia and suggested that this species may be the result of a cassava crop-weed complex, as pointed out in other crops by Harlan and de Wet (1965). Cytogenetics and DNA marker-aided research should be able to provide more insights into the putative ancestors of cassava (Haysom et al. 1994; Nassar 2001).

II. WILD MANIHOT SPECIES: A BOTANICAL REVIEW Procumbent, semiherbaceous subshrubs, shrubs, and trees are found in Manihot. The branching pattern is typically dichotomous or trichotomous, having at the branching point a terminal inflorescence. Bark of the woody species is generally smooth. Many of the species are lacticiferous, and some species, such as M. glaziovii (ceara rubber), are cultivated in Brazil and elsewhere for rubber production (Rogers 1965; Rogers and Appan 1973). This species was used by Storey and Nichols in the 1930s in former Tanganyika (today continental Tanzania) to transfer resistance to CMD (Nichols 1947; Nassar and Ortiz 2007 and references therein). Many species, such as those in section Tripartitae, have their stems adapted to dry periods, die-back to a root crown regularly, and shed their leaves during the dry season. The majority of Manihot species are found on limestone-derived and welldrained soils. All Manihot species are monoecious and a few are dioecious, which make them obligate outcrossers. In many species, they are protogeneous (i.e., pistillate flowers open before staminate flowers of the same inflorescence). Pollination is by insects to whose bodies the sticky pollen adheres. Cross-pollination leads to formation of extremely heterozygous gene pools. Being allopolyploid species, partially apomictic, and having week barriers in addition to the allogamous


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nature have led to the rapid speciation of this group and formation of the large number of species (Nassar 1999, 2000a, 2001a).

III. INTERSPECIFIC HYBRIDS Wild Manihot species have been used in interspecific crosses with cassava because they are source of traits for its genetic enhancement. For example, the interspecific hybrid of M. oligantha with the cultigen showed a high protein content, which reached 4% of peeled roots with low cyanide content (90 mg/kg); that is, doubling protein content of common cassava (Nassar and Dorea 1982). Recently, genes for apomixis from the wild species M. neusana were transferred successfully (Nassar 2000a; Nassar et al. 2000). Another important utilization of wild Manihot species was the finding of some host plant resistance levels to mealybug in M. glaziovii and its transfer to the cassava gene pool through interspecific hybridization (Nassar et al. 1986, Nassar 1996). Nassar et al. (1986) regard M. esculenta–M. glaziovii as a secondary gene pool (GP2) of the cultigen since interspecific hybridization was feasible with some success. This interspecific hybrid could be polyploidized and its fertility restored (Nassar 2004). All wild Manihot species examined cytogenetically have a chromosome number of 2n ¼ 36 (Nassar 1978a,g). Despite this high chromosome number, Manihot species behave meiotically as diploids (Nassar 1978b). Natural hybridization occurs between wild Manihot species and between them and cassava (Nassar 1984, 1989). Barriers within the genus appear to be weak due to the group’s recent evolution. For example, frequent hybridization occurs between M. reptans and M. alutacea in sympatric natural habitats where their population boundaries overlap (Nassar 1980a). Morphological markers such as leaf color and bract size were used to identify this interspecific hybridization. The range of M. reptans has expanded during the past 100 years (Nassar 1984), a situation attributed to the continuing gene introgression of Manihot species. Introgression of M. reptans with germplasm from other species allowed its ecotypes to penetrate and colonize areas where M. reptans alone had previously been unable to succeed. This phenomenon was also noted in other species, such as M. cearulescens (Nassar 1980a). Morphological markers for lobe shape, the presence of stem nodes, flower disk color, fruit color, and fruit shape have been used to identify hybrids in offspring ensuing after controlled crosses between cassava and wild Manihot species as well as in natural hybrids

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between cassava and other Manihot species (Nassar 2000a). Interspecific hybrids of cassava with M. glaziovii, M. pseudoglaziovii, M. aesculifolia, M. pilosa, M. dichotoma, M. pohlii, M. neusana, and M. anomala were obtained through controlled crosses, although their frequency was low. The meiotic behavior of several hybrids (e.g., between cassava and both M. neusana and M. pseudoglaziovii) indicated low hybrid fertility between wild Manihot species and the cultigen (Nassar 1992; Nassar et al. 1995). Interspecific hybrids from crosses between several Manihot species with cassava arose through controlled crosses by insect vectors (Nassar 1980a, 1989, 1994a). Morphological markers often are used to identify interspecific hybrids. For example, the hybrid plants ensuing from the cross between of M. neusana and cassava exhibited dominant phenotypes from cassava, namely ribbed fruit, red color of the flower disk, nodded-stem, and tuberous root. Glabrous stem, setaceous-foliaceous bracteoles, red-creamy color of flower disks, variegated-green color of fruit, and ribbed–no ribbed fruit are simple morphological markers that can be used to recognize interspecific hybridization with cassava. A. Polyploidization The synthesis of new Manihot species may also occur by polyploidization of interspecific hybrids, as in other genera (e.g., Brassica or Triticum-Secale). Two synthesized Manihot species were named Manihot rogersii and Manihot vieiri (Nassar 2003c, 2006a). Polyploid hybrid cassava clones also ensued from 2n gamete-producing parents (Hahn et al. 1990; Nassar 1991; Va´squez and Nassar 1994). Further research with clones Rayong 1, Rayong 60, M. mga, and two hybrids (OMR 3641–1 and OMR 3641–4) allowed to elucidate 2n gamete mechanisms (Ogburia et al. 2002), which agreed with previous results (Nassar and Fritas, 1997). Megasporogenesis in Rayong 1 and Rayong 60 showed a lack of second meiotic divisions after a successful first division that resulted in partly unreduced embryo sacs with 2n eggs. Meiotic abnormalities during microsporogenesis and megasporogenesis are therefore a source for the occurrence of mixoploids (triploids and tetraploids) in cassava breeding programs. B. Chimeral Instability Asexual polyploidization may lead to chimeras. For example, four interspecific hybrids (M. neusana M. esculenta; M. glaziovii M. esculenta; M. aesculifolia M. esculenta; M. pohlii M. esculenta)


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Table 5.1. Frequency of polyploids obtained in four interspecific hybrids between cassava and wild Manihot species. Polyploid frequency (%) ———————————————————— Tetraploids Chimeras Polyploids

Hybrid M. M. M. M.

esculenta esculenta esculenta esculenta

M. M. M. M.

neusana glaziovii pohlii aesculifolia

1 5 2 —

3 — 2 3

Totals samples

4 5 4 —

20 25 20 15

were used for polyploidization with colchicines. Some of their tissues had different ploidy levels on the same stem as a result of sectorial and periclinal chimeras (Table 5.1). The derivative cells of the outermost layer of the tunica form epidermis, whereas the second layer forms the subepidermal tissues and the third layer forms the pith and vascular tissue. 1. Identification of Chimeras. Pollen grain viability, leaf shape, and stem anatomy help to identify chimeras. The polyploid section of the stem in sectorial chimeras has short leaves whereas the diploid side develops narrow and longer diploid form. Pollen viability, leaf shape, and stomata size are used as a selection criterion for any periclinal chimera. Pollen formed from LII layer while leaves are differentiated form the LI layer. In periclinal chimeras, leaf is different, the stomata is enlarged, and pollen viability is much higher than in diploid plants. All the chimeras in interspecific hybrids of M. esculenta M. neusana and M. esculenta M. glaziovii were sectorial, whereas two sectorial and one periclinal chimera were obtained in the cross cassava M. aesculifolia. In sectorial chimeras, pollen viability and size was as in diploids and tetraploids. The pollen size in the periclinal chimeras reflects the ploidy level of this layer (Table 5.2).

Table 5.2. Pollen viability in diploid and tetraploid sectors of cassava chimeras. Pollen viability (%) Interspecific hybrid

Diploid tissue

Tetraploid tissue

18 11 13 15

92 90 93 91

Cassava Cassava Cassava Cassava

M. M. M. M.

neusana glaziovii pohlii aesculifolia

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2. Instability. Scanty information seems to be available about chimeras in cassava, but the use of polyploidy in cassava breeding is frequently reported by Indian breeders (Sreekumari et al. 1999), although they did not indicate occurrence of chimera that could be overlooked or simply ignored. Competition between tetraploid and diploid tissues leads to the loss of desired traits. Only the chimeras in the LlI (periclinal) layer have a chance of transmitting desirable characteristic to their progeny. In other research with chimeral sectors in cassava (Nassar, unpub. data), the stem exhibited diploid level after about 6 months’ growth restoring the normal leaf shape that became narrow, and pollen viability was that of a diploid. The growth rate of tetraploid tissue was slower than that of the diploid, and tetraploid tissue was often overgrown by diploid tissue. It was however possible to propagate tetraploid tissue through somatic selection by cutting the apical buds of the chimeral stem, followed by removal of the lateral shoots growing from the diploid sector and allowing only the tetraploid side branches to grow. One of the main features of asexual polyploidization is fertility restoration in interspecific hybrids. There may be a low percentage of unviable pollen in the asexual polyploids due to formation of 3% multivalents in their tissue, which should not be surprising because quadrivalent formation also occurs in cassava. Fertility restoration in the interspecific hybrids through asexual polyploidization facilitates the use of wild Manihot species for cassava improvement through their use in further crosses (Nassar 2002b; Sreerkumari and Abraham 1997). Their use involves backcrossing the polyploidized interspecific hybrids with cassava followed by selection for the desirable traits in the progeny. Preferential autosyndetic pairing between chromosomes of cassava may result into elimination of the majority of chromosomes of the wild species during meiotic segregation. Selfing of a fertile asexual polyploidy hybrid may also lead to useful recombination between wild Manihot species and cassava. C. Production of Cassava Cultivars Cassava triploids (2n ¼ 54) were produced in India by crossing the cultivated diploids with asexual tetraploids (Sreerkumari et al. 1999). The triploids were characterized by vigorous growth, erect plant type, broad leaves, and stout stem. The triploid showed significantly higher tuberous root yield than the diploids, and they also had higher starch content. One of the triploids proved to be superior in tuber yield, starch content, and especially culinary quality. The Kerala State Variety


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Release Committee officially named ‘Sree Harsha’ a triploid cultivar in 1996. This Indian-bred clone was the first triploid cassava cultivar released to farmers worldwide. Hahn et al. (1990) reported that tetraploid and triploid hybrids were very vigorous and one of the tetraploid hybrids performed as well as the best cultivar in IITA’s uniform yield trials in Nigeria. These authors concluded that such polypoloid hybrids provide greater genetic variation and offer an opportunity to breed radically new cassava cultivars for Africa.

IV. CASSAVA DIVERSITY AS REVEALED BY DNA MARKERS AND GENETICS The largest cassava genebanks are in CIAT, Empresa Brasileira de Pesquisa Agropecua´ria (EMBRAPA, Brazil), IITA, and Central Tuber Crops Research Institute (CTCRI, Trivandrum, India) (Bonierbale et al. 1997). Such collections provide the genetic endowment to cassava breeders worldwide (Kawano 2003), although only a sample of the accessions held in the gene banks were thoroughly assessed for their useful variation. Hence, a core subset comprising 630 accessions from 23 countries was assembled by CIAT (Bonierbale et al. 1997). This core collection provides a means for further assessment of variation, which will assist in identifying valuable traits, and entry points that facilitate access to the entire collection. This genetic resource tool will be enhanced through the use of isozyme and DNA markers, which are among the biotechnology methods that provide a better understanding of diversity in the crop and wild relatives as well as for gaining insights into the cassava genome through gene mapping and quantitative trait loci (QTL) analysis. Isozyzme are still useful genetic markers to study diversity and to identify duplicates in gene banks (Montarroyos et al. 2003; Sumarani et al. 2004). Likewise, DNA markers reveal insights of the relationships between cassava and wild relatives or confirm previous clustering of cultivars regarding their origin and how much Manihot diversity was still available in the crop gene banks and in the core subset (Carvalho and Schaal 2001). The first molecular genetic map of cassava included initially 132 restriction fragment length polymorphisms (RFLP), 30 random amplified polymorphic DNA (RAPD), 3 microsatellites (SSR), and 3 isoenzyme markers (Fregene et al. 1997). The map consisted of 20 linkage groups spanning 931.6 cM (centMorgan) or an estimated 60%

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of the cassava genome and with an average marker density of 1 per 7.9 cM. Since then more markers, especially SSR, were added (Chavarriaga-Aguirre et al. 1998; Mba et al. 2001). More recently, cDNA and genomic sequences are being used as tools to develop single-nucleotide polymorphisms (SNP) in cassava (Castelblanco and Fregene 2006; Lo´pez et al. 2005a), which will allow mapping of expressed sequenced tags (EST) as well as their association with phenotypic characteristics (Anderson et al. 2004). A. Diversity Among and Between Manihot Species Phylogenetic relationships of Manihot species were revealed by 13 probe-restriction enzyme combinations (Haysom et al. 1994). This research with RFLP showed very little variation between accessions of the same species. The authors demonstrated a close relationship between the Mexican species M. chlorosticta and the M. esculenta subsp. flabellifolia accessions, which puts in question the classification of flabellifolia as a true South American wild species. Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was also investigated in cassava and wild Manihot species (Fregene et al. 1994). Nine distinct chloroplast types (three for cassava and six for wild species) were defined by RFLP analysis. Their results suggest that the cultigen arose from the domestication of wild tuberous Manihot species, followed by intensive selection. More recent microsatellite research indicates that genetic variation in the crop is a subset of that found in the wild M. esculenta subspecies (Olsen and Schaal 2001). Their research also confirms their previous report about the southern border of the Amazon basin as the likely site of domestication (Olsen and Schaal 1998) and rejects the closely related M. pruinosaa as a probably ancestor of cassava. Clustering of accessions of each species by amplified fragment length polymorphism (AFLP) data analysis coincided with previous taxonomic classifications (Roa et al. 1997). The authors indicated that a mixed cluster, consisting of M. esculenta subsp. flabellifolia and M. esculenta subsp. peruviana, was most similar to cassava, while M. aesculifolia,M. brachyloba, and M. carthaginensis were more distant as per the AFLP analysis. Species-specific markers, which may be useful in germplasm classification or introgression studies, were suggested by the unique presence of AFLP products in samples of each of the three wild Manihot species. Their AFLP research also showed the occurrence of intraspecific gene pools, greater homogeneity among cassava accessions than among its closest wild relatives.


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B. Landraces and Bred Clones in the Agro-Ecosystems Fregene et al. (2003) studied spontaneous recombination, as assessed by parent-offspring microsatellite relationships, and farmer-selection for adaptation as among the causes for genetic diversity and differentiation in an asexually propagated crop such as cassava. Their research also found the highest diversity among landraces from Brazil and Colombia but that the genetic diversity between South American and sub-Saharan African landraces was comparable. Although there was low differentiation among country samples, the genetic distance was enough to separate Latin American and African accessions, with the latter having a significant landrace substructure. AFLP analysis also identified a considerable number of duplicates in the African accessions, suggesting a sizable percentage of redundancy (Fregene et al. 2000). This analysis also revealed a genetic divergence between African and Latin American accessions, although some overlap was found between them. African landraces resistant to CMD were also found to be genetically differentiated from susceptible landraces and from resistant elite clones bred by IITA in collaboration with national programs in sub-Saharan Africa. AFLP markers were also useful for assigning seedlings to the cultivars from which they originated in research undertaken to understand traditional management of cassava morphological and genetic diversity (Elias et al. 2001). Several molecular marker systems (AFLP RAPD, SSR) were also used to assess genetic diversity in a broad range of cassava landraces and breeding materials in Latin America (Colombo et al. 2000), subSaharan Africa at large (Lokko et al. 2006), and more specifically in Ghana (Asante and Ofei 2003), Malawi (Mkumbira et al. 2003), Mozambique (Zacarias et al. 2004), and Uganda (Balyejusa Kizito et al. 2005). There was a weak genetic structure among Latin American cassava accessions, which was accounted for by the traditional practice of germplasm exchange among smallholders as well as due to recombination between genotypes (Colombo et al. 2000). However, these authors pointed out that their sampling of genotypes was structured according to both temperature and precipitation when using climatic data. An African sample, which included landraces and breeding materials varying in their host plant resistance to CMD and low inbreeding within groups, also showed a weak genetic structure (Lokko et al. 2006). Diversity Arrays Technology (DArT) was recently used for high-throughput genotyping of cassava and wild Manihot relatives

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(Xia et al. 2005). This DNA marker technique uses microarrays to detect DNA polymorphism at several hundred genomic loci in a single assay without relying on DNA sequence information. About 1,000 candidate polymorphic clones were detected on two arrays with the largest frequency of polymorphic clones (Pst1/Taq1 and Pst1/BstN1). This new set of DNA markers showed high polymorphism information content values and was revealed known genetic relationships among the samples. Hence, DArT may provide a cheaper and faster means for marker discovery and analysis than other DNA marker systems, so it may be an option for genotyping large number of samples (e.g., from a gene bank collection). C. Trait Level The most promising results for dissecting complex traits are from interval mapping with RFLP and microsatellite markers (Fregene et al. 2000; Mba et al. 2001) For example, two flanking RFLP and SSR markers in between a dominant gene may provide new sources of resistance to cassava mosaic diseases (Akano et al. 2002). Research at the Royal Veterinary and Agricultural University, Copenhagen, Denmark, also showed that two RAPD markers linked to genome regions coding for the enzyme catalyzing the first committed step of the biosynthesis of cyanogenic glucosides were tested positively in 46 African genotypes (Ortiz 2004). Likewise, a unique AFLP fragment, found in a relatively high frequency in African accessions but absent in the Latin American accessions, was found to be associated with branching pattern (Fregene et al. 2000). DNA marker-aided genetic analysis and mapping was also used for host plant resistance to cassava bacterial blight (Jorge et al. 2000, 2001), root quality traits (Fregene et al. 2001) early root bulking (Okogbenin and Fregene 2002), root yield and plant architecture (Okogbenin and Fregene 2003), and wound-response genes that are expressed during the postharvest physiological deterioration (PPD) of cassava (Corte´s et al. 2002). D. Gene Expression Expressed sequenced tags for starch content and host plant resistance to cassava bacterial blight resistance were the first to be publicly available (Lo´pez et al. 2004). About 37% of sequences were assigned to a putative functional category after sequence analysis, whereas approximately 16% of the sequences were regarded as cassava-specific genes because they did not show any significant similarity with other proteins available in


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the database. A unigene catalog of 5,700 expressed genes in cassava ensued from this research. A cassava cDNA microarray based on a large cassava EST database was used to investigate the gene expression profile in response to cassava bacterial blight (Lo´pez et al. 2005b). Cassava induces dozens of genes, including principally those involved in oxidative burst, protein degradation, and pathogenesis-related (PR) genes in response to bacterial blight, whereas genes encoding proteins that are involved in photosynthesis and metabolism are downregulated. Likewise, cDNA microarray technology was used for a large-scale analysis of the cassava root transcriptome during the postharvest period in an attempt to characterize the genes that show a significant change in expression during the PPD response (Reilly et al. 2007). A total of 72 nonredundant EST with altered regulation during the post-harvest period were found, of which 63 were induced whereas 9 were downregulated. The upregulated and PPD-specific EST appear to be involved in cellular processes such as: reactive oxygen species turnover; cell wall repair; programmed cell death; ion, water, or metabolite transport; signal transduction or perception; stress response; metabolism and biosynthesis; and activation of protein synthesis. A bacterial articifical chromosome (BAC) was developed to facilitate the positional cloning of resistance genes in cassava (Tomkins et al. 2004). Two libraries were constructed by using a cassava clone bred by IITA with host plant resistance to CMD (from M. glazovii) and bacterial blight and a cassava landrace from Ecuador available at the CIAT gene bank as the source of resistance to white fly. A BAC library screening with the 12 resistance gene candidates (RGC) classes as probes led to the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons (Lo´pez et al. 2003). This identification and characterization of RGC may provide new markers tightly linked to resistance (R) gene loci. Such genetic tools can assist in marker-aided breeding and in high-resolution gene mapping for further map-based cloning of R-genes in cassava. Serial analysis of gene expression (SAGE) was used to analyze the gene expression pattern of host plant resistance to CMD by using bulks of resistant and susceptible genotypes drawn from a genemapping progeny (Fregene et al. 2004). Several genes were expressed during systemic acquired resistance (SAR) in plants and other genes were involved in cell-to-cell and cytoplasm-to-nucleus virus trafficking. This research further showed that a WRKY transcription factor was associated with the region bearing the dominant CMD gene.

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V. TRAIT TRANSFER A. Useful Traits Some of the wild species, including Manihot tree types, show tuberous roots (Rogers and Fleming 1973; Nassar 1978a). This wide variation also includes adaptation to limestone soils (e.g., in M. pentaphylla) or poor acid soils with high aluminum (Al) content (e.g., in M. procumbens), or other interesting traits, such as dwarf habit (e.g., in M. pusilla; Nassar and Fitchner 1978), or drought tolerance and high starch content (e.g., in M. angustiloba; Rogers and Appan 1973; Nassar and Cardenas 1986). M. glaziovii, or ceara rubber, has been used extensively in cassava breeding, especially for CMD in Africa (Nassar and Ortiz 2007 and references therein). Chromosomes of cassava and ceara rubber are similar enough for pairing although there are some abnormalities, as found in F1 hybrids among both species (Magoon et al. 1970). Such pairing allowed random transmission of genetic material from the male gamete.

B. Apomixis Apomixis means seed formation without fertilization. In cassava, it is an alternative to reproduction by cuttings, as normally practiced by farmers (Nassar 2002a). Vegetative propagation by cuttings leads to accumulation of viral and bacterial diseases that affect tuberous root yields and may affect the survival of superior genotypes. The use of apomictic plant in propagation will therefore avoid systemic pathogens and avoid the genetic segregation in the ‘‘progeny’’. Plant-produced stems through apomixis from a contaminated clone will be free from viruses and bacteria, and can begin a new clone cycle. If apomixis was found or had been introduced into the superior Brazilian clones. such as Guaxupe and Vassourinha, they would have been preserved for a longer time. According to Nassar (2002b), the use of apomixis for preserving superior genotypes and for avoiding some pests will also provide benefits to international cassava programs that export routinely their germplasm. Recently a polyploid type was synthesized artificially by chromosome doubling of a hybrid (Nassar 2006b). This polyploid had 29% multiembryonic sacs in the ovules examined whereas sacs were absent in the diploid type. It was further observed that apomixis occurs in this polyploid, whose progeny showed the maternal characteristics (Nassar 2007). Facultative apomixis was discovered in the wild cassavas M. dichotoma and M. glaziovii (Nassar 1994a; Nassar et al. 1998). It


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was noted earlier in M. neusana—a species with resistance to bacterial wilt and stem borers (Nassar 1985). Interspecific hybridization was carried out to transfer these useful genes to the cultigen pool (Nassar 1989). For example, apomixis genes were successfully transferred to cassava by hybridizing this cultigen with the wild species M. glaziovii (Nassar and Collevatti 2005a). Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava (Nassar and Collevatti 2005b). All sibs were identical with each other and with their maternal progenitor. The authors concluded that apomixis also played an important role in Manihot speciation. Further research aided by microsatellite markers has confirmed the facultative apomictic nature of cassava but also highlighted the high influence of the environment on this trait (Nassar et al. 2006). Apomixis was detected anatomically using a ‘‘clearing’’ method (Nassar et al. 1996) whereby unpollinated pistillate buds were collected, fixed, dissected, and then examined according to Herr (1992). These anatomical ovlular studies showed that the embryo was formed by apospory from a somatic cell in the nucellus. The megasporogenesis in ovules with aposporous development proceeds normally up to a certain moment when nucellar cells enlarge and the nuclei divides to form aposporous embryo sacs (Nassar et al. 1998a,b,c). These aposporous embryo sacs appear to develop faster than sexual embryo sacs, probably because they are not delayed by meiotic division (Asker 1979; Nogler 1984). In some cases, development of apospory embryo sacs from cells within the sexual one was noted. Both the aposporous and sexual embryo grew in parallel and finally coexisted. This observation confirms results from previous research (Nassar 1995), where two seedlings grew side by side; one was apomictic and the other had a sexual origin. Nogler (1984) reported that in Potentilla, aposporous and sexual processes coexisted in one individual ovule that produced several embryos. This study documents the survival of two aposporous embryo sacs beside a sexual one, and being all of them at a developed stage in the ovule. A cytogenetical study showed that out of the 25 individuals examined, 13 plants were sterile, and the percentage of pollen viability ranged from 4 to 15% (Table 5.3). Two plants were aneuploids (2n þ 1) while the rest were normal (2n). The 12 normal plants were highly fertile and had pollen viability ranging from 92% to 97%. The embryonic study revealed that all of the sterile plants were partially apomictic whereas the fertile plants were sexual. Sterility apparently leads to apomixis. Sterility was caused by consistent defects of

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N.M.A. NASSAR AND R. ORTIZ Table 5.3. Chromosome number, pollen viability and apospory in apomictic cassava offspring. Clone 1 2 3 4 5 6 7 8 9 10 11 12 13

Chromosome number

Pollen viability (%)

9.2 6.8 4.9 2.0 4.7 6.1 15.6 4.1 8.3 5.6 4.1 4.6 4.7

1.2 1.5 1.7 1.6 1.1 1.2 1.3 1.4 1.7 1.3 1.8 1.4 1.3

Apospory (%) 2n 2n þ 1 2n 2n þ 1 2n 2n 2n 2n 2n 2n 2n 2n 2n

meioses due to lack of pairing. All sterile plants showed asynapsis in meiotic metaphase. Formation of univalents ranged from 4 to 6 per cell. The irregular chromosome segregation in these sporocytes should lead to genetically unbalanced and aborted gametes. It seems that this sterility triggers certain genes controlling apomixis (Nassar 2001b). C. Carotenoids The World Health Organization (WHO) recommends that the average requirement of b-carotene for adults should be between 2.4 mg and 3.5 mg. Vitamin A deficiency, a serious problem in several areas of the developing world, results in progressive eye damage. Pro-vitamin A carotenoids are a cheap source since they are found abundantly in plants (Graham and Rosser 2000). Selecting high-carotene-content cassava clones may therefore contribute significantly to reducing vitamin A deficiency in among the resource poor of the developing world. A recent survey of carotene contents in the roots of 2,457 landraces and bred-clones of cassava shows a wide range, varying from 0.102 to 1.040 mg pre 100 g1 fresh tissue, which correlated positively with color intensity and cyanogenic potential (Cha´vez et al. 2005). The range of carotene content in cassava was also assessed to select clones derived from wild species that are rich in carotene and have good


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palatability. In this regard, the colorimetric method for parenchyma tissue was used to identify cassava clones such as UnB-400, which showed 236 mg g1 of lutein compared to zero in other cultivars. The content of this clone was 2.2 mg g1. Its tuberous roots will help meet the daily adult requirements if 500 g of cassava are eaten daily. This clone was one of the most outstanding in palatability tests. It was easily cooked within 5 to 10 min turning to a very soft mass like a cream. The clone showed very low cyanide content, as judged by its taste. Another interesting finding was the content of both trans-b-carotene and lutein in leaves of clones UnB-400 and ICB-300. The transb-carotene content of this clone was 27.40 mg g1. ICB-300 had almost 20 mg g1 of this type of carotene. This clone is a hybrid between cassava and M. oligantha. It has 5% protein compared to 1.5% to 2% of common cassava (Nassar and Dorea 1982). The lutein amount in UnB400 and ICB-300 were 3081 and 9108 mg, respectively, which were about 4 to 12 times larger on carotenoid content than that of cassava clones. Due to its 4% protein content in the roots, 20 mg g1 of trans-bcarotene, and 9108 mg g1 of lutein in the leaves, the hybrid ICB 300 is an excellent source for improving the diet of cassava eaters. UnB-400 is also a very good source of vitamin A precursors. These results show the need for assessing cassava interspecific hybrids for carotene content because previous research by Nassar et al. (2004) showed that cassava hybrids deriving from M. dichotoma had double the carotene content (22 mg kg1) compared to cassava (13 mg kg1). D. Amino Acids Cassava roots are poor source of protein in spite of its quality and the proportion of amino acids therein. Essential amino acids in cassava are arginine, histidine, isoleucine, leucine, phenylalanine, threonine, tryptophan, and valine. The essential amino acids profile of cassava is deficient in sulfur-containing amino acids (methionine, cystine, and cysteine) (Bailey 1961). Osuntokun et al. (1968) pointed out that both cysteine and cystine are involved in the cyanide detoxication. Cyanide is produced when cyanogenic glucoside-linamarine present in cassava is hydrolyzed by linamarinase or by acid. The cysteine is mainly detoxificated by conversion to thiocyanate; in the process it reacts with cysteine and cystine. Excessive detoxication may be responsible for the low concentration of sulfur-containing amino acids. If cultivars can be bred with a higher quantity of amino acids, such as methionine and lysine, in the tuberous roots, it would enhance the value of cassava as a

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food or feed. Only about 60% of total nitrogen in cassava is derived from amino acids, and about 1% of it is in the form of nitrates and hydrocyanic acid. The remaining 38% to 40% of total nitrogen remains unidentified (Diasolua et al. 2002, 2003). According to Close et al. (1953), the protein of processed cassava includes the highest percentage of glutamic acid and the lowest of methionine (1%). Nassar and Sousa (2007) assessed amino acid compositions of Manihot proteins from the cassava cultivar UnB 101, an interspecific hybrid between cassava and M. oligantha (ICB 300, diploid), and its offpring (ICB 300 Progeˆnese 4, ICB 300 Progeˆnese 9, ICB 300 Progeˆnese 3, and ICB 300 Progeˆnese 10). Sample extracts were dialyzed against water to remove free amino acids, salts, monosaccharides, and other small molecules. Tryptophan could not be analyzed since it is degraded upon acid hydrolysis. By adding the amounts of the analyzed amino acids, it was possible to determine the protein content for each sample (Table 5.4). The interspecific hybrid ICB 300 offspring 3 Raiz showed the highest amount of protein (1.654 g 100 g1 of sample powder), followed by ICB 300 Diploid (1.454 g 100 g1) and ICB 300 Progeˆnese 9 (0.922 g 100 g1). The other samples (ICB 300

Table 5.4. Amino acid (AA) profile (g per 100g sample mass) in peeled roots of cassava cultivar UnB, its inter-specific hybrid with Manihot oligantha ICB 300 diploid, and its offspring (Progeˆnese 3, Progeˆnese 10, Progeˆnese 4, Progeˆnese 9). AA Ala Arg Asp Cys Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Tyr Val Total

UnB 01

Progeˆnese 3

0.020 0.037 0.016 0.027 0.039 0.012 0.000 0.008 0.016 0.010 0.014 0.016 0.000 0.012 0.008 0.000 0.019 0.254

0.093 0.261 0.146 0.029 0.222 0.078 0.038 0.068 0.131 0.098 0.041 0.129 0.054 0.088 0.061 0.000 0.115 1.654

Progeˆnese 10 0.017 0.061 0.023 0.026 0.044 0.012 0.010 0.010 0.013 0.020 0.004 0.058 0.000 0.013 0.007 0.000 0.027 0.344

Progeˆnese 4

Progeˆnese 9

ICB 300 Diploid

0.019 0.082 0.033 0.025 0.065 0.015 0.010 0.010 0.000 0.019 0.000 0.000 0.000 0.018 0.013 0.000 0.025 0.336

0.040 0.320 0.052 0.026 0.151 0.037 0.027 0.018 0.041 0.034 0.019 0.065 0.000 0.033 0.022 0.000 0.039 0.922

0.098 0.108 0.137 0.025 0.221 0.075 0.036 0.069 0.127 0.079 0.037 0.120 0.066 0.078 0.066 0.000 0.112 1.454


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Progeˆnese 10 Raiz, ICB 300 Progeˆnese 4, and UnB 01 Raiz) were poorer in protein content (0.350 g 100 g1). Essential amino acids were more concentrated in Progeˆnese 3 Raiz (His, Leu, Lys, Met, Phe and Val) and ICB 300 Diploid (lIe and Thr), with low or undetectable amounts in the other cultivars. Hence, Progeˆnese 3 Raiz and ICB 300 Diploid would be more interesting for human consumption due to their nutritional traits. Progeˆnese 3 and 9 showed equal protein amounts and doubling that of cassava. These results show the potential to select for high protein in the derived cassava hybrids. The biological value (Block and Michell equivalent) of the total cassava protein is 48%. The crude protein content of roots appears to be relatively stable and constant with plant maturity. Signification variation in protein content (ranging from 0.95% to 6.42%) was observed in a sample of accessions (149) from the CIAT gene bank (Ceballos et al. 2006). Cassava breeders have also pursued the increase of protein content in the tuberous roots by interspecific hybridization with wild species, namely M. saxicola and M. mefanobasis. Over a period of 10 years beginning 1932 and ending with the Japanese occupation of Java in 1942, Bolhuis (1953) carried out a program of cassava breeding for increased protein content in roots. Crosses with M. saxicola yielded a few seedlings with as much as 2% protein in fresh roots. Jennings (1957) reported that the roots of the F1 progeny of M. esculenta M. melanobasis possessed approximately twice as much protein as their cassava parent. The offspring were lost and not cultivated anywhere, probably because of poor root yield. Barros and Bressani (1967) reported cassava cultivars with high protein content (7%). It could be, however, that the nitrogen referred to in such cultivars was either protein or the breakdown of cyanogenic glucosides. Hence, cassava breeders should be aware that cultivars of high nitrogen content could be bitter with high glucoside content. Humidity when drying the materials is another factor that interferes when assessing protein as total nitrogen. Excessive drying of the root powder may increase drastically percentage of nitrogen by threefold.

VI. OUTLOOK Cassava cultivars are deficient in many economic characteristics, such as resistance to pests and tolerance to drought, and have low protein content (Nassar and Dorea 1982; Nassar and Grattapaglia 1986). Such features can be attributed to the mode of evolution in the species and modifications of the allogamy system of the plant (Nassar and O’Hair

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1985). Interspecific barriers to hybridization between cassava and wild Manihot species are weak and can be easily overcome (Nassar et al. 1995). The cultigen pool could therefore be broadened by interspecific hybridization with wild Manihot relatives that possess desired genes (Nassar 1994b, 1996; Nassar and Grattapaglia 1986). Furthermore, cytogenetic research suggests that while polyploidy offers a means for rapid speciation of the Manihot genus, apomixis provides an opportunity for perpetuating hybrid genotypes that may be better adapted to target environments (Nassar 2000b). The advances in cassava genomics and the ensuing knowledge and molecular breeding tools will facilitate further incorporation of the wild Manihot and cassava landrace genetic resources into the breeding populations of this crop. This enhanced genetic endowment of the breeding materials will allow further selection of cassava clones with valuable traits. After extensive testing on station, on farm, and with farmers (Manu-Aduening et al. 2006), this will lead to new cultivar releases with a broaden genetic diversity. However, the occurrence of wild Manihot in natural habitats seems to be decreasing; a few are threatened with extinction (Nassar 2006a), and genetic erosion may occur in landraces elsewhere (Willemen et al. 2007). Hence, governments in centers of Manihot diversity together with international aid and other investors interested in biodiversity conservation should spend their resources to preserve this genetic endowment for the betterment of a crop that feeds the developing world, especially in resource-poor areas.

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6 Breeding Roses for Disease Resistance Vance M. Whitaker and Stan C. Hokanson University of Minnesota Department of Horticultural Science 1970 Folwell Avenue St. Paul, MN 55108 USA

I. INTRODUCTION II. CAUSAL PATHOGENS A. Black Spot Disease B. Powdery Mildew Disease C. Other Diseases III. RESISTANCE SCREENING A. Black Spot B. Powdery Mildew C. Other Diseases IV. BREEDING A. Resistance Genes B. Breeding Methods V. MOLECULAR TOOLS A. Markers and Mapping B. Candidate Gene Approaches C. Other Approaches 1. Transformation 2. Somatic Hybridization VI. FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION The cultivated rose (Rosa hybrida L., Rosaceae) is arguably the world’s most famous ornamental plant. Roses are utilized in the florist industry as both cut stems and potted specimens. Garden roses such as hybrid teas, shrubs, polyanthas, and other types with various flower colors and forms Plant Breeding Reviews, Volume 31 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 277

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fit a wide range of landscape niches. Rose hips possess high levels of vitamin C and cancer-preventing compounds in addition to their ornamental attributes (Wen et al. 2006). Even rose prickles have visual beauty in their diverse colors and types. As a result, roses are also the world’s most economically important ornamental crop. As of 2003, 8 billion cut stems, 80 million potted specimens, and 220 million landscape roses were sold annually (Roberts et al. 2003). In the United States alone in 2005, the wholesale value of cut flower roses was $40 million, with the wholesale value of imports reaching $277 million (USDA 2006). Low-maintenance roses for landscape use is one market that is growing in popularity since roses bloom over an extended period and great diversity can be found for traits such as flower color, plant habit, and fragrance (Lonnee 2005). Disease susceptibility poses the greatest challenge for producing and maintaining quality roses for all market niches but especially lowmaintenance landscape types. A litany of microbes are pathogenic on roses, several of which are capable of serious damage. Black spot disease, caused by Diplocarpon rosae Wolf, is considered the most severe disease of landscape roses due to the potential for quick defoliation leading to compromised plant health and unsightly appearance (Dobbs 1984). Powdery mildew of roses, caused by Podosphaera pannosa (Wallr.: Fr.) de Bary, is another significant and widespread disease of roses that causes damage in landscapes and especially in greenhouses (Horst 1983). The general disease susceptibility of cultivated rose, combined with its importance as an ornamental plant, makes host resistance a worthy breeding goal. Furthermore, concerns over pesticide use in general also motivate the search for more natural methods of disease control. Development of durable genetic resistance as a control strategy should prove to be more efficient and environmentally sustainable than chemical pesticide application. Therefore, substantial research has been conducted in recent years using new molecular tools to elucidate the biology of rose pathogens, develop methods for resistance screening, and devise strategies for resistance breeding. Here we examine the extant literature on the biology and life history of the major pathogens causing disease on rose including fungi, bacteria, nematodes, and viruses. The review focuses on the pathogens causing black spot and powdery mildew diseases due to their primary significance. We then review efforts to screen for resistance genes in roses, the utilization of genes in breeding efforts, and the use of molecular approaches such as markers, candidate genes, and genetic transformation. Finally, we discuss the future prospects for breeding and development of disease-resistant roses.

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II. CAUSAL PATHOGENS A. Black Spot Disease Rose black spot is caused by an infection of the fungus Diplocarpon rosae (Marssonina rosae anamorph), an ascomycete fungus in the Dermateaceae. This organism is obligate to the genus Rosa and thus does not infect any other plant taxa. D. rosae is hemibiotrophic, which means that it is parasitic on living host tissue but also has limited ability for saprophytic growth (Blechert and Debener 2005). Diplocarpon rosae is mainly spread through asexual spores called conidia. Conidia are produced in abundance from acervuli within infection sites on leaves and stems. These conidia overwinter on dormant stems and fallen leaves until they are disseminated to new growth in the spring via water splash (Horst 1983). Although the younger, upper leaves are more susceptible than older leaves, the lower leaves usually are infected first, presumably because they are most affected by water splash. Free water is necessary for the fungus to germinate and directly penetrate the epidermis of rose leaves and stems, even when humidity is 100% (Horst 1983). On susceptible rose genotypes, subcuticular hyphae radiate from the infection site followed by branching intercellular hyphae that give rise to intracellular haustoria (Blechert and Debener 2005). Symptoms may appear to the naked eye in as little as 4 days from infection, with acervuli rupturing the leaf surface in 10 to 14 days (Horst 1983). As lesions expand in size, characteristic yellowing of leaf tissue occurs and is soon followed by defoliation. The damage to plant health and appearance that results from defoliation is what makes black spot the most serious disease of roses in the outdoor landscape (Dobbs 1984). There are challenges to culturing and storing D. rosae that are pertinent to breeders who wish to perform controlled inoculation experiments. Fungal hyphae grow slowly across culture media, and spores produced in culture may not be virulent. To ensure the accuracy of inoculation tests, care must be taken to maintain pathogenicity when storing isolates long term and when growing inoculum in culture plates. The fungus is commonly isolated and cultured on potato dextrose agar (PDA), yeast malt extract agar (YMEA), and biomalt media. However, cultures begin to lose pathogenicity after six months at 23 C (DrewesAlvarez 1992). Refrigeration does not solve this problem, as cultures stored at 1 C to 2 C declined gradually and lost all infectivity after 13 months (Palmer et al. 1996b). Researchers have resorted to maintaining single-spore isolates by freezing infected leaf pieces. Results at 15 C to 20 C are better than at 1 C to 2 C, with conidia on leaf surfaces

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remaining viable for a year (Knight and Wheeler 1978a; Yokoya et al. 2000; Leus 2005). Yet cryogenic storage appears to be the most effective method for preserving black spot isolates long term. Castledine et al. (1982) succeeded in freezing conidia for 24 hours in liquid nitrogen (196 C) with no loss of viability. Subsequently, Whitaker et al. (2007b) successfully stored infected leaf pieces for several months both in the gas phase of liquid nitrogen and at 80 C. These low-temperature treatments were superior compared to 20 C for three different isolates. Conidia harvested from culture plates have also been stored in glass beads above liquid nitrogen (Carlson-Nilsson 2002), although the effectiveness of this method has not been reported. It is advisable to periodically reinfect isolates to susceptible rose cultivars in order to maintain pathogenicity. Spencer and Wood (1992a) bypassed long-term storage strategies altogether by reinoculating isolates to greenhouse-grown roses every 2 to 3 months. Walker et al. (1996) found that conidia grown in culture on MEA did not infect leaf discs excised from ‘Frensham’ rose leaves grown in vitro, even when the leaf surface was mildly abraded. Yet the same conidia successfully infected ex vitro grown ‘Frensham’ leaves at an average rate of 61.7%. The conidia produced on these ex vitro leaves were then capable of infecting ‘Frensham’ leaves grown in vitro. The authors suggested that conidia grown in culture may have been less virulent. Based on these results, the authors subsequently stored all isolates on ex vitro leaves. Evidently, a totally in vitro system is not effective for maintaining virulent D. rosae isolates. Diplocarpon rosae appears to be a diverse species, as indicated by phenotypic observations and genetic studies. Conidial morphology and colony color are quite variable among isolates when grown in culture (Wenefrida and Spencer 1993; Whitaker et al. 2007b). Genetic analysis of the internal transcribed spacer (ITS) region of rDNA by restriction fragment length polymorphism (RFLP) analysis separated 10 D. rosae isolates into three groups with distinct RFLP patterns (Lee et al. 2000). Preliminary studies by British researchers have revealed genetic diversity in a collection of isolates by using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, concluding that AFLP was a sensitive and reproducible marker system appropriate for D. rosae (Drewes-Alvarez 2003). AFLP analysis of 50 isolates collected from 14 locations throughout eastern North America revealed significant diversity among isolates (Fig. 6.1). Over 20% of AFLP fragments were polymorphic, reflecting a higher level of diversity than has been found in AFLP studies of some other fungi in the Dermataceae family, including Pyrenopeziza brassicae


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Fig. 6.1. Polymorphisms between five different Diplocarpon rosae single spore isolates collected from eastern North America, as revealed by AFLP. Pairs of lanes for each isolate represent duplicate selective amplifications.

(pathogenic on crucifers) and Tapesia yallundae (pathogenic on wheat). Analysis of the marker data for D. rosae showed considerable diversity among isolates within each collection site and did not reveal any clustering of isolates based on geographic location (Whitaker et al. 2007a). It is possible that the commercial distribution of roses throughout North America could contribute to this phenomenon, through the movement of D. rosae on infected vegetative propagules. The results suggest that screening for resistance in one location could be as effective as screening in multiple locations in this region. However, field screening within even a single location is problematic because of uneven disease progression due to environmental factors and nonuniform distribution of pathogenic races. This problem can be avoided by artificially inoculating with known races in the greenhouse and lab.

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Although the diversity of D. rosae does not appear to be geographically structured in eastern North America, a random amplified polymorphic DNA (RAPD) analysis of 15 isolates collected from locations in Canada, France, Sweden, and the United States exhibited some clustering based on geography (Carlsson-Nilsson 2002). It is not known to what extent sexual recombination contributes to diversity, but the sexual stage of D. rosae has been observed only twice in England and twice in North America (Horst 1983). Conclusions about sexual recombination based on these limited observations should be guarded, since recent genetic studies have revealed recombination in several apparently asexual species of fungi in the Ascomycota (Taylor et al. 1999). Genetic diversity of D. rosae is also reflected in a diversity of pathogenic races. Races are determined by inoculating multiple isolates to a collection of host genotypes. Isolates that can be differentiated from one another based on their host range are called races or pathotypes. In 1977, the previously resistant rugosa rose cultivar ‘Martin Frobisher’ suddenly became infected with black spot (Bolton and Svejda 1979). Further inoculation tests in Ontario, Canada, revealed the presence of four different races of the fungus (Svejda and Bolton 1980). In 1992, seven races were discovered in a small, seven-county area in Mississippi (Spencer and Wood 1992a, 1992b). Meanwhile, five races were discovered in Germany (Debener et al. 1998). Four races were differentiated in Belgium (Leus 2005), and four races were described in England (Yokoya et al. 2000). Recently three races were differentiated from among 14 isolates collected in eastern North America (Whitaker et al. 2007c). These three races are the only described races from North America that are known to be maintained long term in a virulent state. The loss of other collections is presumably due to the aforementioned difficulties in storing this fungal species. Pathogenic races described in Europe and North America have not yet been compared, although a standard host differential set is currently being established. B. Powdery Mildew Disease Powdery mildew fungi are classified in the family Erysiphaceae, in the class Ascomycetes. The Erysiphaceae is comprised of 13 genera and approximately 650 species, although detailed studies utilizing modern techniques have not been applied systematically within the family (Braun et al. 2002). Powdery mildews are obligate, biotrophic fungi, meaning they can survive only on cells in specific living hosts. Despite their restrictive host specificity, powdery mildews are ubiquitous,


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infecting all crop plants worldwide with the exception of pines and their relatives (Horst 1999). Most powdery mildew infections are restricted to the epidermal surface, appearing as masses of white hyphae that exist in mats on the leaf surface (Bushnell 2002). Hyphae are attached to the leaf by pegs that penetrate the epidermal cell walls. At these attachment points, haustoria form, which allow the fungus to absorb nutrients from the leaf tissue. The characteristic powdery white appearance associated with a powdery mildew infection results from vegetative hyphae or conidiophores. The hyphae grow out from the leaf and produce chains of asexual spores or conidia. Sexual spores occasionally are produced in spherical structures, ascomata, which appear as reddish-brown dots in the hyphal mats. Powdery mildew disease of rose is incited by an infection of the fungal organism Podosphaera pannosa (Wallr.: Fr.) de Bary. Prior to revisions in the generic system (Braun and Takamatsu 2000), the organism was known as Sphaerotheca pannosa var. rosae (Wallr.: Fr.) Le´v., which was differentiated from S. pannosa var. persicae Woron., a Prunus-infecting isolate. Morphological and DNA sequence data did not differentiate the two species (Takamatsu et al. 1998; Saenz and Taylor 1999), and they were subsequently considered to be a single species that is capable of infecting rose and Prunus species. Powdery mildew disease of rose is the most widely occurring disease of rose worldwide (Horst 1983). While occurring on garden-grown roses, the disease occurs most frequently and has the largest impact on greenhouse-grown roses (Linde and Shishkoff 2003). Powdery mildews propagate primarily through vegetative means, via asexual spores or conidia. In the outdoor environment, infections occur in autumn on dormant buds where the pathogen overwinters. When plant growth resumes the following spring, the fungus on the newly emergent foliage produces conidia that are dispersed via wind. Conidia land on newly emergent plant tissue. Germination and initial development of the fungus is enhanced by elevated humidity but impeded by the presence of free water (Sivapalan 1993). In the greenhouse, infections generally begin with the introduction of infected plants or via conidia through vents, fans, insects, human hands, or clothing. Infections spread rapidly in the highly conducive environment created in the greenhouse. Once an infection of powdery mildew is established in a greenhouse, it is difficult to eradicate. An eradication effort typically entails disposal of all rose plants, settling of all conidia, disinfecting all greenhouse surfaces, and establishment of a more rigorous prevention protocol.

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Conidia germinate optimally (within 2 to 6 hours) at a temperature of 23 C and 100% humidity (Xu 1999). Temperatures above 30 C were reported to prevent germination (Xu 1999). In humid conditions, conidia can survive for up to 3 months at low temperatures, 0 C to 3 C (Price 1970). Conidia germination and fungal growth can occur in relative humidity as low as 50%, although at decreased levels (Rogers 1959; Pathak and Chorin 1969). Other environmental factors that promote powdery mildew infection and disease progression include shade, closely spaced plantings, and profuse foliage growth, all of which serve to reduce air circulation and/or promote increased humidity (Linde and Shishkoff 2003). In optimal conditions conidia can germinate, colonize, and produce new conidia in 4 to 10 days (Linde and Shiskoff 2003). The fungus is capable of producing sexual fruiting bodies in late summer if both mating types are present. The fruit bodies called ascocarps are formed by the sex organs of the fungus, the gametangium and the ascogonium, on lateral branches of the mycelium. The ascocarps contain 4 to 8 ascospores that are capable of infecting young rose tissue. In field observations reported by Price (1970), ascocarps were rare and were found in only 5% of cases. However, cleistocarps (equivalently termed ascocarps) were reported in over 700 rose species and cultivars inspected over three seasons (Price 1970). The presence of ascocarps offers the opportunity for genetic recombination and the formation of new races of the pathogen. After germination, an appressoria develops at the end of the germination tube, which attaches the mycelium to the rose plant surface by a fine slime layer (Hajlaoui et al. 1991). A penetration peg emerges through a pore in the appressorium and enters the cuticle and underlying epidermal cell wall. In the epidermal cell, the penetration peg enlarges to form the haustorial neck. From the center of the attachment of the appressoria, multilobed, globose mature haustoria are formed (Hajlaoui et al. 1991). The haustoria serve to absorb nutrients for the fungus from the rose host. Haustoria continue to form as hyphae extend along the leaf surface. In addition to the environmental factors just noted, symptoms of powdery mildew infection in rose also can be influenced by the host tissue infected, age of the tissue infected, as well as host genotype. Rose leaves are most readily infected in the first 3 days after unfurling (Linde and Shishkoff 2003). Infections at this stage typically result in twisted, stunted, or distorted leaves. Powdery mildew can spread quickly on young leaves, leading to shriveling and defoliation. Infections on young leaves often result in dark reddish spotting on


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the leaf before appearance of the characteristic powdery white hyphal growth. Older leaves are less susceptible and infection progression is slower, often resulting in a patchy occurrence of the fungus. Young rapidly extending stem tissue can become infected, often where a thorn attaches. The infection generally will persist as the stem matures, resulting in irregular powdery patches of fungus on the stem. Fungal infections also can occur on the flowers. Generally infections start on unopened flowers on the calyx of unopened flower buds. The infection often spreads to the petals as the flowers open, leading to distorted, poor-quality flowers (Horst 1983). Numerous claims of powdery mildew resistance for specific cultivars are made in popular and nursery trade literature, but durable resistance to the disease is seldom seen. Initially, inoculations and observations of rose cultivars and species revealed little difference between species and few cultivars with broad resistance (Mence and Hildebrandt 1966; Atkiss 1978). Subsequent inoculations with single conidial isolates have confirmed the presence of multiple races of powdery mildew (Bender and Coyier 1984; Linde and Debener 2003), but not all tests have differentiated races (Leus et al. 2002, 2005). C. Other Diseases Other fungal diseases include, but are not limited to, botrytis blight caused by Botrytis cinerea Pers. ex Fr., Cercospora leaf spot caused by Cercospora puderi B.H. Davis and Cercospora rosicola Pass., downy mildew caused by Peronospora sparsa Berk., rust caused by various species of the genus Phragmidium, and spot anthracnose caused by Sphaceloma rosarum (Pass.) Jenkins (Horst 1983). Botrytis blight is an important disease of many crop plants. It is significant on roses mainly because of its detrimental effect on flower bud appearance, although it also can cause blighting of canes (Horst 1983). Botrytis cinerea is biologically amenable to controlled inoculation experiments. Isolates can be grown easily and maintained in culture, but loss of pathogenicity is known to occur and should be considered (Pie and Brower 1993). Cut roses often are infected during storage and shipping because of cool, moist conditions. Optimum conidial germination of occurs at 15 C, and lesions appear on petals as early as 8 to 12 hours postinoculation when relative humidity is maintained at 100% (Horst 1983; Pie and Brouwer 1993). Finding genetic resistance is increasingly important, since populations of B. cinerea have developed resistance to fungicides (Gullino and Garibaldi 1996).

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Downy mildew causes few problems in low-humidity environments, but under cool conditions with humidity greater than 85%, leaf abscission can be more serious than for black spot disease (Horst 1983). Downy mildew symptoms include purplish or reddish-brown irregular spots on the upper leaf surface, with mycelia and conidial growth occurring on the lower leaf surface. Free water or high humidity is required for spore germination, with spores germinating at a high rate between 2 C and 18 C (Xu and Pettitt 2003). Conidia may be stored in frozen water suspensions (15% DMSO) for 6 months at 20 C without complete loss of virulence (Schulz and Debener 2005). Opinions differ as to whether P. sparsa overwinters as mycelia on or within stem tissue. Xu and Pettitt (2003) report that oospores appear to be the main source of overwintering inoculum and that winter sanitation of diseased leaves is an effective control measure. However, polymerase chain reaction (PCR) and microscopic assays as well as fungicide trials have confirmed the presence of perennial infections of P. sparsa within rose stem tissue (Aegerter et al. 2002). For this reason, chemical treatment and/or sanitation of propagation source plants may be necessary. Spot anthracnose is continually spread by water-borne conidia during the summer months. Occasionally its incidence can be as serious as a black spot infestation under humid conditions. Spot anthracnose is commonly mistaken for black spot but can be visually distinguished by the purplish cast of young lesions, the centers of which ultimately turn gray or white and fall away, leaving a shot-hole appearance (Horst 1983). Ethylene production is increased in rose leaflets infected with this disease (McClellan 1953). Other than this, little information has been published on the biology of this disease, which is surprising considering its prevalence. Crown gall is caused by the bacteria Agrobacterium tumefaciens Conn., which typically enters the plant through stem wounds. Infection results in masses of undifferentiated tissue that can reach several centimeters in diameter on susceptible roses, resulting in serious damage if located at the root-shoot junction (Horst 1983). Agrobacterium tumefaciens can remain latent in rose tissues at temperatures below 10 C but will remain viable and cause symptoms when higher temperatures are reached. Wounding of stem tissue should be minimized during cultivation and handling, and pruning tools should be disinfected to prevent spread (Gullino and Garibaldi 1996). Isolation from roses can be performed by macerating galls and growing the gall extract on peptone-yeast-glucose agar (LPGA) medium (Pionnat et al. 1996). However, not all isolated strains can cause tumors on rose.


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Only strains containing the Ti plasmid, a circular strand of DNA, are pathogenic. The Ti plasmid contains two elements necessary for tumorigenesis, the transferred DNA (T-DNA) and vir genes. The T-DNA, which encodes plant growth hormones and other compounds, is transferred into the plant cell nucleus and incorporated into the host genome. The vir genes mediate the transfer and incorporation process (Sheng and Citovsky 1996). Therefore, virulent strains may be detected by confirming the presence of the Ti plasmid. This has been accomplished in roses with PCR, using primers specific to portions of the vir genes and T-DNA (Pionnat et al. 1996). Nematodes pathogenic to roses are distributed worldwide and can cause symptoms of dwarfing, reduced vigor, wilting, and chlorosis. Horst (1983) lists an astounding 18 genera that have been found on roses. The two most important species infecting roses are Meloidogyne hapla Chitwood (root-knot nematode) and Pratylenchus vulnus Allen and Jensen (root-lesion nematode) (Voisin et al. 1996). Races of M. hapla have been reported based on an inoculation of isolates obtained from Canada and France to Rosa indica (Wang et al. 2004). Nematode infestation is most problematic in field production nurseries. Methyl bromide fumigation of the soil has been an effective control measure in the past. However, this chemical is being phased out by law because of its harmful environmental effects, and alternative fumigants currently are being tested for their effectiveness (Schneider et al. 2005). Like nematodes, viruses can cause symptoms of stunting, chlorosis, and general decline. Though there is no mandatory virus-free certification program, the Foundation Plant Services (FPS) at the University of California, Davis, and Florida Southern College provide virus-tested material (Manners 1993). By rule, new All-America Rose Selections winners are required to enter the FPS program (Foundation Plant Services 2007). At least 11 different virus diseases are reviewed by Horst (1983). Among the important causal viruses affecting rose are rose mosaic virus (RMV), strawberry latent ringpot virus (SLRV), and rose streak virus (RSV). A previously undescribed filamentous virus of roses, preliminarily named rose yellow mosaic virus (RoYMV), was discovered in 2004. Rose yellow mosaic virus causes severe yellow sectoring and mosaic symptoms on leaves and necrotic ringspots on the stems of at least one genotype (Fig. 6.2). Nucleotide and amino acid sequence comparisons to previously described viruses yielded no close matches (Lockhart and Olszewski 2007). For purposes of breeding, development of robust methods for controlled inoculation of the major viruses and rating of symptoms would be beneficial.

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Fig. 6.2. Disease symptoms on leaves of Rosa ‘Ballerina’ infected with the newly described filamentous virus RoYMV. (Source: Courtesy of Ben Lockhart, University of Minnesota.)

III. RESISTANCE SCREENING A. Black Spot For purposes of genotype evaluation and breeding, resistance to rose black spot disease can be broadly categorized into race-specific resistance and partial resistance types. The aforementioned inoculation tests that have differentiated D. rosae pathogenic races in North America and in Europe have done so on the basis of clearly discerned compatible and incompatible interactions. For example, Debener et al. (1998) rated interactions susceptible (compatible) if the fungus grew outside the original spore suspension droplet and if spore-bearing acervuli were formed on the leaflet surface. Resistant (incompatible) reactions, however, were characterized by exhibiting neither of these symptoms. Such resistance can be conferred by rapid host cell death called the hypersensitive response (HR), which was documented in ‘Allgold’ rose by Kuklinski (1980) and in R. wichuriana and R. roxburghii by Wiggers et al. (1997). Blechert and Debener (2005) observed two types of HR in species roses, one in which single cells died and one in which larger cell clusters died. The latter response was presumably due to a delay in HR until after some fungal development within the host tissue had already occurred. They also reported another type of resistance in which no fungal structures penetrated below the


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leaf cuticle. Similarly, Reddy et al. (1992) observed the death of spores on the leaf surfaces of resistant species and concluded that spore development halted at germination on these species, possibly due to germination inhibitors exuded from the leaf epidermis. This result is consistent with reduced conidial germination on the leaflet surface of the resistant ‘Allgold’ (Knight and Wheeler 1978b). Partial resistance is an incomplete form of resistance characterized by suppression of pathogen colonization after infection. This type of resistance is generally considered to be race nonspecific. Blechert and Debener (2005) described multiple interaction types on 34 different rose species, separating susceptible reactions into five different classes based on hyphal morphology, location, and length. In some susceptible reactions acervuli were produced, but subcuticular and short-distance hyphal growth was restricted. Kuklinski (1980) described black spot infections in which haustoria formed but were retarded in development or became enclosed in a ‘‘convoluted’’ membrane. It appears that the cuticle can also play a role in partial resistance, and it may be advisable in some cases to abrade the leaflet surface prior to inoculation in order to eliminate this variable (Castledine et al. 1981). On a macroscopic level, partial resistance reactions can be observed according to the timing of development as well as size and number of black spot lesions. Xue and Davidson (1998) formalized these observations into five measures, or ‘‘components,’’ of partial resistance that were used to screen roses from the Agriculture and Agri-Food Canada breeding program in Morden, Manitoba. These are incubation period (IP), leaf area with symptoms (LAS), number of lesions (NL), lesion length (LL), and sporulation capacity (SC). Four of the components, LAS, IP, NL, and LL, were positively correlated with one another, although SC was not significantly correlated with IP, NL, and LL. From this analysis, the authors concluded that, in light of limited resources, LAS and SC were the two most useful components, although SC is more difficult to measure than the visual rating of LAS (Fig. 6.3). Leaf drop may be considered another component of partial resistance. It is not well correlated with lesion development (Kuklinski 1980; Whitaker et al. 2007c), since some cultivars may retain their leaves despite severe infection, while other mildly infected clones drop their leaves rapidly. In one example, rooted cuttings of a diploid rose accession (H71) dropped all their leaves only 12 hours after inoculation and subsequently grew disease-free shoots (Svejda and Bolton 1980). Such a mechanism may effectively sanitize the plant by rapid removal of future inoculum sources. The production of ethylene by D. rosae combined with auxin degradation may contribute to leaf

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Fig. 6.3. Rose leaflets infected with D. rosae. The partial resistance rating for the center leaflet (lesions highlighted) was 20.3% leaf area with symptoms (LAS), as measured with Assess Software. (Source: APS Press, St. Paul, MN.)

yellowing and abscission (Horst 1983; Kazmaier 1961). Ethylene has been implicated in the premature defoliation that occurs as a result of several diseases and is known to be produced by fungal and bacterial pathogens (Agrios 1997). If leaf abscission in response to black spot infection is correlated with general ethylene sensitivity, ethylene applications may be used to screen against early leaf drop. Single-spore isolates are important for studies of both race-specific and partial resistance. Without the ability to isolate and produce inoculum from single spores, inoculation results may be inconsistent, depending on what combination of races is present when spores are collected in the field. Palmer and Seminuk (1961) collected spores from the field because of their immediate need for large amounts of inoculum. However, interpretations of their results were limited because the races they inoculated were unknown. Producing inoculum from single spores in culture may be worth the wait, since large numbers of spores can be produced from cultures on PDA in 7 to 8 weeks, with tens of millions of spores produced from each plate (Xue and Davidson 1998). If fewer inocula are required, single-spore isolates produced in culture may be inoculated to surface-sterilized leaves. The infected leaves may be frozen and subsequently thawed to harvest spores. Detached leaf assays and leaf disc assays with single-spore isolates have been used successfully for determining races and identifying the presence and absence of major resistance genes (Debener et al. 1998; Von


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Malek and Debener 1998; Yokoya et al. 2000). Whole-plant inoculations have been used so far in studies of partial resistance (Xue and Davidson 1998; Whitaker et al. 2007c). Since detached leaf assays are efficient and can be performed in the lab, they would also be convenient for screening partial resistance. Jenkins (1955) reported a near-perfect correlation coefficient (r ¼ 0:97) between a comparison of lesion sizes in detached leaflet and whole-plant assays for four cultivars and one species inoculated with three isolates of D. rosae. This suggests that finer distinctions between levels of susceptible reactions also may be measured using detached leaves. Other research, however, raises the question of whether detached leaf assays are accurate for measuring partial resistances. Walker et al. (1996) documented susceptibility on detached leaflets of ‘Alberic Barbier’ rose, which was noted to be resistant in the field. In addition, the growth of D. rosae was repressed by cell suspensions from ‘Alberic Barbier’ root callus. From this it was suggested that diffusible substances produced away from leaves could have a role in resistance. Palmer et al. (1966a) reported that the deterioration of detached leaflets made rating impractical for some rose genotypes. They also noted that this method may not accurately measure the effects of vigor in whole plants. Knight and Wheeler (1978a) commented on a lack of whole plant realism in leaf disc assays, but suggested that leaf disc assays could serve as a complement, rather than a replacement, to field trials. It also should be noted that the defoliation response of the plant to infection is important and can be determined only using whole plants. Therefore, the correlation between whole plant and detached leaflet methods should be tested before the latter is utilized for genetic studies of partial resistance against multiple races. Such experiments are currently under way at the University of Minnesota (unpublished data). Black spot screening in commercial breeding programs still is carried out in the field (Noack 2003). Yet Carlson-Nilsson (2002) compared controlled inoculation data and field data and concluded that greenhouse inoculations of whole plants could be used as a substitute for field trials. Greenhouse inoculations have advantages over natural field inoculations, since it takes several years for pathogen pressure to build up in field plots (Carlson-Nilsson 2000). Also, correlations between disease ratings among years can be low due to nonuniform spread of inoculum and climatic differences (Leus 2005). Greenhouse screening is only effective when high humidity is maintained for at least 24 hours after inoculation. This can be accomplished using plastic tents. Leus (2005) found that for seedling selection in the greenhouse, it is best to

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apply two or three repeated inoculations. They also found that an inoculum concentration of only 2 102 conidia/ml was sufficient when inoculations were repeated. Although race-specific resistance and partial resistance are useful categories in terms of breeding and genetics, they do not directly describe the underlying biology of plant defense. Plant response to pathogen attack involves a myriad of pathogen receptors, signal transduction pathways, and defense-related gene products. Some defenses are constitutive, but other defense compounds are induced only after pathogen attack. For example, polyphenol and phytoalexin production increase during black spot infection (Saunders 1967). Two categories of inducible resistance are called systemic acquired resistance (SAR) and induced systemic resistance (ISR). They are differentiated by the nature of their signaling cascades. SAR is dependent on salicylic acid as a signal molecule, and ISR is dependent on jasmonic acid and ethylene signaling (Van Loon 1999). Several families of pathogenesis-related (PR) proteins have been identified that are actively involved in resistance in many species. Interestingly, PR proteins are associated with SAR but not with ISR (Van Loon 1999). Several families of PR proteins (PR-1, PR-2, PR-3, and PR-5) accumulate in D. rosae– infected rose leaves, and all except PR-1 are systemically induced in uninfected upper leaves (Suo and Leung 2002a). It also appears that the expression of two specific types of PR proteins, b-1,3-glucanase and chitinase, contribute to the reduction of black spot symptoms on in vitro rose plants. These antifungal compounds are known to degrade fungal cell walls into short oligosaccharides, which may serve as signals for other downstream defense responses (Suo and Leung 2001a). Screening for differences in induced resistance may be possible by inducing SAR with synthetic compounds such as acibenzolar-Smethyl (BTH), which is thought to affect the SAR signal transduction pathway downstream of salicylic acid (Ryals et al. 1996). Pathogenesis related proteins can be induced within 3 days of BTH treatment, and BTH treatment prior to inoculation of in vitro plantlets reduces black spot symptoms in rose (Suo and Leung 2001b, 2002b). Systemic acquired resistance may be a mechanism underlying the partial resistance phenotype in rose. If so, BTH applications to selected genotypes would distinguish their potential for partial resistance. B. Powdery Mildew In 1966, Mence and Hildebrandt tested the resistance of various rose cultivars and species using polysporous inoculum on detached leaves.


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Their results allowed several initial insights into the resistance of roses to P. pannosa and implications for screening methods. Inoculations were performed by gently brushing clean leaflets with infected leaflets. Although they did not utilize single-spore isolates, host-specificity was nonetheless apparent, as isolates collected from R. virginiana were not infective on some cultivars. Degree of resistance was rated for susceptible genotypes according to the percentage of leaf area covered by sporulating colonies. It was noted that susceptibility decreased dramatically with leaf age. Some roses may become resistant more quickly than others as their leaves age. For instance, ‘Queen Elizabeth’ became resistant at a faster rate that ‘Christopher Stone’ (Mence and Hildebrandt 1966). Therefore, in order to avoid the genotype-specific effects of aging, leaves must be inoculated within a few days of unfolding. The cuticle was deemed to have little role in resistance since abrasion had a negligible effect on susceptibility (see also Conti et al. 1985), although conflicting evidence on this point has been presented for other genotypes (Ferrero et al. 2001b). Rosa rugosa leaflets were susceptible only when inoculated on the abaxial surface; all other genotypes were effectively inoculated on both surfaces. Since this initial study, resistance has been identified in other commercial cultivars as well as in species such as R. multiflora, R. wichuriana, R. laevigata R. rugosa hybrids, and others from sections Caninae (R. agrestis and R. glutinosa) and Pimpinellifoliae (R. foetida var. persiana) (Ferrero et al. 2001a; Linde and Debener 2003; Linde and Shishkoff 2003). Chinese accessions of R. sterilis and R. chinensis are reported as resistant, with different accessions of R. roxburghii exhibiting a wide range of symptoms (Wen et al. 2006). Screening methods have been improved over the years, and single-spore isolates are now utilized for the differentiation of races and screening of populations for resistance genes (Bender and Coyier 1984; Linde and Debener 2003; Leus et al. 2006). Unlike black spot disease, germination of P. pannosa conidia on the leaf surface does not appear to be related to host resistance and is not a good indicator of susceptibility (Conti et al. 1985). Instead, resistance mechanisms are initiated only during or after penetration. Host cell death is a common means of resistance and appears to be associated with the accumulation of phenolic compounds (Conti et al. 1986) and hydrogen peroxide (Dewitte et al. 2007). The HR is localized to single cells in some resistant genotypes, but a delayed HR may affect multiple cells and be visible to the naked eye (Linde and Shishkoff 2003). Although necrosis of groups of cells can be indicative of resistant genotypes (Wen et al. 2006), browning of multiple cells also can occur

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in conjunction with sporulation and mycelial development (Dewitte et al. 2007). Therefore, the occurrence of visible browning is not by itself a sufficient indicator of resistance. In other cases, resistance mechanisms of roses such as R. laevigata anemoides may include papillae formation and abnormal haustoria in the absence of any cell reaction or collapse (Dewitte et al. 2007). Therefore, most rating systems measure the degree of sporulation on infected leaves, since this is the most reliable and most easily observed indicator of susceptibility. If disease measures such as percent of leaf area covered with conidiophores are to be used, care should be taken to quantify spore density during inoculations and apply inoculum evenly. This is not possible when using infected leaves or camel’s-hair brushes to apply P. pannosa conidia. Therefore, settling towers that use compressed air to disperse dry conidia have been developed. Linde and Debener (2003) used such an inoculation tower to distribute approximately 2 conidial/ mm2 onto detached leaflets. Spores were captured and measured by placing microscope slides alongside the leaflets. Leus (2005) encountered high variability and low reproducibility of disease scores using an inoculation tower and recommended several replicate inoculations when using this method. A disadvantage of this strategy is that inoculum density cannot be fixed beforehand. This is because spores are released into the tower by using compressed air to blow them off infected leaves. In addition, only a limited number of genotypes may be inoculated simultaneously. Maintaining high humidity around inoculated leaflets is advisable for promoting optimum infection. Incubating inoculated leaflets at 95% relative humidity (RH) promotes significantly more sporulation than incubating at 50% RH (Hural and Coyier 1985). However, free water has a negative effect on infection if present during the first 6 hours after inoculation. Although germination of conidia is not affected, the penetration of the germ tube is inhibited by free water (Perera and Wheeler 1975). Yan et al. (2006) were able to develop a liquid spore suspension inoculation method that avoids the negative effects of free water on infection. This was accomplished by spraying young cuttings with a concentration of 103 to 104 conidia/ml and immediately increasing the temperature of a climate controlled compartment to 28 C for 15 minutes to promote evaporation. They then lowered the temperature to 22 C for the duration of the experiment. This method is advantageous for determination of partial resistance because it allows the uniform application of a predetermined concentration of spores on a large number of plants at the same time. The partial resistance


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components rated were the percentage of leaf area covered with symptoms, latent period (LP), and rate of symptom development (RSD). The RSD was calculated as the ratio of the percent leaf area at 11 days postinoculation to the time interval from the first appearance of lesions until 11 days postinoculation. As with black spot, one could ask whether detached leaf assays are suitable for screening partial resistance to powdery mildew. The data for answering this question conclusively are not yet available. In one instance, lines containing an antimicrobial transgene differed in their disease scores when inoculated as detached leaves versus whole plants (Li et al. 2003). Of seven lines tested, three differed widely in their disease scores between the two assays. However, different inoculation methods were used for the two assays, and spore concentrations were not quantified in the whole-plant assay. Leus et al. (2003) developed a greenhouse screening protocol in which susceptible plants of ‘Pfander’s Canina’ were planted intermittently among seedlings. Conidia from these plants were dusted onto the seedlings. Increased infection in this greenhouse experiment resulted in much higher selection intensity based on powdery mildew resistance compared with greenhouses not containing the ‘Pfander’s Canina’ plants (Leus 2005). Disease ratings from both greenhouse treatments were well correlated with field infections. From this study, the authors recommend an initial screening of young seedlings in the greenhouse followed by screening of advanced selections with the most virulent pathotypes (Leus et al. 2003). As with BTH, treatment with 2,6-dichloroisonicotinic acid (INA) also can be used to chemically induce SAR. This approach was taken by Higwegen et al. (1996) to compare the powedery mildew resistance of two roses, ‘Madelon’ and ‘Sonia’. Conidia were dusted on plants 4 days after INA application, and ratings were performed 18 days postinoculation. Both cultivars exhibited decreased colony formation and spore production in response to INA as well as early necrosis, haustorial encasement, and polyphenol production. Interestingly, ‘Sonia’ had increased partial resistance after INA treatment compared to ‘Maledon’, exhibiting slightly less colony formation and a marked decrease in sporulation capacity. Leus (2005) examined the effectiveness of SAR against powdery mildew by applying BTH and Milsana1 (an extract of giant knotweed, Reynoutria sachalinensis) to cuttings of ‘Excelsa’. Both treatments significantly decreased infection. Leus (2005) also demonstrated the presence of ISR in roses against powdery mildew, which can be induced by nonpathogenic soil bacteria, specifically Pseudomonas strains. These strains, when applied to the roots of rose cuttings prior to

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inoculation, caused a decrease in disease symptoms similar to BTH. These methods make it possible to evaluate both SAR and ISR in roses and could be used to screen for non–race-specific resistance against powdery mildew. C. Other Diseases Botrytis cinerea screening can be accomplished by spray inoculation methods with conidia that are produced in culture. Evidently, no complete resistance to this fungus has been found, but small differences in resistance have been found among cultivars that are indicative of partial resistance. Pie and Brouwer (1993) observed that some cultivars had reduced disease severity that was due to decreased hyphal growth after germ tube penetration. Unfortunately, symptoms still were visually evident, and it was suggested that a form of resistance that prevented penetration would be more effective. Hammer and Evensen (1994) attributed the resistance of one cultivar to a decrease in germ tube penetration. However, the difference in disease severity among genotypes was small and noticeable only at high inoculum concentrations. Discovery of more effective resistances against B. cinerea would be valuable. Cercospora leaf spot usually is considered a minor fungal disease of roses, but infestations can cause significant spotting and defoliation of roses under hot, humid conditions in the United States (Hagan et al. 2005). In a 5-year field study of the resistance of rose cultivars to fungal diseases in Alabama, few cultivars were susceptible to both black spot and leaf spot. Interestingly, all cultivars with high resistance to black spot were infected with Cercospora leaf spot. Among those cultivars susceptible to leaf spot, significant differences in resistance were observed that were consistent over all years (Hagan et al. 2005). These differences may provide a genetic basis for breeding. The results also raise questions about the extent of correlation between resistance to some pathogens and susceptibility to others. Downy mildew may be artificially inoculated by spraying conidia of Peronospora sparsa on leaf surfaces. Maximum infection can be obtained when temperature is maintained at or below 18 C and leaf wetness is maintained for 120 hours (Xu and Pettitt 2003). Schulz and Debener (2007) artificially inoculated detached leaves and rated downy mildew symptoms using a 5-step scale based on conidiophore production on the lower leaf surface. They reported that 13 of 84 wild species tested were resistant based on this scale, 6 of which are from section Caninae. Although oogonia are formed primarily within


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necrotic lesions, conidiophores usually are present at the lesion margins and even within the green, apparently uninfected portions of the leaf (Xu and Pettitt 2003). Therefore, when screening for resistance, extralesion formation of conidia should be considered. Detection of conidia or other pathogenic structures prior to visible formation of symptoms is possible with a PCR-based assay specific to the pathogen’s internal transcribed spacer (ITS) region (Aegerter et al. 2002). A protein-based approach using enzyme-linked immunosorbent assay (ELISA) is also effective for early detection. ELISA results correlated well with the visual estimations of conidiophore production in the field (Schulz and Debener 2007). Because initial downy mildew symptoms may be easily confused with those of black spot or powdery mildew, methods for early diagnosis are quite useful. Although rose rust species (Phragmidium spp.) are usually specific to certain roses, some rose hosts may be infected by multiple rusts. For example, P. tuberculatum and P. mucronatum could not be distinguished on some rose species in a European survey using morphological characters, and it was suggested that molecular methods of identification would be more successful (Leus and Van Huylenbroeck 2007). Importantly, field ratings of rust infection revealed marked differences between rose species that may serve as a basis for resistance breeding efforts. For instance, R. rubiginosa had a low disease index whereas R. canina was severely infected. Spot anthracnose symptoms can be rated with methods similar to methods used for black spot, but care must be taken not to confuse young spot anthracnose lesions with black spot lesions. Uggla and Carlson-Nilsson (2005) used a simple percentage rating scale to assess spot anthracnose in field plots over 2 years. Families from interspecific crosses within section Caninae were compared. Families with R. rubiginosa as the female parent differed significantly depending on the male parent. These differences offer a sound basis for selection among families and the first evidence for variation in combining ability for resistance to spot anthracnose. Inoculations of A. tumefaciens can be performed by pricking rose stems with a bacteria-laden needle or applying bacterial solution to vertical stem cuts. After 6 to 8 weeks, crown galls can be rated by measuring percent incidence and gall diameter (Aloisi et al. 1998; Zhou et al. 2001). Aloisi et al. (1998) established that there are significant differences in resistance among rose genotypes. Genotypes of R. indica and R. multiflora exhibited a large range of symptoms, ranging from 100% of plants affected for some genotypes to only 4% of plants affected for others. A separate test utilizing both in vitro and

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in vivo inoculation techniques indicated a range of susceptibility for R. multiflora genotypes and also confirmed several cultivars including ‘Double Delight’ and ‘Fire’ as highly resistant (Zhao et al. 2005). Suo and Leung (2001b) reported differences among cultivars, with ‘Madam Isaac Pieriere’ exhibiting 70% gall formation as compared to 25% for ‘Alexander’. Screening for induced resistance to crown gall also may be possible using chemical compounds. Crown gall incidence of in vitro inoculated plantlets of ‘Madam Isaac Pieriere’ was reduced by 26% after pretreatment with BTH (Suo and Leung, 2001b). Whether the acquired resistance was systemic or merely local was not clear. Due to the belowground location of nematode infection, inoculation of plants with nematodes is more difficult than for foliar pathogens. Nevertheless, French researchers have successfully inoculated rose rootstocks of various rose species with the root-knot nematode Meloidogyne hapla, demonstrating a wide range of genetic resistance (Voisin et al. 1996). Tomato plants with galled roots were grown in the same pot with the rose genotypes for 2 months until the top part of the tomato plant was removed. After 90 days, symptoms were assessed. Based on root gall ratings and counts of nematodes extracted from roots, R. manetti genotypes were the most resistant, followed by R. canina. Rosa multiflora genotypes were variable in their resistance. IV. BREEDING A. Resistance Genes The genetic nature of disease resistance is important in rose breeding. Resistance may be conferred by single genes or by many. Alleles may be dominant or recessive. Certain genes may confer race-specific resistance, while others may be non–race specific. These and other aspects of host genetics and biology determine which breeding strategies will be most effective. The first black spot resistance gene to be described is Rdr1, which is a major, race-specific resistance gene (R gene) (Von Malek and Debener 1998). Genetic studies were carried out with a tetraploid line developed from a resistant R. multiflora that was chromosome doubled and crossed to the tetraploid ‘Caramba’. Segregation ratios of resistant to susceptible progeny from selfing (35:1), backcross to the susceptible parent (5:1), and crosses to three different susceptible cultivars (5:1) were consistent with a single, dominant gene in duplex (RRrr) configuration. Discovery of this R gene was the first direct evidence


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of a gene-for-gene interaction in any rose pathosystem. The region around this gene was fine-mapped using AFLP markers, and bacterial artificial chromosomes (BACs) spanning this locus were identified using genomic DNA from R. rugosa (Kaufmann et al. 2003). Another gene, Rdr2, was discovered that is effective against a different isolate of black spot than Rdr1. By examining its segregation in two diploid populations, it was determined that it is tightly linked to Rdr1 (Hattendorf et al. 2004). Rdr1 resides within a cluster of 8 sequences, each containing a nucleotide binding site and a leucine-rich repeat. Because it lies in such a cluster, identification of the actual gene may require separate transformation of susceptible roses with each candidate sequence (Debener 2005). Another resistance gene in roses is Rpp1, which confers racespecific resistance to powdery mildew and gives evidence of gene-forgene interactions in the rose–powdery mildew pathosystem (Linde and Debener 2003). In a backcross to the susceptible parent, a 1:1 segregation ratio indicated that this trait is conferred by a single dominant gene. Transgressive segregation occurred in which the disease index of some susceptible progeny far exceeded that of the susceptible parent, possibly due to minor alleles with negative effects. Considerable transgressive segregation for powdery mildew resistance was also observed by Yan et al. (2006), indicating heterozygosity for resistance genes in the parents. Major R genes typically confer complete resistance but only against a limited number of fungal races. These resistances can be compromised quickly through pathogen mutation or migration, which has occurred for rose black spot (Bolton and Svejda 1979; Yokoya et al. 2000). Yet single dominant genes have the advantage of being easy to select for using detached leaf assays and can be selected for early in the breeding process. They also may be combined together in single genotypes in a pyramiding scheme to produce broader resistance. To date, a genepyramiding approach for disease resistance has not been demonstrated in rose. Partial resistance to disease in plants generally is assumed to be controlled by many genes and to be non–race specific (Simmonds 1991). Therefore, searching for quantitative trait loci (QTL) is a logical fist step in the genetic analysis of partial resistance. Two QTL for powdery mildew resistance were identified in a population of diploid F1 plants arising from a cross between a cultivated diploid rose and R. wichuriana (Dugo et al. 2005). Disease symptoms were scored on a simple 0 to 2 scale over one field season, and the two QTL Pm1 and Pm2 accounted for 45.2% and 24.9% of phenotypic variability

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respectively. In a larger study that included multiple disease scores, inoculation methods, and both field and greenhouse assessment, QTL were analyzed in a diploid population (Linde et al. 2006). In order to focus on the genotypes with the most extreme disease ratings, a selective genotyping scheme was employed in which 170 of the 270 phenotyped plants were included in the analysis. This method was effective, resulting in the discovery of 28 QTL contributing to powdery mildew resistance, while saving time and labor necessary to genotype the entire population. Partial resistance to powdery mildew has utility in breeding as demonstrated on a progeny mean basis (Leus 2005). Although one progeny had a mean disease rating exceeding 80%, another consistently exhibited less than 30% infection. These results were consistent in both the field and greenhouse. This approach is a practical way to gain information about the combining ability of the parents and their potential to transmit partial resistance in other crosses. No QTL for black spot resistance have been described, but continuous distributions of traits among some progenies are indicative of polygenic control (Carlson-Nilsson 2000; Uggla and Carlson-Nilsson 2005). Resistance to black spot and powdery mildew in roses has a clear quantitative aspect, but one should not assume that partial resistance in roses is always quantitative and non–race specific. Partial resistance to wheat leaf rust (Rubiales and Niks 1995) and leaf blast of rice (Zenbayashi et al. 2002) is conferred by single genes. Race-specific partial resistance has been documented in several pathosystems (Parlevliet 2002), including the discovery of isolate-specific QTLs for barley leaf rust (Qi et al. 1999), clubroot of Brassica sp. (Rocherieux et al. 2004), and apple scab (Durel et al. 2003). QTL studies with multiple races of rose pathogens could be conducted to determine if these exist for rose powdery mildew or black spot. Some evidence already exists, as Linde et al. (2006) reported a QTL for powdery mildew race 9 that was not detected in the greenhouse and field tests with polysporous inoculum. In addition, some QTLs for powdery mildew occurred in regions that contained resistance gene analogs (RGAs), although most QTLs did not colocalize with RGA clusters. This concept is further supported by an inoculation of two singlespore isolates of powdery mildew to a tetraploid rose population arising from a cross between two partially resistant parents (Yan et al. 2006). The isolates differed in their overall pathogenicity on this population. Moreover, the population exhibited race specificity for partial resistance, resulting in a highly significant genotype isolate interaction. Similarly, in an inoculation of 12 rose genotypes with 14


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single-spore isolates of black spot, Whitaker et al. (2007c) demonstrated that partial resistance for some rose genotypes varied with isolate. Recently multiple inoculations confirmed the race dependence of lesions size for at least one rose genotype (unpublished data). Genetic studies are currently under way to determine the inheritance of this trait. Genetic analyses of resistance have been conducted for spot anthracnose, crown gall disease, and nematode infection. Resistance to spot anthracnose in a single study appeared to be quantitative, which is indicated by family mean differences for progenies with different male parents (Uggla and Carlson-Nilsson 2005). However, these results must be interpreted with caution, as multiple pathotypes may have been present in the field. The genetic basis of resistance to crown gall was studied in self and F1 progenies of the resistant ‘PEKcougel’ and the susceptible ‘Dukat’ (Zhou et al. 2001). Disease incidence in the ‘PEKcougel’ self-pollinated progeny was significantly lower than for the other progenies, and a continuous distribution of resistance in all progenies indicated multigenic control. Similarly, resistance to the nematode M. hapla in R. multiflora and R. indica is controlled by several minor genes, which was inferred from monomodal distributions of progenies from a partial diallel crossing scheme (Wang et al. 2004). Although race-specificity of resistance was discovered in R. indica, crosses that would have allowed analysis of this resistance were not reported. B. Breeding Methods About 130 species of roses are cataloged worldwide (Zlesak 2006). Yet most of the recurrent-flowering or reblooming rose cultivars in our gardens are tetrasomic tetraploids (2n ¼ 4x ¼ 28Þ originating from 10 to 15 mostly diploid (2n ¼ 2x ¼ 14) species (De Vries and Dubois 2001). Various fertility barriers, reviewed by Gudin (2000) and Zlesak (2006), limit the parental germplasm base and restrict the size of progeny populations in this highly heterozygous, outbreeding species complex. Since cultivated germplasm is derived from such a small proportion of known species, introgression of disease resistance traits from species roses has potential. However, this usually involves transmission of desirable traits from the diploid to the tetraploid level. The transfer of disease resistance traits across ploidy levels may be accomplished by several methods. Colchicine doubling of wild species followed by crossing with cultivated tetraploids is the most-used method (Von Malek and Debener 1998), although decreased fertility in

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the original 4x induced plant can occur (Zlesak et al. 2005). Furthermore, inbreeding depression of hybrids may result after successive generations of backcrossing. To avoid this result, different cultivated varieties can be used for each backcrossing cycle rather than using a single clone as the recurrent parent (Debener 2000). A slightly different approach is to cross two wild rose species and then chromosome double the resultant seedlings, which are called amphidiploids. Basye (1990) pioneered this work by doubling R. banksiae R. laevigata hybrids with colchicine. The same methods were later used to create amphidiploids from other species (Ma et al. 1997). It was determined that these amphidiploids are more fertile than the original diploid hybrids, although they still have only ‘‘low to moderate’’ fertility. Some have been successfully hybridized with commercial varieties to transmit resistance into cultivated forms (Byrne et al. 1996). Polyploidization with trifluralin is also effective, as demonstrated in Rosa chinensis minima, and promises to be a cheaper and safer alternative to colchicine (Zlesak 2005). Oryzalin is also a desirable compound for spindle inhibition since it is poses less carcinogenic hazard to animal cells. It may not induce chromosomal abnormalities and DNA mutations in plants, as does colchicine (Kermani et al. 2003). Oryzalin may be used for chromosome doubling of shoot meristems and nodal sections of desirable genotypes, as demonstrated by Kermani et al. (2003). This method allows the doubling of chromosomes in clones as opposed to seedlings. Tetraploid plants were recovered at a rate of 66% from a 1-day treatment of nodal sections with 5 mM oryzalin. Importantly, chromosome doubling significantly increased pollen viability for the tetraploid forms of four diploids and for the hexaploid form of a triploid hybrid. Doubling of diploids and triploids with low fertility, therefore, has potential for introgression of resistance. However, one intriguing instance provides reason for caution. A diploid hybrid from a cross of ‘Martin Frobisher’ ‘Mistress Quickly’, resistant to three races of D. rosae, became susceptible to all three races after polyploidization to tetraploid forms (Allum and Roberts 2005; A.V. Roberts, pers. comm.). Chromosomal loss and/or mutation could be the cause of this phenomenon and would be difficult to rule out. Other possible explanations are epigenetic changes resulting from polyploidization that could alter expression of R genes or gene dosage effects. Experiments to further elucidate the effect of ploidy on susceptibility are currently being conducted. An alternative to chemical induction of polyploids is sexual polyploidization, which has the potential to preserve fertility and


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prevent inbreeding depression by maintaining heterozygosity. One way to increase ploidy level by sexual means is through exploitation of unreduced gametes. Crespel et al. (2002a) demonstrated the presence of both first-division restitution (FDR) and second-division restitution (SDR) mechanisms in rose dihaploids that produced fertile 2n gametes. Three segregating populations that resulted from crosses of the dihaploids with rose species yielded an average of 66% polyploidy progeny. AFLP analysis of the parents and progeny revealed that some FDR events occurred with no meiotic crossover (FDR-NCO), contributing 100% of parental heterozygosity to the progeny. Polyploids may be produced by crossing two different 2n gamete-producing genotypes with each other or by crossing normal diploid or polyploid roses with 2n gamete-producing genotypes (El Mokadem et al. 2002). Detailed information on the rate of incidence, heritability, and genetic transmission of unreduced gamete production is not yet available and would be of considerable value to rose breeders. A second sexual polyploidization approach involves creating a ploidy bridge by crossing wild rose species with tetraploids to generate triploids, which are subsequently crossed with cultivated tetraploid germplasm to produce tetraploids. Triploid roses are generally considered to be sterile, but Leus (2005) showed that, although pollen viability of triploids is low compared to tetraploids, a small percentage of pollen (0–10%) is still functional. Crosses between male triploids and female tetraploids primarily produced tetraploid offspring (98%), suggesting a competitive advantage for diploid pollen over monoploid pollen. By utilizing AFLPs and microsatellite markers, Leus (2005) demonstrated that DNA from diploid parents was transmitted to tetraploid progeny by this method. When selecting species for use in the introgression of disease resistance genes, it is important to note that members of the same species may differ widely in their resistance. Palmer et al. (1966a) reported differences in black spot resistance between two different clones of R. multiflora and between two clones of R. wichuriana. Similarly, two different clones of R. wichuriana differed widely in an inoculation with German isolates, with one clone resistant to all five races and the other susceptible to all races (Debener et al. 1998). Until the 1970s, R. rugosa was considered to be resistant to black spot, but ‘Martin Frobisher’ was suddenly overcome by a new race in Ottawa (Bolton and Svejda 1979), with susceptibility of R. rugosa later demonstrated in Europe as well (Debener et al. 1998). Although R. spinosissima is thought to be quite disease resistant, one clone was found to be susceptible to three North American races of black spot,

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with up to 48% of the leaf area affected (Whitaker et al. 2007c). For powdery mildew, two clones of R. multiflora exhibited different race specificities when inoculated with P. pannosa (Linde and Debener 2003). For crown gall, R. indica and R. multiflora clones varied widely in their resistance to A. tumefaciens biovar 1 (Aloisi et al. 1998). And finally, Voisin et al. (1996) found marked differences among R. multiflora species for resistance to the nematode M. hapla. Therefore, species should be screened with multiple pathogenic isolates prior to selecting clones for breeding purposes. A significant drawback to introgression of disease resistance traits from wild genotypes is the time frame involved. Ten or more years are needed to develop cultivars starting from species roses, as several generations of crosses are required to eliminate the wild background. A potentially quicker approach would be the detection and utilization of disease resistance traits already present in cultivated germplasm. Another advantage of this approach is that polyploidization can be avoided altogether by utilizing resistant varieties. Inoculations designed to differentiate pathogenic races of black spot (Svejda and Bolton 1980; Spencer and Wood 1992a,b; Debener et al. 1998; Yokoya et al. 2000; Leus 2005; Whitaker et al. 2007c) and powdery mildew (Linde and Debener 2003) have identified race-specific resistances in commercially available cultivars. Although a number of modern cultivars are triploid, many are fertile tetraploids as well (Leus 2005). The utility of such resistances will depend on whether they can be pyramided together effectively to combat multiple pathogenic races. Roses included in these inoculation tests that are wild species or selections that are closely related to wild species tend to have broader resistance in that they are usually resistant to more fungal races than the cultivars. Ultimately, the approach taken must depend on the goal of the breeding effort. If the breeder aims to introduce hardy, landscape-type roses, wild species can be used with less extensive backcrossing (Debener 2000). The gene pool of florist roses is much narrower, and a scheme that utilizes resistances from cultivated germplasm would be most practical. Disease resistance traits do not exist in a vacuum. Flower color and form, vigor, scent, stem quality, and other traits are vital in commercial breeding programs. Variable offspring originating from highly heterozygous parents means that large progenies are required in order to obtain the optimal combination of traits. In a typical commercial breeding scheme presented by Noack (2003), at least 100,000 seedlings are grown yearly, of which about 3% are selected after the first year for replicated testing. Since most repeat blooming roses flower as young seedlings, there is heavy early selection for floral


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characters. More resources and time are required to assess diseaseresistance characteristics, so gain from selection for disease resistance may be very slow (Zlesak 2006). Since early rouging of seedlings conserves valuable resources, marker-assisted selection (MAS) could be a way to conduct early selection for resistance, concurrent with selection for floral traits. This would require that markers linked to disease-resistance traits are available and could be applied in a costeffective manner (Noack 2003). Early screening of black spot and powdery mildew resistance can be performed by inoculating in a greenhouse. On the surface, inoculation appears to be a lower-cost approach compared to MAS. However, the presence of race specificity for major genes and QTLs means that multiple inoculations with different races should be performed. This consideration might lower the relative cost of MAS. In addition, resistance conferred by major genes can obscure the presence of partial resistance QTLs, which can be detected using molecular markers. V. MOLECULAR TOOLS A. Markers and Mapping Discovery of DNA markers closely linked to resistance genes and QTLs may facilitate MAS and is usually a prerequisite for gene discovery and cloning. Comparing different genetic maps within and outside of plant species also can speed up the discovery of new markers and resistance gene candidates. Mapping of disease-resistance genes in roses began with the discovery of markers linked to Rdr1 via bulk segregant analysis (Michelmore et al. 1991) of a segregating tetraploid population (Von Malek et al. 2000). Seven AFLP markers linked to the gene were identified, one of which was converted to a sequenced characterized amplified region (SCAR) marker and localized on a diploid map of rose (Fig. 6.4). This diploid map was constructed previously using 60 F1 plants in a double-pseudotest cross design and was the first linkage map of rose (Debener and Mattiesch 1999). Bulk segregant analysis with AFLP also was used to find markers linked to powdery mildew resistance gene Rpp1 (Linde et al. 2004). An AFLP marker closely linked to Rpp1 was converted into SCAR marker, which was found to be unlinked to the Rdr1 locus in a diploid population segregating for both genes. Although Rpp1 was localized on linkage group 3, Rdr1 was localized to linkage group 1. The powdery mildew QTLs of Linde et al. (2006) were mostly clustered on linkage groups 3 and 4. The cluster colocalizing with a locus for the presence/absence of prickles on linkage group 3 accounted for around

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Fig. 6.4. Linkage groups 1 of the rose chromosome map for population 94/1, shown separately for the female (A1) and the male (B1) parent. The black arrow indicates the position of the black spot resistance gene Rdr1. Black diamonds indicate the position of resistance gene analog (RGA)–derived markers. (Source: Courtesy of Anja Hattendorf, University of Hannover, Germany.)

65% to 80% of genetic variation for powdery mildew resistance. One QTL, located at the top of linkage group 6, was detected in a controlled inoculation with powdery mildew race 9. The most advanced map of rose to date includes AFLP markers as well as RFLP markers and other types including 74 SSR, 24 protein kinase (PK), and 51 resistance gene analog (RGA) markers (Yan et al., 2005). Some clustering of RGA sequences was observed, and RGAs were found mostly on linkage groups 1, 2, 4, 5, and 7 (Fig. 6.5). Linkage

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Fig. 6.5. Genetic linkage maps of diploid rose parents P117 (A) and P119 (B). Disease-resistance loci, protein kinase (PK) markers, and resistance gene analog (RGA) markers are indicated in bold.(Source: Courtesy of Oene Dolstra, Wageningen, The Netherlands.)

308 Fig. 6.5. (Continued)


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group 6 contained only one RGA marker and no PK markers. Importantly, the SSR and RFLP markers will serve as anchor points for integration of this map with other rose maps. As marker and map information for R genes and QTLs increases, the feasibility of markerassisted selection will also increase. Two additional rose maps have been constructed that do not include map locations for resistance genes but that may help facilitate future efforts. Crespel et al. (2002b) evaluated the traits of recurrent bloom, double flowers, and thorn density in a population of diploid roses using AFLP. Despite the complexity of inheritance in tetraploids, Rajapakse et al. (2001) constructed a low-density map from a tetraploid F2 population, which may serve as a useful starting point for the mapping of important traits in cultivated roses. Marker information can be used to select for desirable genomic regions; it also can be used to select against undesirable genomic regions. Debener et al. (2003) demonstrated this idea in the context of breeding for black spot resistance by using AFLP markers in a backcrossing strategy to select against the genome of the wild donor species (Rosa multiflora) in progenies segregating for Rdr1. The species (donor) parent and the cultivated parent initially were screened for genotype-specific markers. Plants of the BC1 generation containing Rdr1 were tested again for the presence of these markers. These plants segregated for 32% to 82% of the donor genome. Plants with the least amount of the donor genome were selected as parents for the next backcross generation. Molecular marker screening proved to be more effective than morphological screening for reduction of the wild species’ genetic background in both the BC1 and BC2 generations. Using markers for efficient backcrossing may allow breeders to reduce the number of backcrosses required to eliminate undesirable alleles, thereby saving time and expense (Debener 2000). B. Candidate Gene Approaches Major resistance genes (R genes) encode proteins that function in pathogen perception, acting as receptors (either directly or indirectly) for products of pathogenic avirulence (Avr) genes. The combination of R and Avr genes in the host and fungus determine the race specificity of the interaction. By examining the DNA sequence of R genes from various plant species, researchers have gained insights into the structure of these genes. This knowledge enables the discovery of new genes. By far the most abundant class of R genes is the NBS-LRR class, so named for its nucleotide binding site (NBS) domain and its C-terminus

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leucine-rich-repeat (LRR) domain. The NBS may be involved in signal transduction and contains several conserved motifs; the LRR apparently functions in pathogen recognition (Young 2000). Many of these proteins contain an N-terminus Toll/interleuikin receptor (TIR) domain. The non-TIR class of NBS-LRR proteins may contain coiled-coiled (CC) or leucine-zipper (LZ) motifs. Although sequences of NBS-LRR genes may be quite divergent, the presence of highly conserved amino acid motifs within the NBS domain allow the use of a degenerate PCR approach in isolating these sequences (Leister 1996; Pan et al. 2000), which are commonly called resistance gene analogs (RGAs). Xu et al. (2005) recently employed degenerate RGA primers for the study of powdery mildew resistance in chestnut rose (Rosa roxburgii). Different sets of primers were found to preferentially amplify the TIR class and the non-TIR class from a resistant parent. In all, 34 RGAs that contained continuous open reading frames (ORFs) were cloned and sequenced. A subset of these sequences were converted into sequence tagged sites (STS), cleaved amplified polymorphic sequence (CAPS), and RFLP markers and used in a bulk segregant analysis to identify linked markers in an F1 population. These markers were then used to characterize the entire F1 population (n ¼ 109). Segregation of resistance indicated the presence of more than one gene. Three markers were found to be linked to a gene (CRPM1) that explained 72% of the variation for resistance. Sequences from cloned RGAs may be used to develop molecular markers, as in Xue et al. (2005), but an alternative strategy called NBSprofiling targets R genes while at the same time generating polymorphic markers (Van der Linden 2004). This AFLP-like approach utilizes digestion and ligation steps, followed by two PCR steps using a degenerate RGA primer and an adapter primer. The goal is to anchor one end of the marker within a conserved motif of an R gene NBS region while generating marker diversity based on length polymorphisms and sequence polymorphisms within restriction sites. The NBSprofiling strategy was used to develop RGA markers for the rose map constructed by Yan et al. (2005). A much more extensive survey of RGAs from multiple rose genotypes was conducted using seven degenerate primer pairs to amplify NBSLRR sequences from both genomic and cDNA (Hattendorf and Debener 2007). Three primer pairs specifically amplified TIR RGAs, three primer pairs amplified LZ RGAs, and one primer pair amplified both types. A library of 7,000 clones was constructed and selected RGAs were sequenced. Sequences with at least 80% overall sequence similarity


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were grouped into the same families, and 40 different families were identified. Southern blot analyses showed that RGAs were organized genomically into single-copy, low-copy, and multicopy loci. In one diploid population (97/7), 37 RGAs are located on a single linkage group, while other linkage groups contained no RGAs (Hattendorf et al. 2004). Clustering of R genes is a common phenomenon in plant species. In another population (94/1), several RGAs are scattered on linkage group one, some proximal to Rdr1 but none tightly linked (Fig. 6.4). Expression studies showed the upregulation of some RGAs in response to black spot attack, but further experiments will be needed to identify which are functional RGAs (Hattendorf and Debener 2007). This survey demonstrates the breadth of RGA diversity in the rose genome and provides valuable sequence resources for construction of more PCR primers that target rose RGAs. Xu et al. (2007) applied the degenerate PCR approach to find candidate genes coding for members of the PR-2 and PR-5 families. PR2 genes code for b-1,3-glucanases; PR-5 genes code for osmotin. Based on single nucleotide polymorphisms (SNPs) among gene family members, single nucleotide-amplified polymorphisms (SNAP) markers were developed. One marker was linked to a minor QTL that accounted for 12% of variation in powdery mildew resistance. This approach holds promise for developing markers for other classes of PR proteins, including the PR-1 and PR-3 families, which are already known to exist in rose (Suo and Leung 2002a). C. Other Approaches 1. Transformation. Transformation of roses has been accomplished with both particle bombardment and Agrobacterium tumefaciens followed by regeneration via somatic embryogenesis. Since somatic embryogenesis in roses is time consuming, development of transgenic plantlets requires a minimum of a year. Nevertheless, methods of transformation are being improved, and transgenes with antimicrobial properties already are being utilized in a research context (Dohm 2003). The first transgene for disease resistance in rose was a rice chitinase gene under the control of the CaMV 35S promoter that was transformed into ‘Glad Tidings’ using a biolistic approach (Marchant et al. 1998). For the best-performing transformant, black spot lesion diameters were reduced by 43%. Among transformed plants, the level of chitinase activity explained 96% of the variability in black spot lesion diameter. Reduction in lesion diameter was most pronounced at later stages of

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infection. Since this Class I chitinase is thought to be produced in the vacuole, activity against penetrating fungal hyphae is presumed to increase with increased fungal invasion. Agrobacterium-mediated transformation was employed to transform ‘Pariser Charme’ and ‘Heckenzauber’ with other PR protein genes, namely a Class II chitinase, a b-1,3-glucanase, and a Type I ribosomeinhibiting protein (RIP) from barley (Dohm and Debener 2001). Expression of the chitinase and glucanase in the cytosol had no effect on black spot infection rates, but apoplastic accumulation of the RIP protein decreased infection by 40% on average. Expression of a T-4 lysozyme had no effect. Somaclonal variation during regeneration produced some morphological deviation and individuals with reduced fertility. Marchant et al. (1998) recovered only phenotypically normal plants, possibly because the callus phase was shorter for biolistic transformation. Li et al. (2003) transformed ‘Bucbi’ (Carefree BeautyTM) with a different PR protein gene in the defensin class, namely a cysteine-rich antimicrobial protein (AMP) gene called Ace-AMP1 that originally was isolated from onion seeds. Out of seven transgenic roses, six showed decreased hyphal spread and sporulation of S. pannosa in both detached leaf and whole-plant inoculations. Powdery mildew symptoms were decreased by approximately 50% for the most resistant transformants. Transformation of scented geranium with the same gene, Ace-AMP1, reduced botrytis blight symptoms, suggesting that this gene has potential to confer resistance to B. cinerea in rose, although this hypothesis has not yet been tested (Bi et al. 1999). 2. Somatic Hybridization. Another technology for the introduction of disease-resistance genes is somatic hybridization through protoplast fusion, which has potential for transfer of resistance from species into susceptible cultivars or for introgression of nonhost resistance from other genera in the Rosaceae. Successful protoplast fusion was demonstrated in Mottley et al. (1996) by the self-fusion of a Rosa persica xanthina hybrid. Resultant plants were doubled from the diploid to the tetraploid level, which was confirmed by chromosome counts as well as guard cell lengths and chloroplast numbers. In a later study, protoplast fusions for the purpose of disease resistance were carried out between rose species and cultivars (Schum and Hofmann 2001). Fused calli were obtained for hybridizations of ‘Heckenzauber’ þ R. wichuriana and of ‘Pariser Charme’ þ R. wichuriana, which were confirmed by AFLP analysis. However, at the time of publication, no plantlets had been regenerated successfully from the hybrid calli.


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Squirell et al. (2005) completed the next step of producing putatively intergeneric cell lines of Rosa þ Prunus, which was confirmed by RAPD analysis, although regenerated plantlets contained RAPD markers from Rosa alone. Rosa þ Rubus fusions were also performed that resulted in plants with phenotypes strikingly different from Rosa, although once again RAPD markers could not confirm their status as intergeneric hybrids. It was suggested that asymmetric fusion, which assimilates only a part of the non–rose donor genome, might prove more successful. Since crosses of roses with other genera within the Rosaceae have not been successful, and since transgenic roses may encounter political and social obstacles, protoplast fusion has advantages as a breeding technique (Squirell et al. 2005). However, more work is needed to refine current protocols and to identify genotypes with increased capacity for regeneration (Schum and Hofmann 2001).

VI. FUTURE PROSPECTS During the last two decades, knowledge of the general biology, diversity, and culture of rose pathogens and host mechanisms of resistance to these pathogens has greatly increased. Building on this foundation, genetic studies of resistance have begun in earnest. The discovery and analysis of race-specific genes for black spot (Rdr1) and powdery mildew (Rpp1) are an excellent start, but they comprise only a small fraction of the race-specific factors that are known based on racetest comparisons of rose genotypes (Debener et al. 1998; Linde and Debener 2003). Therefore, the door is open for further genetic characterization of race-specific resistance for black spot and powdery mildew. An even wider door exists for genetic studies of resistance to other fungal diseases such as downy mildew and spot anthracnose. And nothing is known about the inheritance of viral resistance in roses or even methods for scoring virus symptoms. Once multiple genes against any pathogen have been identified, they may be used in a pyramiding strategy to gain broad resistance. A gene pyramiding scheme for resistance has not yet been demonstrated for rose, either for major genes or QTLs. In order to explore the diversity of resistance genes for any pathogen, it will be necessary to more firmly establish the diversity of pathogenic races worldwide. Since race collections of black spot have already been established and maintained on two continents, D. rosae is probably the first rose pathogen for which a standard host differential set will be established (Debener et al. 1998; Yokoya et al. 2000; Whitaker et al. 2007c).

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Less is known about the genetics of partial resistance, although it generally appears to be polygenic. Isolate-specific affects on levels of partial resistance and an apparent isolate-specific QTL for powdery mildew demonstrate the importance of determining race-specific effects (Linde et al. 2006; Yan et al. 2006). Prior knowledge of the isolate specificity of QTLs would increase the potential effectiveness of a marker-assisted breeding strategy. Examining the inheritance of partial resistance using defined pathogenic isolates would be advisable, whether conducting QTL studies or other quantitative analyses. For practical purposes, breeders are concerned with determining which genotypes are useful parents for resistance. Combining ability studies based on progeny means are useful for such determinations and could be conducted. For example, some apple cultivars have been identified as desirable parents because they possess general combining ability for resistance to powdery mildew (Podosphaera leucotricha Salm.) (Bus et al. 2005). As markers and maps continue to be developed for other important crops of the Rosaceae, rose breeding programs can take advantage of this genomic synergy. Comparison of Prunus (almond, cherry, peach, etc.) and Malus (apple) maps reveals consistent marker order and a high level of synteny between their genomes. Smaller regions of conservation even were found between Prunus and Arabidopsis (Dirlewanger et al., 2004). Of 46 gene-specific primer pairs (20–24 bp) representing 18 genes from Fragaria (strawberry, subfamily Rosoideae), over half amplified PCR products of the expected size from Malus (subfamily Maloideae) and Prunus (subfamily Prunoideae) (Sargent et al. 2007). Transfer of markers from Fragaria to Rosa should be at least as efficient, if not more efficient, since Fragaria and Rosa are both part of the Rosoidae. As markers for disease-resistance genes from other rosaceous crops continue to be identified, they could have application for rose breeding via comparative mapping. Marker-assisted selection for pyramiding of resistance genes and early selection is being applied increasingly in apple for powdery mildew resistance and other traits (Dirlewanger et al. 2004). As genomic resources for rose and for other rosaceous crops such as transferable SSRs, expressed sequence tags (ESTs), microarrays, and physical maps continue to accumulate, candidate gene approaches also will become more widespread. Although rose has a shorter generation time than most woody crops, it is still very long compared to many crop species. A transgenic approach to disease resistance could be advantageous in that it would reduce the amount of crossing and backcrossing necessary to obtain resistance in highly cultivated forms such as hybrid teas. However, the advantages


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and disadvantages must be weighed carefully. The process of transformation, regeneration of desirable transgenic lines, and field testing of transgenics may be no less time consuming and expensive than traditional breeding approaches. Through asexual propagation, desirable transgenic lines of roses could be increased quickly for commercialization. Since rose (except in the context of rose hips) is not a food product, the public may be more amenable to transgenic roses in light of the environmental benefits of disease control not involving pesticides. Overexpression of PR proteins from other species has been demonstrated to confer partial resistance phenotypes to fungal diseases in garden roses. In addition, these resistances have the potential for effectiveness against a broad spectrum of pathogens. However, a difficulty with this approach is that many potential candidates from other crops are protected by patent law and may be difficult or expensive to utilize commercially (Dohm 2003). Since the presence of PR proteins such as b-1,3-glucanase and chitinase has been detected in rose (Suo and Leung 2001a), this problem possibly could be avoided by cloning these genes from rose and overexpressing them in transgenic lines. Indeed, b1,3-glucanase and osmotin genes already have been cloned from R. roxburghii (Xu et al. 2007). Major resistance genes also would be good candidates for a transgenic approach if their race specificity is well characterized and if they were used wisely. In the future when multiple R genes have been cloned from rose, these could be introduced simultaneously into susceptible varieties to achieve broad resistance to multiple pathogens and/or multiple pathogenic races. Transgenes designed to induce RNA interference (RNAi) have been introduced successfully in other plant species to confer resistance to crown gall and root-knot nematode and could also be applied to rose. Tumorigenesis in plant roots infected by A. tumefaciens is dependent on the oncogene region of the bacterial T-DNA. Transgenic Arabidopsis thaliana and Lycopersicon esculentum (tomato) carrying constructs for expression of double-stranded RNA (dsRNA) effectively eliminated expression of the iaaM (auxin production) and ipt (cytokinin production) oncogenes, thereby halting tumorigenesis (Escobar et al. 2001). This method also has been used to generate crown gall–resistant apple trees and is expected to be a highly durable resistance mechanism (Viss et al. 2003). Resistance to root-knot nematodes has been achieved in A. thaliana through transformation with a dsRNA corresponding to a nematode parasitism gene (Huang et al. 2006). After piercing the transgenic plant root with its stylet, the nematode ingests the dsRNA and the nematode parasitism gene is silenced. Because the target gene is highly conserved and is essential for pathogenesis, the

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transgenic plants were resistant to four different Meloidogyne species (including the important rose pathogen M. hapla). Such a breadth of resistance to root-knot nematode had never been achieved using natural genetic resistances. Genomic approaches utilizing marker-assisted selection and transgenes will not replace but rather complement traditional breeding for disease resistance in roses. Resistance is available in the species germplasm and also among cultivars. Although acquisition of wild germplasm will continue to be important in rose breeding, characterization of the race specificity and genetic transmission of existing resistances is of first importance. This will be accomplished through more comprehensive pathotype characterizations, reliable disease screening methods, and genetic studies. Continued refinement of polyploidization methods also could improve resistance breeding. The use of molecular markers to eliminate nondonor contributions and to select for resistance genes in the context of breeding programs is promising. The decreasing cost of marker technology and other molecular tools along with public demand for nonchemical disease control will mean that conventional strategies and genomic approaches will progress hand in hand. Rose breeders will continue to glean new information from advances in other crops that ultimately will result in disease resistance that is effective and long lasting.

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Sheng, J., and V. Citovsky. 1996. Agrobacterium-plant cell DNA transport: Have virulence proteins, will travel. Plant Cell 8:1699–1710. Simmonds, N.W. 1991. Genetics of horizontal resistance to diseases of crops. Biol. Rev. 66:189–241. Sivapalan, A. 1993. Effects of water on germination of powdery mildew conidia. Mycol. Res. 97:71–76. Spencer, J.A., and O.W. Wood. 1992a. Resistance of selected rose cultivars to variants of Marssonina rosae in Mississippi. J. Environ. Hort. 10:235–238. Spencer, J.A., and O.W. Wood. 1992b. Response of selected Old Garden Roses to seven isolates of Marssonina rosae in Mississippi. J. Environ. Hort. 10:221–223. Squirrell, J., Z. Mandegaran, K. Yokoya, A.V. Roberts, and J. Mottley. 2005. Cell lines and plants obtained after protoplast fusion of RosaþRosa, RosaþPrunus, and RosaþRubus. Euphytica 146:223–231. Suo, Y., and D.W.M. Leung. 2001a. Elevation of extracellular b-1,3-glucanase and chitinase activities in rose in response to treatment with acibenzolar-S-methyl and infection by D. rosae. J. Plant Physiol. 158:971–976. Suo. Y., and D.W.M. Leung. 2001b. Induction of resistance to Diplocarpon rosae and Agrobacterium tunefaciens by acibenzolar-S-methyl (BTH) in rose. J. Plant Dis. Prot. 108(4):382–391. Suo, Y., and D.W.M. Leung. 2002a. Accumulation of extracellular pathogenesis-related proteins in rose leaves following inoculation of in vitro shoots with Diplocarpon rosae. Scientia Hort. 93:167–178. Suo, Y., and D.W.M. Leung. 2002b. BTH-induced accumulation of extracellular proteins and blackspot disease in rose. Biol. Plant. 45:273–279. Svejda, F.J., and A.T. Bolton. 1980. Resistance of rose hybrids to three races of Diplocarpon rosae. Can. J. Plant Path. 2:23–25. Takamatsu, S., T. Hirata, and Y. Sato. 1998. Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae). Mycoscience 39:441–453. Taylor, J.W., D.J. Jacobson, and M.C. Fisher. 1999. The evolution of asexual fungi: reproduction, speciation and classification. Annu. Rev. Phytopath. 37:197–246. Uggla, M., and B.U. Carlson-Nilsson. 2005. Screening of fungal diseases in offspring from crosses between Rosa sections Caninae and Cinnamomeae. Scientia Hort. 104:493–504. USDA. National Agricultural Statistics Service. 2006. Floriculture Crops 2005 Summary. http://www.nass.usda.gov. Van der Linden, G., D. Wouters, V. Mihalka, E. Kochieva, M. Smulders, and B. Vosman. 2004. Efficient targeting of plant disease resistance loci using NBS profiling. Theor. Appl. Genet. 109:384–393. Van Loon, L.C., and E.A. Van Strien. 1999. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol. 55:85–97. Viss, W.J., J. Pitrak, J. Humann, M. Cook, J. Driver, and W. Ream. 2003. Crown-gall resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes. Mol. Breed. 12:283–295. Voisin, R., J.C. Minot, D. Esmenjaud, Y. Jacob, G. Pelloli, and S. Aloisi. 1996. Host suitability of rose rootstocks to the root-knot nematode Meloidogyne hapla using a high-inoculum-pressure test. Acta Hort. 424:237–239. Von Malek, B., and T. Debener. 1998. Genetic analysis of resistance to blackspot (Diplocarpon rosae) in tetraploid roses. Theor. Appl. Genet. 96:228–231.


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7 Plant Breeding for Human Nutritional Quality Philipp W. Simon Vegetable Crops Research Unit United States Department of Agriculture Agricultural Research Service Department of Horticulture University of Wisconsin Madison, Wisconsin 53706 USA Linda M. Pollak Corn Insects and Crop Genetics Research Unit United States Department of Agriculture Agricultural Research Service Department of Agronomy Iowa State University Ames, Iowa 50011 USA Beverly A. Clevidence Food Components and Health Laboratory United States Department of Agriculture Agricultural Research Service Beltsville Agricultural Research Center Beltsville, Maryland 20705 USA Joannne M. Holden and David B. Haytowitz Nutrient Data Laboratory United States Department of Agriculture Agricultural Research Service Beltsville Agricultural Research Center Beltsville, Maryland 20705 USA


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I. INTRODUCTION II. SOURCES OF NUTRIENTS A. Assessing Food and Nutrient Consumption by Individuals in the United States 1. Shortfall Minerals: Magnesium, Calcium, and Potassium 2. Total Dietary Fiber 3. Shortfall Vitamins: Vitamin C, Vitamin E, and Vitamin A 4. Carotenoids 5. Anthocyanins B. Resources for Accessing Food Intake and Nutrient Composition of Foods III. PROGRESS IN BREEDING FOR NUTRIENT CONTENT AND COMPOSITION A. Breeding for Macronutrients 1. Protein and Protein Quality 2. Carbohydrates 3. Fatty Acids B. Breeding for Micronutrients and Phytonutrients 1. Minerals 2. Vitamin C 3. Vitamin E 4. Carotenoids 5. Anthocyanins IV. PLANT BREEDING STRATEGIES FOR INCREASING INTAKE OF SHORTFALL NUTRIENTS LITERATURE CITED

ABBREVIATIONS AI CGIAR CSFII DRI DW EAR ERS FNDDS GI MDGs NDL NHANES NIR NLEA NSP PUFA QPM QTL

Adequate intake Consultative Group on International Agricultural Research Continuing Survey of Food Intake by Individuals Dietary reference intakes Dry weight basis Estimated average requirement Economic Research Service Food and Nutrient Database for Dietary Studies Glycemic index Millennium Development Goals Nutrient Data Laboratory National Health and Nutrition Examination Survey Near infrared Nutrition Labeling and Education Act of 1990 Nonstarch polysaccharides Polyunsaturated fatty acids Quality protein maize Quantitative trait locus

7. PLANT BREEDING FOR HUMAN NUTRITIONAL QUALITY

RDA RS SR UL

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Recommended dietary allowance Resistant starch Standard reference Tolerable upper intake level

I. INTRODUCTION With the advent of agriculture came a more reliable and abundant source of food and the nutrients that food supplies. Yet it only became obvious in the last several centuries that food is a complex mixture of components, each with independent roles in contributing to particular aspects of health. The first vitamins were described with the discovery of vitamin A in 1913 (McCollum and Davis 1913) and several others thereafter, only a decade after Mendel’s work was rediscovered to provide a more scientific foundation for plant breeding. Differences in crop color due to carotenoids (yellow versus white maize) were soon associated with differences in vitamin A nutrition (Steenbock 1919). With this report it might have been expected that plant breeders would include the improvement of nutritional quality as a basic breeding goal for all food and feed crops. In fact, yellow maize virtually replaced white maize by 1970 because it was recognized to be a better animal feed (Troyer 1999). Yet the numerous challenges in producing the crop and delivering an acceptable commodity to the consumer must be met before nutritional quality comes to the attention of plant breeders. Breeding for staple crop yield and processing quality, in fact, generally has been successful in providing a supply of essential macronutrients: carbohydrates, protein and fats. Likewise, breeding to maintain an adequate supply of vegetables and fruits generally has provided consumers in the developed world with an adequate supply of micronutrients and phytochemicals to maintain health. Yet even with our abundant supply of food today, dietary intake of certain essential nutrients falls short in the United States and other developed countries. Looking globally, the nutritional status for much of the lessdeveloped world continues to improve, but food supply for several nutrients remains precarious in many regions and inadequate in some of them. Humans have consumed a very broad range of plants over the history of agriculture. Major staple crops of the past, such as tef (Eragrostis tef (Zuccagni) Trotter) and quinoa (Chenopodium quinoa Willd.), or vegetable crops, such as salsify (Tragopogon porrifolius), scorzonera (Scorzonera hispanica), and celeriac (Apium graveolens), are barely

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known beyond a very small fraction of the current civilizations. Within the bounds of the crops that are widely grown today, broad genetic diversity in nutritional quality is found in primitive or locally specialized cultivars, and an even broader range of diversity often can be found in the wild relatives of crops. Plant breeders play a key role in determining what we eat, since the breeding stocks they develop from this breadth of germplasm are at the beginning of the dietary food chain. Nutritional scientists play a parallel role in determining nutritional requirements, and in gathering a wealth of data describing what we eat, where our nutrients come from, and where we fall short in the intake of specific nutrients. Working together, plant breeders and nutritionists can play a key role in sustaining the supply of dietary nutrients in the crops we breed where intake is adequate and in developing strategies for increasing intake of those nutrients where intake is inadequate. This chapter provides plant breeders with information about nutrient requirements and consumption of selected nutrients and phytonutrients in the United States, because of the extensive U.S. database available. Intake of several nutrients falls below recommended levels, and these nutrients, in particular, are described in more detail from both the crop source and the plant breeding perspective. Beyond those shortfall nutrients in the United States, deficiencies for other nutrients are prevalent in other parts of the world, and breeding progress for these nutrients is also reviewed.

II. SOURCES OF NUTRIENTS Approaches for improving human health through diet involve both those activities that require action on the part of consumers—such as education, motivation, and behavior change—and those that are passive or require no action on the part of consumers—such as changing the food supply. The former has met with limited success. However, plant scientists can improve nutrient intake by enhancing the nutrient content of commonly consumed foods, such as by selecting for nutrient-rich cultivars and by enhancing nutrient content of foods through nutritionoriented breeding programs. This section focuses on commonly consumed, plant-based foods that supply shortfall nutrients and selected phytonutrients in the U.S. food supply. The report of the 2005 Dietary Guidelines Advisory Committee (Dietary Guidelines Advisory Committee 2004) identified nutrients that are likely to be consumed in inadequate amounts and referred to these as shortfall nutrients. For adults, the shortfall nutrients identified are


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vitamins A, C, and E, calcium, magnesium, potassium, and fiber. For children, shortfall nutrients are vitamin E, calcium, magnesium, potassium, and fiber. Adequacy of selected shortfall nutrients for adults, as determined by the 2005 Dietary Guidelines Advisory Committee, was assessed using the most recent food survey available at that time, the Continuing Survey of Food Intake by Individuals (CSFII), 1994 to 1996, of the U.S. Department of Agriculture (USDA), the predecessor of the current What We Eat in America survey. The probability of adequate intake for men and women was, respectively, for vitamin A, 47% and 48%; for vitamin C, 49% and 52%; for vitamin E, 14% and 7%; for magnesium, 36% and 34%, and for calcium, 59% and 46% (Dietary Guidelines Advisory Committee 2004). Folate, also identified as a nutrient with a high prevalence of inadequate intake, is not discussed here, largely because CSFII 1994 to 1996 data were collected before 1998 when the Food and Drug Administration required fortification of enriched grains with folic acid. It should be noted, however, that folate may continue to be a nutrient of concern (Dietary Guidelines Advisory Committee 2004). The benchmark for determining shortfall is the Estimated Average Requirement (EAR) for those nutrients where an EAR has been determined. The EAR is the average daily nutrient intake level estimated to meet the requirements of half the healthy individuals in a particular life stage and gender group (Institute of Medicine 2006). This is a lower benchmark than the Recommended Dietary Allowance (RDA), which is the average daily dietary nutrient intake level sufficient to meet the nutrient requirements of nearly all (97% to 98%) healthy individuals in a particular life stage and gender group. For calcium, potassium, and fiber, there is not sufficient evidence to establish an EAR. When scientific data are not sufficient for calculating an EAR, a reference intake of less certainty, the Adequate Intake (AI), may be used. The AI is the recommended average daily intake level based on observed or experimentally determined approximations or estimates of nutrient intake by a group (or groups) of apparently healthy people that are assumed to be adequate. Collectively, the EAR, RDA, AI, and the UL (tolerable upper intake level) comprise the Dietary Reference Intakes (DRIs). For simplicity, DRIs for pregnant and lactating women, often higher than their age-matched counterparts, are not discussed in this chapter. In Table 7.1, EAR and RDA values are displayed by gender and life stage group for vitamin C, vitamin E, and magnesium; AI values are displayed for calcium, potassium and total dietary fiber. There are no DRIs for carotenoids or flavonoids. It should be noted that DRIs apply

330

39 56 60 60 60 60

Females 9–13 y 14–18 y 19–30 y 31–50 y 51–70 y >70 y

z

45 75 90 90 90 90

39 63 75 75 75 75 9 12 12 12 12 12

9 12 12 12 12 12

5 6

EAR

Vitamin E (mg/day)

11 15 15 15 15 15

11 15 15 15 15 15

6 7

RDA

200 300 255 265 265 265

200 340 330 350 350 350

65 110

EAR

240 360 310 320 320 320

240 410 400 420 420 420

80 130

RDA

Magnesium (mg/day)

1.3 1.3 1.0 1.0 1.2 1.2

1.3 1.3 1.0 1.0 1.2 1.2

0.5 0.8

Calcium (g/day) AI

A partial listing from Dietary References Intakes: The Essential guide to Nutrient Requirements (IOM 2006).

45 65 75 75 75 75

15 25

13 22

RDA

Children 1–3 y 4–8 y Males 9–13 y 14–18 y 19–30 y 31–50 y 51–70 y >70 y

EAR

Vitamin C (mg/day)

4.5 4.7 4.7 4.7 4.7 4.7

4.5 4.7 4.7 4.7 4.7 4.7

3.0 3.8

Potassium (g/day) AI

Estimated average requirement (EAR) and recommended dietary allowances (RDA) for selected nutrients.z

Demographic group

Table 7.1.

26 26 25 25 21 21

31 38 38 38 30 30

19 25

Total dietary fiber (g/day) AI


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to healthy individuals. Needs of special populations (e.g., the ill and malnourished) may differ; additionally, values may need to be adjusted based on lifestyle and physiological factors (e.g., athletes, smokers). In the United States, plant-based foods are major sources of most of the shortfall nutrients. Low intakes of fruits and vegetables, for example, result in low intake of magnesium, vitamin A (as the provitamin A carotenoids), and vitamin C. In addition to providing provitamin A, fruits, vegetables, and also whole grains provide an array of phytonutrients that may be biologically active and hold promise as agents for extending years of healthful life. Plant-based foods also can supply a number of essential nutrients that are in short supply in developing countries. Breeding for provitamin A carotenoids, iron, and zinc is of keen interest as a strategy to alleviate nutrient deficiencies (van den Berg et al. 2000; Gregorio 2002; Khush 2002; King 2002; de Carvalho et al. 2006; Nestel et al. 2006; Graham et al. 2007; Hotz and McClafferty 2007). In this section we identify those foods responsible for providing large percentages of shortfall nutrients and selected phytonutrients for the U.S. population. These data, derived from U.S. food consumption data, may not be directly applicable to the developing world. However, current food intake data in many developing countries is sparse; thus, U.S. data can help identify foods that may be targeted for enhancement of nutrients of concern. In addition, these methodologies for identifying foods and setting priorities can be used in other countries with relevant consumption and composition data sets. Furthermore, numerous cultivars of specific plant-based foods with wide-ranging levels of nutrients and other biologically active components are available worldwide. In 2004 the Convention on Biological Diversity’s Conference of the Parties recognized the linkage among biodiversity, food and nutrition, and the need to enhance sustainable use of biodiversity to combat hunger and malnutrition, and thereby contribute to their Millennium Development Goals (MDGs). Recently, the Food and Agriculture Organization, together with Bioversity International and other partners, has initiated the development of nutrition indicators for biodiversity. Although biodiversity is considered essential for food security and nutrition, and can contribute to the achievement of MDGs through improved dietary choices and positive health impacts, it is seldom included in nutrition programs and interventions. This is due in large measure to insufficient data on the nutritional value of local foods sources from biodiversity and to lack of methods for obtaining, analyzing, and using data on biodiversity in food composition studies and nutritional programs.

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These efforts will emphasize research and development of biologically diverse but underutilized cultivars that may have exceptional nutritional value and fit well within global objectives for sustainable agriculture. For example, underutilized cultivars of deeply colored bananas are being promoted in Pohnpei in Micronesia as newly recognized sources of provitamin A and other carotenoids (Englberger et al. 2003a,b). The international agricultural research centers of the Consultative Group on International Agricultural Research (CGIAR) have a long history of crop germplasm collection and improvement, including attention to nutrient content. In other areas of the world, scientists participating in the CGIAR Biofortification Challenge Program, also known as HarvestPlus, are scanning the compositional characteristics of staple priority crops, such as rice, maize, wheat, cassava, sweet potato, and beans, and developing improved cultivars that deliver more iron, zinc, and b-carotene (Bouis 1996, 2002; Welch 1997, 2002; Welch and Graham 2004). Efforts like these can dovetail with U.S. objectives to develop and promote plant-based products with levels of nutrients superior to conventional types.

A. Assessing Food and Nutrient Consumption by Individuals in the United States The USDA estimates both food availability at the wholesale and retail level [food disappearance as assessed by the Economic Research Service (ERS)] and food consumption by individuals [food survey data from Agricultural Research Service’s What We Eat in America, a component of the National Health and Nutrition Examination Survey (NHANES)]. Details of the process for collecting food consumption data have been published (Raper et al. 2004). Plant scientists may be more familiar with the food availability data system due to its focus on commodities. The ERS Web site includes U.S. per capita, per day, estimates of amounts of individual nutrients available as well as nutrients available from major food groups. What We Eat in America assesses the kinds and amounts of foods consumed by a representative sample of the U.S. population, using 24-hour dietary recall method. Paired with data on the nutrient content of foods, nutrient intake data are obtained on a national average per capita basis. This type of food data collection generates multiple specific entries for a commodity due to varied forms of food processing. For example, rather than considering tomato collectively, individual tomato-based foods are considered (e.g. tomato sauce, catsup, raw tomato, and salsa). Information on nutrient content of individual foods


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can be useful to plant scientists, for example, where cultivars are tailored for specific processing techniques. The source of the nutrient data for the assessment of food and nutrient consumption is the USDA’s National Nutrient Database for Standard Reference (SR) (US Department of Agriculture 2007), which is developed and maintained by the Nutrient Data Laboratory (NDL) at the Beltsville Human Nutrition Research Center, an institute of the Agricultural Research Service (Beltsville, MD). The SR contains data for 7,500 foods for up to 140 components, approximately 2,700 of which are used to develop the Food and Nutrient Database for Dietary Studies (FNDDS; Bodner-Montville et al. 2006). Values for 64 nutrients are provided with this subset and are updated with each release of the database. The version SR20 was released via the Internet in September 2007. USDA’s nutrient database products provide data for amounts of nutrients provided in standard and expected quantitative units per 100 g amount of food, given on the wet-weight or ‘‘as-consumed’’ basis. This may contrast with the nutrient data for crops and animals, which may be presented on the basis of standard units on the dry- or freshweight basis. The SR product is used as the foundation for virtually all other food composition databases by other government agencies, the food industry, and healthcare sectors. Detailed information about the sources of composition data can be obtained from the NDL site (www. ars.usda.gov/nutrientdata). The Key Foods process was developed by NDL using USDA’s food composition and food consumption data to identify and prioritize foods and nutrients for analysis (Haytowitz et al. 2000, 2002). Key Foods are those foods that, in aggregate, contribute 75% of the nutrient intake for selected nutrients of public health importance from the diet. Tables 7.2 to 7.12 are derived from the analysis of food survey data (www.cdc.gov/nchs/nhanes.htm, www.ars.usda.gov/ba/bhnrc/fsrg) for the shortfall nutrients and phytonutrients covered in this chapter generated during the Key Foods process. Data are representative of mean intake for 8,354 individuals 2 years old and older weighted for the population of the United States. The latest Key Foods list of 454 food items was generated using weighted two-day food consumption data from the What We Eat in America component of NHANES 2003–2004 Data Files (National Center for Health Statistics 2006) and food composition data from SR18 (issued in 2005). The USDA’s Key Foods lists for nutrients of public health significance can be found on the NDL Web site (www.ars.usda.gov/nutrientdata). Foods in Tables 7.2 to 7.12 are compiled from food survey data and listed in rank order by their relative contribution to total intake of that

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Source: http://www.ars.usda.gov/nutrientdata.

Milk, reduced fat, fluid, 2% milkfat, with added vitamin A Milk, whole, 3.25% milkfat Coffee, brewed from grounds, prepared with tap water Orange juice, canned, unsweetened Bananas, raw Snacks, tortilla chips, plain, white corn Milk, nonfat, fluid, with added vitamin A (fat free or skim) Peanut butter, smooth style, with salt Alcoholic beverage, beer, regular, all Rolls, hamburger or hotdog, plain Fast foods, potato, french fried in vegetable oil Tea, brewed, prepared with tap water Wheat flour, white, all-purpose, enriched, bleached Snacks, potato chips, plain, salted Tortillas, ready-to-bake or -fry, corn Bread, white, commercially prepared (includes soft bread crumbs)

Food item 3.8 2.8 2.0 2.0 1.8 1.8 1.5 1.4 1.4 1.4 1.4 1.1 1.1 1.1 1.0 1.0

10 3 11 27 146 11 154 6 21 34 3 22 70 72 23

Total nutrient intake (%)

11

Magnesium (mg/100 g)

24.5 25.6 26.6

22.4 23.5

17.1 18.6 19.9 21.3

10.6 12.4 14.2 15.7

6.6 8.7

3.8

Cumulative (%)

4.0 3.8 11.6

98.8 13.3

2.5 62.3 17.3 10.6

47.8 17.6 3.2 36.7

75.0 181.4

91.5

Per capita daily consumption (g)

Table 7.2. Sources of magnesium ranked by their relative contribution to intake of total magnesium based on NHANES 2003–04 all age groups 2 yr and greater.

2.8 2.7 2.7

3.0 2.9

3.8 3.7 3.6 3.6

5.3 4.8 4.7 4.0

7.5 5.4

10.1

Per capita nutrient intake (mg)


335

nutrient by the U.S. population. Percent of nutrient provided is calculated based on nutrient density of the food and amount of the food consumed. Foods listed include either those individual foods that supply 1% or more of a nutrient or those that that collectively supply up to 70% of the intake for a nutrient. It is important to distinguish between foods that are rich sources of a given nutrient and foods that commonly deliver the largest amounts of a given nutrient to consumers. Kale, for example, is a rich source of lutein plus zeaxanthin, providing 18 mg/100 g (Table 7.11), but kale is not a particularly popular food. In contrast, iceberg lettuce, although not a concentrated source of lutein plus zeaxanthin, supplies more of these compounds to the U.S. diet because it is consumed in large amounts relative to kale. In general, highly consumed foods (e.g., apples, orange juice) are identified as the major contributors of certain nutrients on a population basis. However, changes in either the nutrient density of the food or its level of consumption will result in changes in the relative rank of a food. In particular, changes in the level of a nutrient in a food will result in a change in the relative rank without a change in the consumption level. It should be noted that certain fruits and vegetables, while not widely consumed, may be popular among various ethnic groups and therefore could be an important source of a given nutrient to that group. As discussed here, ‘‘good source’’ describes a food that is consumed in quantities sufficient to deliver an important amount of the nutrient in question. 1. Shortfall Minerals: Magnesium, Calcium, and Potassium. Milk is the single food providing the largest percentage of intake of magnesium (Table 7.2), potassium (Table 7.3) and calcium (not shown) in the U.S. diet. However, magnesium and potassium are widely distributed among foods, whereas the milk group provides over 70% of dietary calcium in the U.S. diet. Vegetables are rich sources of magnesium, a component of chlorophyll, but orange juice and bananas are more commonly consumed sources, each contributing about 2% to the average intake of magnesium. Rich sources include halibut (170 mg/1/2 fillet; 107 mg/ 100 g), spinach (boiled; 157 mg/portion; 87 mg/100 g), mature soybeans (boiled; 148 mg/portion; 86 mg/100 g), and various beans (portions in this chapter are 0.24 liter unless otherwise indicated). Whole grains are also rich sources of magnesium, but most is lost during milling. Therefore, if the frequency of intake of whole grains by the population sample is low, then the intake of magnesium attributable to (whole) grains will also be low. In addition to milk products, rich food sources of

336


Milk, reduced fat, fluid, 2% milkfat, with added vitamin A Milk, whole, 3.25% milkfat Coffee, brewed from grounds, prepared with tap water Orange juice, canned, unsweetened Snacks, potato chips, plain, salted Bananas, raw Fast foods, potato, french fried in vegetable oil Milk, nonfat, fluid, with added vitamin A (fat free or skim) Tomatoes, red, ripe, raw Tea, brewed, prepared with tap water Potatoes, boiled, cooked without skin, flesh, without salt Sauce, pasta, spaghetti/marinara, ready–to–serve Milk, lowfat, fluid, 1% milkfat, with added vitamin A Ice cream, vanilla

Food item 5.2 4.1 3.4 3.2 2.5 2.4 2.2 2.2 1.5 1.4 1.3 1.3 1.2 1.0

143 49 175 1642 358 550 156 237 37 328 376 150 199


150

Potassium (mg/100 g)

33.0

32.0

30.7

26.7 28.1 29.5

25.3

15.9 18.4 20.8 23.1

9.3 12.7

5.2

Cumulative (%)

13.8

21.8

8.7

16.4 98.8 10.5

36.7

47.8 4.0 17.6 10.6

75.0 181.4

91.5


Table 7.3. Sources of potassium ranked by their relative contribution to intake of total potassium based on NHANES 2003–04, all age groups 2 yr and greater.

27

33

33

39 37 35

57

84 66 63 58

107 89

137



337

calcium include fortified ready-to-eat cereals, collards (cooked; 357 mg/ portion; 210 mg/100 g), rhubarb (cooked; 348 mg/portion; 145 mg/ 100 g), and sardines (325 mg/93 g portion; 382 mg/100 g). All of the basic food groups contribute potassium to the U.S. diet; milk products and fruits are major contributors (Table 7.3). Tomato, orange juice, and potato are plant-based foods that are both rich sources of potassium and good sources in the U.S. diet. Other plant-based foods that are rich in potassium, but less well consumed, include beet greens (cooked; 1309 mg/portion; 909 mg/100 g), sliced plantains (cooked; 716 mg/ portion; 465 mg/100 g), green soybeans (cooked; 970 mg/portion; 539 mg/100 g), lima beans (cooked; 955 mg/portion; 508 mg/100 g), and sweet potato (cooked; 542 mg/med potato; 475 mg/100 g). For men and women ages 14 and older, a portion of beet greens and green soybeans provide 28% and 20%, respectively, of the AI for potassium. Bananas, the classic food recommended by doctors to patients taking diuretics, contains 422 mg potassium/med (118 g) banana. Thus a medium banana supplies 9% of the AI for men and women age 14 and older (Table 7.1). For men age 31 and older, the group with the highest EAR for magnesium, one portion of orange juice from frozen concentrate supplies 7% (25 mg) of the EAR for magnesium. Similarly, for men and women age 14 and older, orange juice supplies 10% (473 g) of the AI for potassium. A portion of cooked spinach supplies much more; 45% (157 mg) of the highest EAR for magnesium and 18% (839 mg) of the highest AI for potassium. (Recall that EAR and AI for pregnant and lactating women are not considered in this chapter). 2. Total Dietary Fiber. Foods that deliver 1% or more of total dietary fiber to the U.S. diet include fruits (notably banana and apple), vegetables (notably tomato, potato, and lettuce), beans and grains (Table 7.4). Potato- and maize-based snack foods are among the items providing 35% of total dietary fiber. Beans, peas, and lentils are rich, but underutilized, sources of fiber, as are whole grains. Fiber contents of cooked navy beans (19.1 g/portion; 10.5 g/100 g), lentils (15.6 g/ portion; 7.9 g/100 g), black beans (15.0 g/portion; 8.7 g/100 g), and kidney beans (13.8 g/portion; 5.4 g/100 g) are notably high. Fiber content of oatmeal is 2.3 g/100 g and that of whole wheat bread is 1.9 g/slice; 6.8 g/100 g. A portion of navy beans provides 50% of the AI for dietary fiber for males ages 14 to 50 and 73% of the AI for females ages 9 to 18; that is, the age-gender groups that have the highest AI values. Two slices of whole wheat bread supply 10% and 15% of the AI for men and women, respectively, of these ages.

338


Bananas, raw Fast foods, potato, french fried Apples, raw, with skin Rolls, hamburger or hotdog, plain Wheat flour, white, all-purpose, enriched, bleached Bread, white, commercially prepared (includes soft bread crumbs) Beans, pinto, mature seeds, boiled Tortillas, ready-to-bake or -fry, corn Bread, wheat (includes wheat berry) Spaghetti, cooked, enriched Macaroni, cooked, enriched Tortillas, ready-to-bake or -fry, flour Tomatoes, red, ripe, raw Refried beans, canned Potatoes, boiled, without skin, flesh, without salt Lettuce, iceberg (includes crisphead types), raw Snacks, potato chips, plain, salted Snacks, tortilla chips, plain, white corn Beans, pinto, mature seeds, raw Snacks, popcorn, oil-popped, microwaved Peanut butter, smooth style, with salt

Food item 3.1 2.5 2.5 2.5 2.4 1.9 1.6 1.6 1.6 1.6 1.4 1.4 1.3 1.3 1.3 1.2 1.2 1.2 1.1 1.0 1.0

2.4 9 6.3 4.3 1.8 1.8 3.1 1.2 5.3 1.8 1.2 4.4 5.3 15.5 7.9 6


2.6 3.5 2.4 2.1 2.7

TDF (g/100 g)

30.4 31.6 32.7 33.7 34.7

28.0 29.2

16.5 18.1 19.7 21.2 22.7 24.1 25.4 26.7

14.8

3.1 5.6 8.1 10.6 13.0

Cumulative (%)

3.99 3.21 1.04 1.95 2.48

10.53 15.02

2.69 3.77 5.44 12.94 11.81 6.76 16.37 3.66

11.55

17.64 10.61 15.35 17.32 13.26


Table 7.4. Sources of total dietary fiber ranked by their relative contribution to intake of total dietary fiber based on NHANES 2003–04, all age groups 2 yr and greater.

0.18 0.17 0.16 0.15 0.15

0.19 0.18

0.24 0.24 0.23 0.23 0.21 0.21 0.20 0.19

0.28

0.46 0.37 0.37 0.36 0.36

Per capita nutrient intake (g)


339

3. Shortfall Vitamins: Vitamin C, Vitamin E, and Vitamin A. Thirteen food items account for 50% of vitamin C intake (Table 7.5). Orange juice and raw oranges represent three of the five top-ranking sources of vitamin C in the U.S. diet. Orange juice, canned and frozen, represents 23% of vitamin C intake. Collectively, fruits and fruit juices dominate the top ranks of the list of common sources of vitamin C. Broccoli is notable as the only vegetable, botanically speaking, in the list of foods comprising 54% of vitamin C intake. Peppers are notable as concentrated sources of vitamin C. Although other vegetables (e.g., leafy greens) contain appreciable amounts of vitamin C, they are not widely consumed. Banana, containing only 8.7 mg vitamin C/100 g, makes the list due to the popularity of this fruit; per capita daily consumption of banana is 17.6 g. For men and women 19 years and older, a portion of cranberry juice cocktail, fresh or frozen sliced strawberries, cooked broccoli, and orange juice from frozen concentrate supply 129 to 143% and 162 to 178%, respectively, of the EAR for vitamin C. Foods providing vitamin E (as a-tocopherol) to the diet are numerous; 20 foods provide only 38.6% of vitamin E intake (Table 7.6). Common food sources of vitamin E, a fat-soluble nutrient, are dominated by vegetable oils, either directly or indirectly as margarines, spreads, and snack foods. Among common cooking oils, sunflower oil ranks high in vitamin E content, but soybean oil, canola oil, and maize oil are consumed more frequently. Rich but underutilized sources of vitamin E include nuts and seeds; sunflower seeds (dry roasted; 7.4 mg/28 g portion; 26.1 mg/100 g), almonds (7.4 mg/28 g portion; 26.2 mg/100 g), and hazelnuts (4.3 mg/28 g portion; 15.0 mg/ 100 g). Two commonly consumed fruits, tomato (fresh and as sauce) and orange (as juice), are among the foods providing a cumulative 30% of intake of vitamin E. One 28 g portion of dry-roasted sunflower seeds or almonds supplies 62% of the EAR for vitamin E for men and women age 14 and older. Only animal-based foods and fortified foods provide preformed vitamin A (retinol); provitamin A is supplied by plant-based foods and bioconverted to retinol in the body. Vitamin A is a shortfall nutrient in the United States, and vitamin A deficiency is a major public health problem in much of the developing world. It is estimated that in developed countries, such as the United States and Canada, 70% of vitamin A comes from animal sources while 30% is derived from plant-based foods (Institute of Medicine 2001). In contrast, people in developing countries derive about 70% to 80% of vitamin A from plantbased foods (FAO/WHO 1988). Vegetarians and populations with limited access to animal products depend on provitamin A carotenoids

340


Orange juice, canned, unsweetened Orange juice, frozen concentrate, unsweetened, undiluted Cranberry juice cocktail, bottled Added Vitamin C Oranges, raw, all commercial varieties Fruit punch-flavor drink, powder, without added sodium Strawberries, raw Broccoli, boiled, drained, without salt Tomatoes, red, ripe, raw Apple juice, canned or bottled, unsweetened, with added ascorbic acid Peppers, sweet, green, raw Fruit punch drink, with added nutrients, canned Melons, cantaloupe, raw Bananas, raw Peppers, hot chili, green, raw Cranberry-apple juice drink, bottled

Food item 17.9 5.0 3.5 3.4 3.0 2.4 2.4 2.3 2.3 2.1 2.0 2.0 1.7 1.7 1.3 1.1

42.3 99999.9 53.2 121.9 58.8 64.9 12.7 41.6 80.4 29.6 36.7 8.7 242.5 39.5


34.4 137.9

Vitamin C (mg/100 g)

50.0 51.7 53.0 54.1

46.3 48.3

37.6 39.8 42.1 44.2

26.3 29.7 32.7 35.1

17.9 22.8

Cumulative E (%)

4.3 17.6 0.5 2.5

2.3 6.3

3.8 3.2 16.4 4.7

7.6 0.0 5.2 1.8

47.8 3.3


Table 7.5. Sources of vitamin C ranked by their relative contribution to intake of total vitamin C based on NHANES 2003–04, all age groups 2 yr and greater.

1.6 1.5 1.2 1.0

1.9 1.9

2.2 2.1 2.1 1.9

3.2 3.1 2.8 2.2

16.4 4.6


341


Margarine, regular, stick, composite, 80% fat, with salt Snacks, potato chips, plain, salted Peanut butter, smooth style, with salt Salad dressing, mayonnaise, soybean oil, with salt Sauce, pasta, spaghetti/marinara, ready-to-serve Egg, whole, raw, fresh Oil, soybean, salad or cooking Snacks, tortilla chips, plain, white corn Vegetable oil, canola Oil, vegetable, corn, industrial and retail, all purpose salad or cooking Snacks, popcorn, oil-popped, microwaved Orange juice, canned, unsweetened Tomatoes, red, ripe, raw, year round average Salad dressing, italian dressing, commercial, regular Fast foods, potato, french fried in vegetable oil Fast foods, chicken, breaded and fried, boneless pieces, plain Tomato products, canned, sauce Seeds, sunflower seed kernels, oil roasted, with salt added Tomato products, canned, puree, without salt added Peanuts, all types, oil-roasted, with salt

Food item

2.6 2.4 2.3 1.7 1.6 1.6 1.5 1.4 1.3 1.3 1.2 1.2 1.1 1.1 1.1

2.04 0.97 9.21 3.53 17.1 14.3 5.01 0.2 0.54 5 0.78 1.12 2.08 36.33 1.97

1.0

4.0 3.3 2.7

6.74 8.99 5.22

6.94

4.0


6.23

Vitamin E (mg/100 g)

38.6

37.6

35.3 36.5

33.0 34.2

27.8 29.2 30.5 31.8

19.1 21.4 23.1 24.7 26.3

16.7

8.0 11.3 14.0

4.0

Cumulative E (%)

0.94

3.89

3.72 0.21

10.61 7.09

1.95 47.78 16.37 1.76

16.75 1.67 3.21 0.64 0.76

8.73

3.99 2.48 3.48

4.36


Table 7.6. Sources of vitamin E ranked by their relative contribution to intake of total vitamin E based on NHANES 2003–04, all age groups 2 yr and greater.

0.07

0.08

0.08 0.08

0.08 0.08

0.10 0.10 0.09 0.09

0.16 0.15 0.11 0.11 0.11

0.18

0.27 0.22 0.18

0.27


342

P. W. SIMON ET AL.

(b-carotene, a-carotene, b-cryptoxanthin) for vitamin A, and carotenoids thus contribute to vitamin A needs of omnivores. It is estimated that healthy humans derive 1 mg of retinol from 12 mg of b-carotene or from 24 mg of a-carotene or b-cryptoxanthin (Institute of Medicine, 2006). It should be noted, however, that efficacy for improving vitamin A status of anemic children in Indonesia (de Pee et al. 1998) and lactating women in Vietnam (Khan et al. 2007) differed in studies where b-carotene was consumed from fruits rather than from vegetables. In both studies, 1 mg of retinol was derived from 12 mg b-carotene from fruit. The Indonesian children derived 1 mg of retinol from 26 mg b-carotene from green leafy vegetables and carrots (de Pee et al. 1998); similarly, the Vietnamese women derived 1 mg retinol from 28 mg of b-carotene from green leafy vegetables (Khan et al. 2007). It is not clear to what extent food matrix, inhibitors, and health status affect these observations. 4. Carotenoids. Carrot is the major source of a- and b-carotene in the U.S. diet. Collectively, raw, boiled, and frozen-boiled carrots provide 28% of b-carotene and 67% of a-carotene, respectively (Tables 7.7 and 7.8). Vegetables dominate the list for these carotenoids. Pumpkin (canned; 6.9 mg/100 g) and kale (cooked; 8.8 mg/100 g) are rich sources of b-carotene but less popular than those foods in Table 7.7, each of which provides 1% or more of b-carotene to the U.S. diet. Collectively, canned orange juice, frozen orange juice concentrate, and raw oranges provide 60% of dietary b-cryptoxanthin (Table 7.9). Citrus fruits are rich sources of this carotenoid as is orange-fleshed papaya (raw; 0.8 mg/100 g). Tomato-based foods dominate the list of foods providing lycopene, a carotenoid that does not have provitamin A activity, to the U.S. diet (Table 7.10). Pasta sauce accounts for 25% of lycopene intake in the U.S. diet. Raw tomato accounts for 7% of lycopene consumption, trailing catsup, which accounts for 11%. Common food sources of lycopene other than tomato-based foods include watermelon (diced; raw, 4.5 mg/ 100 g) and pink/red grapefruit (1.4 mg/100 g), but these foods do not rank among the foods providing 72% of lycopene intake in the U.S. diet. Dark green leafy vegetables dominate the list of foods providing large percentages of lutein plus zeaxanthin to the U.S. diet (Table 7.11). Spinach, raw, boiled, frozen, and canned, collectively provides 23% of lutein plus zeaxanthin. Individuals who only rarely eat green leafy vegetables might benefit from enrichment of less nutrient-dense food sources with these carotenoids. Egg, which provides 4% of these compounds to the U.S. diet, are notable as an animal source of carotenoids due to the presence of lutein and zeaxanthin in feed ingredients.

343


Carrots, raw Carrots, boiled, drained Sweet potato, baked in skin Melons, cantaloupe, raw Tomatoes, red, ripe, raw Spinach, raw Sweet potato, boiled, without skin Spinach, boiled, drained Lettuce, iceberg (includes crisphead types), raw Carrots, frozen, boiled, drained Broccoli, boiled, drained Lettuce, cos or romaine, raw Sauce, pasta, spaghetti/marinara, ready-to-serve Margarine, regular, stick, composite, 80% fat, with salt Carrot juice, canned Vegetables, mixed, frozen, boiled, drained Catsup Spinach, frozen, chopped or leaf, boiled, drained Collards, boiled, drained, without salt Spinach, canned, drained solids Watermelon, raw Collards, frozen, chopped, boiled, drained Lettuce, green leaf, raw

Food item 18.2 8.1 5.2 4.8 4.0 3.4 2.5 2.5 2.5 1.9 1.6 1.6 1.5 1.5 1.3 1.3 1.3 1.2 1.2 1.1 1.0 1.0 1.0

0.61 9.30 2.08 0.56 7.24 4.81 5.88 0.30 6.82 4.44


8.29 8.33 11.51 2.02 0.45 5.63 9.44 6.29 0.30 8.09 0.93 3.48 0.32

b-Carotene (mg/100 g)

65.6 66.7 67.7 68.7 69.7

60.6 61.9 63.2 64.4

59.3

18.2 26.3 31.5 36.3 40.3 43.7 46.2 48.7 51.2 53.1 54.7 56.3 57.8

Cumulative (%)

0.4 0.3 6.1 0.3 0.4

0.3 1.2 4.2 0.3

4.4

4.0 1.8 0.8 4.3 16.4 1.1 0.5 0.7 15.0 0.4 3.2 0.8 8.7


Table 7.7. Sources of b-carotene ranked by their relative contribution to intake of total b-carotene based on NHANES 2003–04, all age groups.

21.2 20.2 18.4 18.3 18.1

24.7 23.9 23.3 22.2

26.6

334.3 147.9 95.4 87.4 73.5 62.9 46.0 45.5 44.9 35.0 30.1 28.7 28.3


344

P. W. SIMON ET AL.

Table 7.8. Sources of a-carotene ranked by their relative contribution to intake of total a-carotene based on NHANES 2003–04, all age groups 2 yr and greater.

Food item

a-Carotene (mg/100 g)


Cumulative (%)

3.48 3.77

41.9 20.0

41.9 62.0

4.0 1.8

140.3 67.0

0.10

4.9

66.9

16.4

16.5

3.77

4.9

71.8

0.4

16.3

Carrots, raw Carrots, boiled, drained Tomatoes, red, ripe, raw Carrots, frozen, boiled, drained

Per capita Per capita daily nutrient consumption intake (g) (mg)


5. Anthocyanins. Raw fruits, particularly berries, are major contributors of anthocyanins to the U.S. diet (Table 7.12). Fresh and frozen strawberries and blueberries are particularly popular sources, collectively providing 61% of dietary anthocyanins. Red cabbage and beans are the only nonfruit sources among the food items providing 1% or more Table 7.9. Sources of b-cryptoxanthin ranked by their relative contribution to intake of total b-cryptoxanthin based on NHANES 2003–04, all age groups 2 yr and greater.

Food item Orange juice, canned, unsweetened Orange juice, frozen concentrate, unsweetened, undiluted Oranges, raw, all commercial cultivars Carrots, raw Watermelon, raw Corn, sweet, yellow, boiled, drained Carrots, boiled, drained Persimmons, Japanese, raw

b-Cryptoxanthin (mg/100 g)


0.148

49.0

49.0

47.8

70.7

0.321

7.4

56.4

3.3

10.7

0.116

4.2

60.6

5.2

6.1

0.125 0.078 0.161

3.5 3.3 2.9

64.1 67.4 70.3

4.0 6.1 2.6

5.0 4.7 4.2

0.202 1.447

2.5 2.2

72.8 75.0

1.8 0.2

3.6 3.1


Per capita Per capita daily nutrient Cumulative consumption intake (%) (g) (mg)


345

Table 7.10. Sources of lycopene ranked by their relative contribution to intake of total lycopene based on NHANES 2003–04, all age groups 2 yr and greater.

Food item

Total nutrient Lycopene intake Cumulative (MG/100 G) (%) (%)

Sauce, pasta, spaghetti/marinara, ready-to-serve Tomato products, canned, puree, without salt added Catsup Tomato products, canned, sauce Tomatoes, red, ripe, raw Sauce, ready-to-serve, salsa

Per capita Per capita daily nutrient consumption intake (G) (mg)

17.20

24.6

24.6

8.7

1500.7

21.75

13.9

38.5

3.9

846.7

16.71 15.15

11.4 9.2

49.9 59.1

4.2 3.7

693.8 563.8

25.73 10.52

6.9 6.1

66.0 72.1

16.4 3.5

421.1 371.4


of anthocyanins to the U.S. diet. It should be noted that since not all anthocyanins are equally bioavailable (e.g., Kurilich et al. 2005), not only total anthocyanin amount but also the spectrum of individual anthocyanins should be considered when comparing food sources. Comparing across Tables 7.2 to 7.12, some foods are notable for providing significant percentages of several nutrients and phytonutrients. Oranges, which are well known as a popular source of vitamin C, also contribute potassium, b-cryptoxanthin, lutein plus zeaxanthin, and fiber to the diet. Tomato, well known as a source of lycopene, also contributes vitamin C, vitamin E, potassium, a- and b-carotene, lutein plus zeaxanthin, and fiber to the diet. Table 7.13 is from www.ars.usda.gov/nutrientdata and presents nutrient content of selected common plant-based foods. Data are given for comparing foods by weight (nutrients per 100 g of food). Foods from this table are often sources of multiple nutrients. For example, broccoli contains important amounts of vitamins C and E, potassium, b-carotene, lutein plus zeaxanthin, and fiber. Spinach is a rich source of vitamin E, magnesium, potassium, b-carotene, and lutein plus zeaxanthin. B. Resources for Accessing Food Intake and Nutrient Composition of Foods There are several key sources of information on human nutrient requirements and function, food consumption, and sources of nutrients (Table 7.14). The USDA Nutrient Data Lab’s home page provides links to

346

Spinach, raw Spinach, boiled, drained Egg, whole, raw, fresh Orange juice, canned, unsweetened Spinach, frozen, boiled, drained Chicory greens, raw Lettuce, iceberg (includes crisphead types), raw Spinach, canned, drained solids Broccoli, boiled, drained Collards, boiled, drained Squash, summer, all cultivars, boiled, drained Kale, boiled, drained Collards, frozen, chopped, boiled, drained Corn, sweet, yellow, boiled, drained Corn, sweet, yellow, canned, whole kernel, drained solids

Food item 10.3 6.2 4.2 4.1 3.6 3.2 3.1 2.7 2.6 2.6 2.3 2.3 2.2 1.9 1.8

10.6 1.1 7.7 2.2 18.2 10.9 1.0 1.0


12.2 11.3 0.3 0.1 15.7 10.3 0.3

Lut þ Zea (mg/100 g)

47.2 49.5 51.3 53.1

37.5 40.1 42.7 45.0

10.3 16.5 20.6 24.8 28.4 31.6 34.8

Cumulative E (%)

0.2 0.3 2.6 2.3

0.3 3.2 0.4 1.4

1.1 0.7 16.7 47.8 0.3 0.4 15.0

Per capita daily consumption (G)

Table 7.11. Sources of lutein plus zeaxanthin ranked by their relative contribution to intake of total lutein plus zeaxanthin based on NHANES 2003–04, all age groups 2 yr and greater.

29.9 29.3 25.1 23.7

36.3 35.0 33.8 30.4

136.4 81.8 55.4 54.9 48.1 42.7 41.6


347


Snacks, popcorn, oil-popped, microwaved 1.1 Peas, green, frozen, boiled, drained 2.4 Tomatoes, red, ripe, raw 0.1 Lettuce, cos or romaine, raw 2.3 Spinach, frozen, chopped or leaf, unprepared 12.7 Snacks, tortilla chips, plain, white corn 0.5 Beans, snap, green, canned, regular pack, 0.4 drained solids Beans, snap, green, boiled, drained 0.7 Orange juice, frozen concentrate, 0.4 unsweetened, undiluted Broccoli, frozen, chopped, boiled, drained 1.5

54.7 56.3 57.8 59.3 60.6 61.9 63.0 64.0 65.0 66.0

1.6 1.6 1.5 1.4 1.4 1.2 1.1 1.0 1.0 1.0

0.8

1.9 3.3

2.0 0.9 16.4 0.8 0.1 3.2 3.4

12.7

13.7 13.6

21.2 21.0 20.1 19.0 18.0 16.3 14.8

348


Strawberries, raw Blueberries, raw Cherries, sweet, raw Pears, raw Cabbage, red, raw Beans, black, mature seeds, raw Blueberries, frozen, unsweetened Grape juice, canned or bottled, unsweetened, without added vitamin C Plums, raw Strawberries, frozen, unsweetened Raspberries, raw Apples, raw, with skin Cranberries, raw Peaches, raw

Food item 31.0 25.9 9.7 8.2 5.6 2.7 2.2 1.9 1.9 1.7 1.3 1.2 1.1 1.0

12.02 20.61 38.68 0.32 91.88 1.61


33.63 163.52 80.19 12.18 72.98 28.00 94.25 1.05

Total anthocyanins (mg/100 g)

89.1 90.9 92.1 93.3 94.4 95.5

31.0 56.9 66.6 74.9 80.4 83.1 85.3 87.3

Cumulative (%)

0.64 0.35 0.14 15.35 0.05 2.64

3.78 0.65 0.50 2.77 0.31 0.39 0.10 7.57

Per capita daily consumption (G)

Table 7.12. Sources of total anthocyanins ranked by their relative contribution to intake of total anthocyanins based on NHANES 2003–04, all age groups 2 yr and greater.

0.072 0.052 0.049 0.044 0.042 0.036

1.063 0.399 0.337 0.229 0.110 0.090 0.079 0.077


349

0.05 0.18 0.37

2.03 0.29 0.54

36.7 53.2 80.4

28.1 58.8 12.7

99 16 10

9 40 10

5 47 6 33 5

CA

FE)

2.71 0.41 0.27

0.21 0.1 0.34

0.26 0.73 0.28 0.3 0.72


0.1 0.78 0.57 0.66 0.07

8.7 89.2 9.7 5.9 0.7

Banana Broccoli, raw Blueberries Carrots Corn, sweet, yellow, canned, whole kernel, drained solids Melon, cantaloup Orange Peppers, sweet, green Spinach, raw Strawberries Tomato, raw

Vit E

Vit C

Food item

Content (mg)

79 13 11

12 10 10

27 21 6 12 15

MG

558 153 237

267 181 175

358 316 77 320 135

K

5626 7 449

2020 71 208

26 361 32 8285 22

bCarotene

0 0 101

16 11 21

25 25 0 3477 0

aCarotene

0 0 0

1 116 7

0 1 0 125 11

b-Cryptoxanthin

Content (mg)

0 0 2573

0 0 0

0 0 0 1 0

Lycopene

Table 7.13. Nutrient content of 100 g edible portions of selected foods based on the National Nutrient Database for Standard Reference, USDA, Release 20.

12198 26 123

26 129 341

22 1403 80 256 176

Lutein/ zeaxanthin

2.2 2 1.2

0.9 2.4 1.7

2.6 2.6 2.4 2.8 1.9

Total dietary fiber (g)

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Table 7.14. Resources for accessing food intake and nutrient composition of foods. Information source Dietary Reference Intakes: The Essential Guide to Nutrient Requirements presents information on nutrient requirements, functions of nutrients and nutrient interactions

Reference Institute of Medicine 2006

2005 Dietary Guidelines Advisory Committee http://www.health.gov/dietaryguide Report documents the scientific base lines/dga2005/report/ used to develop Dietary Guidelines for Americans. In part, this report addresses shortfall nutrients, food sources of selected nutrients, and dietary patterns for achieving recommended nutrient intakes The USDA Nutrient Data Lab’s home page. provides an easy to use online search. Users can look up nutrient content for up to 140 components in over 7,500 individual food items as well as food sources of 39 traditional nutrients and carotenoids. This site also has data for flavonoids in 385 food items; proanthocyanidins in 205 foods, and isoflavones in 128 foods

http://www.ars.usda.gov/nutrientdata

Food survey data from the NHANES

http://www.cdc.gov/nchs/nhanes.htm

The Food and Nutrient Database for Dietary Surveys are used in the What We Eat in America component of NHANES

http://www.ars.usda.gov/ba/bhnrc/fsrg

Personal assessment of dietary intakes can be performed online

http://www.mypyramid.gov/

the Nutritive Value of Foods (Home and Garden Bulletin No. 72), which includes information on the average nutrient composition of crops as well as links to reports of nutrient content for selected foods. Some nutrients included that may be of interest to plant breeders are those in Tables 7.2 to 7.12, other vitamins and minerals, isoflavones, proanthocyanidins, and oxalic acid.

III. PROGRESS IN BREEDING FOR NUTRIENT CONTENT AND COMPOSITION The longest continuous plant breeding effort has been the University of Illinois project, where maize oil and protein content have been


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modified over 100 cycles of selection (Dudley et al. 1992; Dudley and Lambert 2004). Although improved nutritional quality has not been an immediate goal of this project, it demonstrates the potential for altering the plant composition in these macronutrients. It is interesting to note that early plant breeding texts routinely described the Illinois long-term selection project and sometimes mentioned unpublished information suggesting differences in nutritional quality among cultivars of a crop. Yet breeding for nutritional quality was not mentioned as a primary goal in plant breeding texts through the mid-20th century (e.g., Hayes and Garber 1927; Allard 1960) in spite of the dramatic differences in vitamin A nutritional value between yellow and white maize demonstrated by Steenbock in 1919. A. Breeding for Macronutrients Plant breeders have been extremely successful at improving productivity of agronomic crops. Nutritional quality has received less attention. In crops used primarily as foods, attention has always been paid to traits that impact the quality of the food product, such as bread quality for wheat, but rarely for nutritional quality. Other crops, such as maize, are used primarily for feed and industrial uses in the United States, so their food uses have been comparatively minor and most resources have been used to improving other traits. However, in the industrial forms of sweeteners and starch, corn is found in thousands of processed foods (Corn Refiner’s Association 2006). Historically, breeders have applied selection pressure to traits related to agronomic performance, particularly yield, because these are the traits important to the producer. In some cases, selection for yield does not affect nutritional characteristics, such as for protein composition in soybean (Mahmoud et al. 2006). In other cases, such as for iron, zinc, and selenium concentrations in wheat, selection for yield does impact the nutritional quality (Garvin et al. 2006). Typically, composition or other quality traits are measured late in the breeding process as varieties or lines approach commercialization (Webster, 2002). Rarely have farmers been paid for nutritional or quality factors, so there have not been economic incentives to pay much attention to these traits. However, grain quality, referring to aspects affecting processing requirements and food or cooking quality, is becoming increasingly important and is being driven by an increasing technical marketplace (Wrigley and Morris 1996). Some grain quality changes affect nutritional aspects, such as those that affect protein and protein quality of wheat used for bread making. In light of health problems facing U.S. consumers, breeding for nutritional quality is likely to become more economically beneficial

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(Cordain et al. 2005). Diet is implicated in the cause and severity of many diseases, including cancer, heart disease, and diabetes. Healthier foods that are staples in the diet have the potential to alleviate the incidence and severity of these diseases as well as obesity, which is a causal factor for many chronic diseases. Prevalence of obesity is increasing worldwide, even though certain nutrients are consumed at suboptimal levels in many developing and developed countries. Staple agronomic crops, primarily grains and legumes, are eaten as mature seeds. Nutritional improvements in the seeds can be made through changing the composition and quality of the major components of carbohydrate, protein, and oil and by changing the composition of minor components such as minerals, phytochemicals, vitamins, and antinutritional factors. The mode of inheritance can be through genes with major or minor effect, often a combination of both. There is usually a large environmental effect, especially when the component is present in tiny amounts, such as for phytochemicals. Selection for these components can be very successful, as illustrated in the Illinois LongTerm Selection experiment for protein and oil in maize, recently reviewed in Dudley and Lambert (2004). Selection for over a century (Hopkins 1899) resulted in the extremes of protein and oil content known in maize. Genetic studies identified a large number of genes with small effects responsible for the steady increase in oil content, indicating that recurrent selection would be an appropriate approach to increasing this component (Laurie et al. 2004a,b). Amino acid balance, fatty acid composition, and starch properties, constituents of major components, are important targets for modification because they impact the importance of grain for human health, and some of this value may be captured by farmers and the food industry. A widely used approach has been to use single-gene mutants, but quantitative genetic approaches have been successful when used long term. The most successful approaches involve the use of recurrent selection and mutants together, for example, the development of Quality Protein Maize (QPM) based on the opaque 2 mutant with selection for improved kernel types and the development of high-amylose maize based on the amylose extender mutant (Bjarnason and Vasal 1992; Fergason 1994, 2001). Often breeding is limited by lack of information about nutritional characteristics of the crop germplasm. Metabolite analyses conducted to gain information about natural variation of the composition of conventional crop germplasm for comparison of transgenic crops includes nutrients and antinutrients and can be a resource for planning breeding strategies (Harrigan et al. 2007a,b; Reynolds et al. 2005).


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1. Protein and Protein Quality. Plant proteins provide the majority of those ingested by humans worldwide with cereal grains being the major source. Mixed diets of plant foods can supply adequate amounts of essential amino acids (Millward 1999). Humans and other monogastric animals need to ingest essential amino acids (those the body cannot produce). Grains are limited in certain essential amino acids so human or animal diets composed of one primary grain may be deficient in these amino acids. For example, maize is limited in lysine, methionine, and tryptophan relative to dietary needs. A breeding goal for the nutritional improvement of grains is to increase the level of the limiting amino acids. Breeding for limiting amino acids in maize was recently reviewed by Pollak and Scott (2005). In summary, recurrent selection has been shown to be successful, although major impediments include the lack of inexpensive, high-throughput assays and the time investment required to generate substantial improvements with multiple cycles of selection. Because of these difficulties, breeding programs have used several mutants that alter zeins—the major seed storage proteins. The most widely used mutant, opaque 2, causes the kernels to have elevated levels of the essential amino acids lysine and tryptophan due to a reduced content of zeins (Mertz et al. 1964). Major impediments in using mutants include pleiotropic effects; in the case of opaque 2, these effects give soft kernels (Loesch et al. 1977), making them susceptible to mechanical damage (Lambert et al. 1969), poor germination (Loesch et al. 1978), and reduced grain yield (Lambert et al. 1969; Sreeramulu and Bauman 1970). Thus, much of the effort in breeding programs using mutants is devoted to overcoming adverse pleiotropic effects. Over 30 years, breeders at the International Wheat and Maize Improvement Center (CIMMYT) along with scientists at the University of Illinois, Purdue University, the Instituto Sperimentale per la Ceralicoltura, and other institutions have successfully overcome the adverse pleiotropic effects to create QPM. The development of QPM (Bjarnason and Vasal 1992; Prasanna et al. 2001; Vasal 2001) is an important accomplishment in breeding for nutritional quality. In recognition of their roles in developing QPM, Drs. Surinder Vasal and Evangelina Villegas were awarded the 2000 World Food Prize. The success in maize led to development of high-lysine mutants in other crops, such as barley (Jood and Singh 2001). Nelson et al. (1966) reported that the mutant gene floury-2 caused production of maize endosperm proteins with as high a lysine level as that of the mutant opaque-2, and higher methionine concentration, but subsequent breeding proved difficult due to poor seed quality and reduced grain yield (Pollak and Scott 2005). Another mutant gene,

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P. W. SIMON ET AL.

designated dzr1, is found in a line released as B101 (Hallauer and Wright 1995). Experimental hybrids using inbreds developed by using the progenitor of B101 as the donor parent had 23% to 43% higher methionine levels than control hybrids (Olsen et al. 2003). Nonmutant breeding lines also contained significant variation for methionine and tryptophan, and significant general and specific combining ability effects indicated that methionine content would respond to selection (Darrigues et al. 2005). In contrast to maize, pulses and their relatives had very little genetic variation for the S-amino acids methionine and cysteine (Table and Higgins 1998). 2. Carbohydrates Dietary Fiber. Dietary fiber is a heterogeneous mixture of polysaccharide carbohydrates, such as cellulose, hemicellulose, pectins, gums, mucilages and lignins, plus noncarbohydrates, such as lignin, that are indigestible in the small intestine (Dreher 2001). Insoluble fiber is mainly cell wall components, such as cellulose, lignin, and hemicellulose found in grains, fruits, and vegetables. Health benefits of insoluble fiber include shortening of the bowel transit time and bulkier and softer feces. Noncellulosic polysaccharides, such as pectin, gums, and mucilages, are components of soluble fiber found in fruits; grains, especially oats and barley; and legumes. Soluble fiber delays gastric emptying, slows glucose absorption, and lowers serum cholesterol levels, and is completely or partially fermentable in the large intestine. Fermentation produces shortchain fatty acids that have many health benefits related to blood glucose levels, cholesterol synthesis, immune function, and colon health. In spite of many years of research and nutrition education about potential benefits of dietary fiber to help prevent diseases such as cancer, heart disease, and diabetes, the consumption of complex carbohydrates and dietary fiber has decreased (Slavin 2001). Whole grains and bran are concentrated sources of fiber, compared to fruits and vegetables with their high water content (Dreher 2001), and increasing the amount of soluble or insoluble fiber is a logical breeding goal to improve the nutritional quality. A water-soluble polysaccharide important in oat and barley bran, b-glucan, has become a commercial product that is added to food to increase the soluble fiber content (Whistler and BeMiller 1997). Both an oat fiber extract with concentrated soluble fiber b-glucan (Behall et al. 1997) and whole-grain barley (Behall et al. 2004) were effective in reducing total and low-density lipoprotein (LDL) cholesterol levels in people with slightly elevated cholesterol levels,


355

but barley is not widely consumed in U.S. diets. Oats are more widely consumed as whole-grain cereal; thus the benefits of whole-grain components are available as well as the b-glucans for which a health claim is allowed by the U.S. Food and Drug Administration (FDA 1997). By using flour from oat lines with varying levels of b-glucans and testing in vitro bile acid binding capacity, which is related to cholesterollowering effects, Sayar et al. (2006) concluded that binding capacity occurred from the synergistic interaction of the whole-grain components with b-glucan and lignin (insoluble fiber) having large effects. Barley and oat mixed-linkage (1!3)(1!4)-b-D-glucans may also enhance immune function (Fulcher and Rooney Duke 2002). When a plant component is found to be important for human health, and producers and food companies can capitalize on the value of the component, there may be an opportunity for plant breeders to increase the amount of the component. If the crop contains genetic variability for the component, if selection is effective without detrimental pleiotropic effects, and if there is an easy method to measure the component, then plant breeders can be successful. For the example of b-glucan, Givens et al. (2000) reported that oat cultivars can vary widely in both b-glucan and total nonstarch polysaccharides (NSP), that the variation is due both to genetic and environmental effects, and that due to a poor relationship between b-glucan and soluble NSP concentrations, selection for better health components are best based on b-glucan content. Traditional methods to measure total dietary fiber and components of dietary fiber are expensive and time consuming, thus unsuitable for plant breeding, where many samples need to be measured in a short time frame. Nearinfrared (NIR) transmission and reflectance spectroscopy has shown promise as a rapid screening method for NSP in grains and b-glucan in barleys (Blakeney and Flinn 2005) and for total dietary fiber in barleys (Kays et al. 2005). Estimation of breeding parameters related to inheritance of a component gives breeders information about the success of selection for the component. Breeding parameters measured on b-glucan in oats (Holthaus et al. 1996) indicated that additive gene effects were much more important than dominant gene effects, with no evidence of epistasis. Broad-sense heritability on an individual plant basis was estimated to be 0.55, and a positive correlation between grain yield and b-glucan content was observed. In another study, mean b-glucan content increased from 53.9 to 59.9 g kg1 and from 63.5 to 66.0 g kg1 in two broad-based populations, respectively, from the S0 to S1 generation (Cervantes-Martinez et al. 2002). Their heritability estimates were 0.80

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P. W. SIMON ET AL.

to 0.85 on a line mean basis. Results from these studies indicate that direct selection for b-glucan content in oats should be successful. If direct selection for a plant component is successful, but environmental effects overwhelm the genetic effects, it would be difficult to assign value to the component that could be passed to the farmer, and it would be difficult for food companies to make specific health claims for their product. Barley of varying b-glucan levels grown at three locations during two years had about 49% of the b-glucan variability attributed to year, location, year location, and their interactions with genotype (Hang et al. 2007). As opposed to the findings of Holthaus et al. (1996) in oats, there was a weak negative correlation with seed yield, but positive correlations with protein and percentage plump kernels. For oat genotypes grown during three years at four locations, b-glucan content was also about equally influenced by environmental and genetic effects (Doehlert et al. 2001). Many minor grains and seed crops may have beneficial health properties because of their fiber. These crops are found primarily in the health food market, but are gaining wider acceptance as consumers have more interest in natural and traditional foods. If products were developed to take advantage of their special health benefits from fiber and associated phytochemicals, these crops could play a role in mainstream healthy diets. Native emmer wheat from Italy, a hulled wheat, had significantly more polysaccharide fractions of pericarp fiber than durum wheat, showing that pericarp fiber from hulled wheat was clearly different from modern durum pericarp fiber (D’Antuono et al. 1998). Significant genetic variability in dietary fiber was found for seeds of sunn hemp genotypes (Morris and Kays 2005). Fiber from these seeds could be used as a nutraceutical, along with phytochemicals that have potential as phytopharmaceuticals, as long as toxic alkaloids also found in the seeds are not contaminants. If these components have enough value, plant breeding could possibly play a role in reducing toxins as well as increasing levels of valuable components. Starch. Carbohydrate is packaged and stored in starch granules in plants. As granules, starch is quasi-crystalline and water insoluble but is easily converted to sugar by plant metabolic enzymes and digestible when eaten as food. Granules vary widely in size and shape among plants but give two main types of X-ray diffraction patterns. A-type patterns are characteristic of cereal grain starches, while B-type patterns are characteristic of tuber, fruit, and stem starches. C-type patterns are intermediate and are probably due to mixtures of A- and B-type crystallites, within or among granules within a plant (French 1984).


357

The tissue of grain includes a triploid endosperm regarded as continued development of the female gametophyte from the union of a sperm with the two polar nuclei during double fertilization, a diploid embryo or the new sporophyte, and the pericarp and other outer layers derived from the mother plant of the previous sporophytic generation (Kiesselbach 1980). Some cereal grains, such as those of barley and oats, still carry the hulls or glumes from the mother plant that are not lost through harvesting. Starchy endosperm is normally the largest portion of the cereal grain seed. Starch granules structurally unique to the cereal accumulate in endosperm tissue. In addition, the endosperm contains lipids, storage protein, NSP, and low-molecular-weight phenolic compounds. The nutritional issues of cereal grains are reviewed in Fulcher and Rooney Duke (2002), and those related to carbohydrates are summarized here. Depending on their structure, starch granules digest differently because they present different surface areas to digestive enzymes. The other endosperm components also interact with each other and with the granules to affect digestibility. Excessive heat stress also can cause starch to be less digestible through annealing. Starches more resistant to digestion, called resistant or slowly digestible starches, act like dietary fiber and contribute to gut health. Starch is composed of two glucose polymers, amylopectin and amylose, which differ in their physical properties, chain length, and degree of branching. Amylopectin is more highly branched and normally constitutes about 70% to 80% of the starch granule by weight; amylose is mostly linear and constitutes 20% to 30% of the granule. The ratio of amylopectin to amylose, chain length, and degree of branching are all under genetic control (Young 1984). Much of the breeding effort for unique starch properties has involved changing the relative proportions of these two polymers. Maize with a waxy1 mutant allele was discovered in China in the early 1900s (Collins 1909). The Waxy1 gene is required for the biosynthesis of amylose, and mutant alleles of waxy1 produce waxy starch that is 100% amylopectin. The starch became commercially important during World War II when tapioca starch, which had similar properties of making clear gels resistant to recrystallization, was unavailable from the East Indies. Fergason (1994; 2001) reviewed the breeding of waxy maize, which has largely been conducted in the private sector. This breeding effort has led to the development of waxy hybrids with nearly the yield of their non-waxy counterparts. Other crops, such as wheat, sorghum, and rice, have waxy counterparts, but demand is much less than for maize because applications are

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still being developed. Considerable variation for waxy alleles was found in a survey of recent U.S. hard winter wheat germplasm (Shan et al. 2007). Waxy soft white spring wheat (Morris and King 2007) and durum wheat (Grant et al. 2004) lines have been developed that have properties that may lead to healthier foods. For example, waxy wheat flour could substitute for shortening in bread while providing similar crumb softness and shelf life (Bhattacharya et al. 2002). In contrast to waxy starch gels, high-amylose-starch gels are opaque and strong. Vineyard and Bear (1952) reported that a maize mutant amylose extender (ae) allele increased the ratio of amylose to amylopectin starch fractions, but the total starch content remained the same. The breeding of high-amylose maize was also reviewed by Fergason (1994, 2001). Mutant ae alleles do not confer 100% amylose starch but rather a range of amylose percentages depending on the genetic background and environment. An unknown number of genetic modifiers interact with ae alleles affecting the values of amylose. In addition, the relative amylose content must be measured by chemical analysis, adding significant expense and time to the breeding program. Because environment has a major influence on amylose levels, the separation of genetic versus environmental effects is important in order to make genetic progress. In spite of these limitations, selection and breeding have increased the amylose content of high-amylose hybrids from approximately 50% to well over 70% (Boyer and Hannah 1994). Advances in breeding for amylose content can come from new breeding methods, cheaper and more rapid methods of analysis, or new modifier genes. Campbell et al. (1997, 1999, 2000) developed a NIR transmittance spectroscopy calibration that had limited precision and thus could not replace chemical analysis, but could be used for initial screening of large sample sizes. Campbell et al. (2002) then developed a relatively rapid and inexpensive wet chemistry method that was used to identify genotypes with high apparent amylose concentrations. Another important finding of this study, supported by a previous study of Argentine landraces (Robutti et al. 2000), was that exotic germplasm might be an important source of new modifying factors. Resistant starch (RS), considered to function as a dietary fiber, is defined as the fraction of dietary starch that escapes digestion in the small intestine (Sajilata and Singhal 2006). RS contributes to a healthy diet because of its resistance to digestion in the small intestine and partial fermentation in the colon (Cummings et al. 1996). This results in a reduced glycemic index (GI), which has been linked to control of type II diabetes, promotion of health-promoting bifida bacteria in the intestines (and thus, perhaps, to reduced colon cancer incidence), and


359

enhanced satiety to help with weight maintenance and control of obesity and its associated health risks (Thompson 2000). RS formation increases with increased amylose content of the starch. Studies using high-amylose maize show that GI and insulin response are related to the amount of RS in the food, and increase with increasing amylose content of maize starch (Behall et al. 1988; Behall and Hallfrisch 2002). Both cancer and diabetes (National Cancer Institute 2001) are causally linked to obesity. The current obesity epidemic, diabetes, and related diseases affect U.S. racial and ethnic populations disproportionately. For example, the incidence of diabetes among Hispanics, the population group growing most quickly in the United States, is almost twice the incidence of non-Hispanic whites (Centers for Disease Control and Prevention 2004). In the nation, after heart disease, cancer is the number two and diabetes the number six cause of death (National Center for Health Statistics 2007). Maize is widely consumed in this country and is a staple grain in ethnic Hispanic diets. Studies reviewed by Plate and Gallaher (2005) show that maize as a food has many health benefits by having a lower GI and higher levels of phytochemicals than other widely consumed grains. Those foods made with high-amylose maize and foods that are heated and cooled to allow starch retrogradation, in other words maize foods with higher levels of RS, have especially low GI. Another benefit of high-amylose maize may be its increased antioxidant activity as compared to typical maize (Li et al. 2007). In contrast to maize, rice is considered to be highly digestible with digestibility increasing as amylose content increases. Benmoussa et al. (2007) found amylopectin fine structure variability to be related to starch digestion. Pasting breakdown measured by rapid viscoanalyzer was correlated to starch digestibility, and could be used as a screening tool by rice breeders to develop more slowly digestible cultivars. 3. Fatty Acids. Oil is a concentrated source of energy that increases the caloric content of the seed. Forces driving demand for certain fatty acids include functional properties and health benefits. Reduced consumption of saturated fat is appropriate for most of the U.S. population for control of blood lipids associated with cardiovascular disease. It is recommended (HHS/USDA 2005) that dietary fat come primarily from sources rich in polyunsaturated and monounsaturated fatty acids. This has shifted oil consumption to more highly unsaturated oils, such as soybean and sunflower oils, and to monounsaturated oils, such as canola and olive oils. In recent years, consumption of trans fatty acids, primarily produced by partial hydrogenation of polyunsaturated fatty acids, has been

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identified as an additional factor producing undesirable blood lipid profiles. Because trans fatty acids are resistant to oxidation and have certain desirable functional characteristics in food processing, the rush to replace trans fats has created demand for new sources of oils that have desirable functional properties and minimal amounts of highly oxidizable polyunsaturated fatty acids (Tarrago-Trani et al. 2006). A breeding success story occurred when a major oilseed crop used around the world, canola, was developed from rapeseed by eliminating C22:1 erucic acid and lowering glucosinolates in the meal, both believed to be toxic to humans and animals. This transformation is described by Busch et al. (1994). Modification of the fatty acid content to reduce linolenic acid (C18:3), increase oleic acid (C18:1), and decrease saturated fatty acids was reviewed by Scarth and Tang (2006). Sunflower has been modified by induced mutation and selection to increase the level of the saturated fatty acid stearic (C16:0) to produce semisolid oil at room temperature for production of margarine-type products (Osorio et al. 1995; Po´rez-Vich et al. 2004). Of the two major saturated fatty acids in food oils, stearic acid is preferred over palmitic (C14:0) because stearic may have a neutral effect on serum cholesterol concentrations (Pearson 1994). Soybean fatty acid compositions have been modified in many ways by using mutants, including development of genotypes with reduced linolenic oil that have better oxidative stability than conventional soybean oil, reducing or eliminating the need for hydrogenation (Ross et al. 2000). Although soybean with lower linolenic acid content also had lower levels of total tocopherol content, or other antioxidants (tocotrienols), enough variation was found within lines to make breeding for both low-linolenic acid and acceptable tocopherol levels feasible (McCord et al. 2004). Maize oil is relatively stable to oxidative changes during storage because it has a small amount of linolenic acid and high amounts of tocopherols (Dupont et al. 1990), and is considered a premium frying oil. Because wide ranges of fatty acid compositions can be found in adapted (Dunlap et al.1995a) and exotic germplasm (Jellum 1970; Camussi et al. 1980; Dunlap et al. 1995b), it is also possible to develop specialty maize oils with a unique fatty acid pattern. Poneleit and Bauman (1970) determined that progress could be made in selecting for both fatty acid quantity and quality using breeding systems that exploit additive genetic variance. Duvick et al. (2006) utilized germplasm introgressed with genes from Tripsacum, a wild relative of maize, to alter fatty acid content in various lines. Lines were developed that had elevated levels of oleic acid; others had either elevated or lowered saturated fatty acids.


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The lines with elevated saturated fatty acid composition had better oxidative stability than traditional maize oil (Shen et al.1999). B. Breeding for Micronutrients and Phytonutients Plant breeding efforts targeting improved micronutrient content and composition began in the 1940s and 1950s with research describing the inheritance and development of tomato breeding stocks high in provitamin A carotenoids and vitamin C (Lincoln et al. 1943; Lincoln and Porter 1950; Tomes et al. 1953). Similar research leading to the development of darker orange, and consequently high provitamin A, carrots began in the 1960s (Gabelman and Peters 1979). Since then genetic improvement to increase levels of specific micronutrients has been pursued primarily in several vegetables. In the last decade there has also been a large effort in evaluating and breeding for increased phytonutrient and micronutrient content in fruit crops and in increasing micronutrient content for several staple food crops (Tables 7.15, 7.16, and 7.17). For many crop species, there have been surveys of diversity in micronutrient content and composition in diverse germplasm with no published follow-up effort toward genetic or breeding studies. Breeding projects that have released germplasm selected for improved nutrient control or composition are relatively uncommon. In addition to classical breeding efforts for nutrients, there have been several transgenic efforts to improve nutritional quality. ‘‘Golden rice’’ is the best-known example (Ye et al. 2000). We restrict this review to classical breeding efforts utilizing naturally occurring genetic variation. 1. Minerals. Plant breeding strategies to increase content and bioavailability of minerals in staple crops have been developed and initiated with a CGIAR initiative over the last decade (Bouis 1996). This field of study is complex not only because it is a relatively new breeding goal, but also because of mineral interactions with each other, several variations, and numerous other compounds in the soil and in the plant (Frossard et al. 2000). A significant genetic component of iron and zinc content of edible plant parts has been noted, but parallel investigations for calcium are not widely reported for many plant species and even less is known about magnesium. As progress is made in breeding for crop yield, mineral content usually is reduced. Furthermore, breeding for improved mineral use efficiency usually does not alter mineral content of edible plant parts (Welch and Graham 2004). Success in breeding for higher mineral content must consider not only mineral concentration but also organic components in plants that can be

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abundant and either reduce (phytate, phenolic compounds) or increase (vitamin C) bioavailability (Frossard et al. 2000). With these numerous considerations, breeding plants for improved mineral nutritional value is a complicated goal that needs expertise of not only plant breeders and nutritionists but also soil scientists. From 3- to in excess of 10-fold variation in iron and zinc content of several staple crops was observed and summarized by Frossard et al. (2000) with significant genetic component for iron content variation in rice but more complex interaction between genotype and environment for maize, beans, wheat, and yams. Patterns for zinc inheritance follow a similar pattern to iron. Little information for calcium or magnesium inheritance is published. Mineral content currently found in major source crops (Table 7.15) is on the lower end of mineral content across the broader range of variation reported. Genetic variation for mineral content has not been evaluated for many crops, but recent studies do exhibit wide variation for magnesium content of wheat (Roussel et al. 2005; Oury et al. 2006; Sipos et al. 2006), and a broad range of calcium, iron, and zinc content were observed across a range of Andean potato cultivars (Andre et al. 2007). It is difficult to estimate the potential for breeding crops with higher content and bioavailability of shortfall minerals until more information about the genetics and heritability of mineral content has been reported. 2. Vitamin C. Nearly all dietary vitamin C comes from plant sources, and broad variation among breeding stocks or cultivars has been reported for several crops (Tables 7.5 and 7.16). Current vitamin C content of most crops in the U.S. marketplace is roughly in the middle of the range of content reported in the literature, although potential for breeding higher vitamin C content of tomatoes and apples would seem to be promising, as much higher content is reported in genetic studies (Table 7.16). As scurvy was common a century ago, tomato was noted as a ready, albeit variable, source of vitamin C. Research investigating the genetic basis of vitamin C content of tomato were first reported in the 1940s. Lincoln et al. (1943) noted fourfold variation among commercial cultivars and up to 1194 ppm in red-fruited tomato interspecific crosses with S. pimpinellifolium. Bhatt et al. (2001) noted a similar level of variation among diverse breeding stocks requiring a challenging breeding scheme with predominantly nonadditive genetic variance. Rousseaux et al. (2005) identified several QTL in an interspecific cross with S. pennellii but large environmental efforts. The inheritance patterns of vitamin C content have also been evaluated in apple, strawberry, and eggplants, and QTL noted in the


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Table 7.15. Current content and range (ppm) of variation for selected minerals in major crop sources.z Nutrient/ Food

Current content

Range

Magnesium Oranges Bananas Peanuts Wheat

100 273 1760 224

89–133 261–458 1800–1900 190–1890

Potatoes Corn Carrots Apples

206 263 117 54

153–245 152–277 80–231 52–58

Zinc Wheat

12

5–43

Potatoes

3

2–4

Iron Potatoes Wheat Beans Soybean Rice Calcium Potatoes Apples Potassium Oranges Bananas

References Miller-Ihli 1996 Miller-Ihli 1996; Wall 2006 Pennington et al.1995 Pennington et al.1995; Sipos et al. 2004; Roussel et al. 2005; Oury et al. 2006 Pennington et al. 1995 Pennington et al. 1995 Pennington et al.1995; Nicolle et al. 2004 Pennington et al.1995; Miller-Ihli 1996; Oraguzie et al. 2003 Pennington et al.1995; Sipos et al. 2004; Oury et al. 2006 Pennington et al. 1995; Andre et al. 2007

4 36 21 22–44 13

3–16 20–88 10–92 16–97 12–30

Pennington et al. Pennington et al. Pennington et al. Moraghan 2004 Pennington et al.

60–150 62

50–835 26–74

Pennington et al. 1995; Andre et al. 2007 Pennington et al. 1995; Miller-Ihli 1996; Oraguzie et al. 2003

1811 3580

1610–2010 3510–4780

1995; Andre et al. 2007 1995; Oury et al. 2006 1995; Moraghan 2004 1995;Choi et al. 2007

Miller-Ihli 1996 Miller-Ihli 1996

z Values are on fresh weight basis unless indicated. DW ¼ Dry weight basis; oil indicates content of oil. Current content from USDA Nutrient Data Lab, http://www.ars.usda.gov/nutrientdata.

former two crops (Lerceteau-Kohler et al. 2004; Davey et al. 2006; Prohens et al. 2007), providing breeders information and tools for developing cultivars higher in vitamin C. Evaluation of genetic variation in vitamin C levels of potato, pepper, and muskmelon have also been reported (Howard et al. 2000; Davies et al. 2002; Burger et al. 2006; Dhillon et al. 2007), and inheritance patterns are complex. Interestingly, relatively little is known about the genetics of vitamin C in citrus, which is by far the most plentiful source in the U.S. diet (Table 7.5).

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P. W. SIMON ET AL.

Table 7.16. Current content and range (ppm) of variation for vitamin C and vitamin E in major crop sources.z Nutrient/ Food

Current content

Range

Vitamin C Oranges Strawberries

533 590

413–627 451–1274

Broccoli Tomatoes

892 127

222–944 84–1194

Apples Peppers

46 800–1260

14–160 151–2024

Melons

181–369

14–431

Bananas Potatoes Eggplants Carrots Squash

87 74–132 13 59 35–55

45–127 110–153 10–22 16–69 27–77

Papaya

618

190–700 DW

151 4 (8 oil)

146–162 oil 3–68 (4–564 oil)

Wheat Corn

3 7

1–10 DW 6–66 DW

Canola

174 oil

(147–230 oil) 71–108

Tomatoes

5

1–22

Vitamin E Peanuts Soybeans

Sunflower oil 41 Carrots 6 Squash 1 Peppers 3–13 Safflower 35 oil

12–1858 2–7 0–42 10–108 64–99 DW

References Dhuique-Mayer et al. 2005 Lerceteau-Kohler et al. 2004; Dodds et al. 2007 Vallejo et al. 2003; Singh et al. 2004 Lincoln et al. 1943; Abushita et al. 2000; Bhatt et al. 2001; Markovic et al. 2002; Rousseaux et al. 2005; Lenucci et al. 2006 Davey et al. 2006 Gupta and Yadav 1984; Yadav et al. 1987; Howard et al. 2000 Eitenmiller et al. 1985; Burger et al. 2006; Dhillon et al. 2007 Wall 2006 Davies et al. 2002; Andre et al. 2007 Prohens et al. 2007 Nicolle et al. 2004 Sirohi and Yayasani 2001; Pandey et al. 2002 Selvaraj et al. 1982 Jonnala et al. 2006 Dolde et al. 1999; McCord et al. 2004; Ujiie et al. 2005; Scherder et al. 2006; Dwiyanti et al. 2007; Nishiba et al. 2007; Rani et al. 2007 Dolde et al. 1999; Moore et al. 2005 Combs and Combs 1985; Egesel et al. 2003 a,b Abidi et al. 1999; Dolde et al. 1999; Leckband et al. 2002; Marwede et al. 2004 Abushita et al. 2000; Lenucci et al. 2006; Frusciante et al. 2007 Dolde et al. 1999; Velasco et al. 2004 Nicolle et al. 2004 Winkler 2000;Tadmor et al. 2005 Kanner et al. 1979 Velasco et al. 2005

z Values are on fresh weight basis unless indicated. DW ¼ Dry weight basis; oil indicates content of oil. Current content from USDA Nutrient Data Lab, http://www.ars.usda.gov/nutrientdata.


365

3. Vitamin E. Vitamin E comes from a diverse range of sources in the U.S. diet, and genetic or breeding efforts have been under way in most oil and cereal crops as well as for several vegetables (Table 7.16). a-Tocopherol is the major isomer contributing to vitamin E activity in most vegetable oils. The most advanced breeding efforts have been in soybean where high a-tocopherol cultivars were bred (Ujiie et al. 2005), but tocopherol levels are also evaluated in some breeding programs for canola, peanut, sunflower, safflower, maize, wheat, and rice. Tomato and squash genetic evaluations have also been reported. Quantitative inheritance patterns are observed for tocopherol levels in all crops with significant nongenetic variation and nonadditive gene action noted (Bergman and Xu 2003; Egesel et al. 2003a; Marwede et al. 2004; Velasco et al. 2004; Dwiyanti et al. 2007; Rani et al. 2007). QTL influencing tocopherol level in soybean, maize, and tomato have been identified. Much higher vitamin E content was reported in studies evaluating diverse cultivars than that in the marketed commodity for most crops (Table 7.16), thereby indicating potential to improve levels that consumers realize in their diet in the future. 4. Carotenoids. Since the carotenoids impart orange, red, and yellow color to crops, and color is an important aspect of consumer appeal, much attention has been paid to genetic evaluation and breeding for carotenoid content of many vegetables and fruits. Those carotenoids that have provitamin A and phytonutrient properties (Table 7.17) have received even greater attention in efforts to biofortify vegetables, fruits, and more recently staple crops. Genetic and breeding efforts have been advanced for carrot, tomato, pepper, potato, muskmelons, squash (including pumpkin), sweet potato, maize, cassava, and lettuce, primarily focusing on total carotenoid content, b-carotene, and lycopene (Fonseca and Simon 1994; de Carvalho et al. 2006). More recent research efforts have considered the prospects for genetically increasing lutein and b-cryptoxanthin content. Uniform orange storage root color has been a trait of interest to carrot breeders for over a century (Simon 2000). Yellow core (xylem) color occurs frequently in older open-pollinated cultivars and is viewed as a defect, so that elimination of yellow core color has been a major breeding objective. Breeding has been ongoing since the 1950s to understand the genetic factors that control color among orange, yellow, and white carrot cultivars. This effort led to the descriptions of the Y and Y2 genes that control these traits (reviewed by Buishand and Gabelman 1979; Gabelman and Peters 1979). Carotenoids of orange carrots consist primarily (in excess of 90%) of the provitamin A carotenes: a- and

366

P. W. SIMON ET AL.

Table 7.17. Current content and range (ppm) of variation for nutritionally important carotenoids and anthocyanins in major crop sources.z Nutrient/ Food

Current content

Range

b-Carotene Carrots

80

0–300

Sweet potatoes

107

1–226

Melons Tomatoes

0–20 4

0–50 1–77

Spinach

55

9–83

Lettuce

3–43

1–91

Broccoli Squash

4 2–29

2–91 1–74

Mangoes Cassava

5 0

7–27 0–8

Chickpeas Corn

0 1

0–1 DW 0–11 DW

Carrots

38

0–380

Squash Peppers

7

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Cereals (Handbook of Plant Breeding, Volume 3)

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Annual Plant Reviews, Plant Mitochondria

Plant Breeding and Biotechnology

Plant-Pathogen Interactions (Annual Plant Reviews S.) (Annual Plant Reviews)

Molecular Plant Breeding (Cabi)

Plant Hormone Signaling (Annual Plant Reviews, Volume 24)

Plastids (Annual Plant Reviews, Volume 13)

Root Development (Annual Plant Reviews, Volume 37)

Biology of the Plant Cuticle (Annual Plant Reviews, Volume 23)

Annual Plant Reviews, Plant Polysaccharides: Biosynthesis and Bioengineering (Volume 41)

Light and Plant Development (Annual Plant Reviews, Volume 30)

Plant Cell Separation and Adhesion (Annual Plant Reviews, Volume 25)

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