Plant Breeding Reviews (Volume 35)

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PLANT BREEDING REVIEWS Volume 35

Plant Breeding Reviews is sponsored by: American Society of Horticultural Science International Society for Horticultural Science Society of American Foresters National Council of Commercial Plant Breeders

Editorial Board, Volume 35 I. L. Goldman C. H. Michler Rodomiro Ortiz

PLANT BREEDING REVIEWS Volume 35

edited by

Jules Janick Purdue University

Copyright Ó 2012 by Wiley-Blackwell. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing. 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 support, 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 978-1-118-09679-6 (cloth) ISSN 0730-2207 Printed in the United States of America eBook ISBN: 978-1-118-10048-6 oBook ISBN: 978-1-118-10050-9 ePub ISBN: 978-1-118-10049-3 10 9 8 7 6 5 4 3 2 1

Contents

Contributors 1. Dedication: Molly M. Jahn Plant Breeder and Geneticist

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1

I. L. Goldman I. Biographical Sketch II. Research Program III. Teaching IV. Administration V. Awards and Recognition VI. The Woman Literature Cited Selected Publications of Molly M. Jahn Germplasm Releases and Patents

2. History, Evolution, and Domestication of Brassica Crops

1 5 7 7 9 9 10 10 16

19

Shyam Prakash, Xiao-Ming Wu, and S. R. Bhat I. Introduction II. Archetypes and Evolution of Basic Genomes and Derived Allopolyploids III. Ethnobotany, Origin, and Domestication IV. Concluding Remarks Acknowledgments Literature Cited

21 25 36 67 70 71

v

vi

CONTENTS

3. Melon Landraces of India: Contributions and Importance Narinder P. S. Dhillon , Antonio J. Monforte, Michel Pitrat, Sudhakar Pandey, Praveen Kumar Singh, Kathleen R. Reitsma, Jordi Garcia-Mas, Abhishek Sharma, and James D. McCreight

I. Introduction II. First Contribution of Indian Melon Germplasm to the U.S. Melon Breeding Programs III. Useful Traits from Indian Melons IV. Genetic Diversity V. Melon Breeding VI. Future Role of Indian Melon Germplasm and Conclusions Acknowledgments Literature Cited

4. Transgenic Vegetable Crops: Progress, Potentials, and Prospects

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88 90 92 120 123 130 133 133

151

Jo~ ao Silva Dias and Rodomiro Ortiz I. World Vegetable Production II. Case for Transgenic Vegetables III. Case Studies IV. GM Vegetables and Integrated Pest Management V. Outlook Literature Cited

5. Millets: Genetic and Genomic Resources

153 154 164 218 221 224

247

Sangam Dwivedi, Hari Upadhyaya, Senapathy Senthilvel, Charles Hash, Kenji Fukunaga, Xiamin Diao, Dipak Santra, David Baltensperger, and Manoj Prasad I. Introduction II. Nutritional Quality and Food, Feed, Medicinal, and Other Uses III. Domestication, Phylogenetic, and Genomic Relationships

251 269 277

CONTENTS

IV. Assessing Patterns of Diversity in Germplasm Collections V. Identifying Germplasm with Beneficial Traits VI. Genomic Resources VII. Enhancing Use of Germplasm in Cultivar Development VIII. From Trait Genetics to Association Mapping to Cultivar Development Using Genomics IX. Conclusions and Future Prospects Acknowledgments Literature Cited

vii

284 300 316 321 332 344 347 347

Subject Index

377

Cumulative Subject Index

379

Cumulative Contributor Index

401

Contributors

David Baltensperger, Soil and Crop Sciences, Texas A&M University, 2472 TAMU, College Station, Texas 77840, USA S. R. Bhat, National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India Narinder P. S. Dhillon, Present address: AVRDC-The World Vegetable Center, East and Southeast Asia, P.O. Box 1010 (Kasetsart), Bangkok 10903, Thailand Xiamin Diao, Lab of Minor Cereal Crops, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Haidian, Beijing 100081, Peoples Republic of China Sangam Dwivedi, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru PO, Hyderabad 502324, AP, India Kenji Fukunaga, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan Jordi Garcia-Mas, Institut de Recerca i Tecnologia Agroaliment aries, Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB), Ctra de Cabrils Km 2, E-08348 Cabrils, Spain I. L. Goldman, Department of Horticulture, University of Wisconsin–Madison, 1575 Linden Drive, Madison, Wisconsin 53706, USA Charles Hash, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru PO, Hyderabad 502324, AP, India James D. McCreight, U.S. Department of Agriculture, Agricultural Research Service, U.S. Agricultural Research, 1636 East Alisal St., Salinas, California 93905, USA Antonio J. Monforte, Instituto de Biologıa Molecular y Celular de Plantas (IBMCP), Universidad Politecnica de Valencia–Consejo, Superior de Investigaciones Cientificas, Ciudad Politecnica de la Innovacioń, Edificio 8E, Ingenierio Fausto Elio s/n, 46022 Valencia, Spain Rodomiro Ortiz, Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, P.O. Box 101, SE-230 53 Alnarp, Sweden Sudhakar Pandey, Indian Institute of Vegetable Research, P.B. No. 01, PO– Jakhini (Shahanshahpur), Varanasi 221 305, India Michel Pitrat, Institut National de la Recherche Agronomique (INRA), UR1052, Genetique et Amelioration des Fruits et Legumes, BP 94, F-84143 Montfavet Cedex, France ix

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CONTRIBUTORS

Shyam Prakash , National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India Manoj Prasad, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, JNU Campus, PO Box 10531, New Delhi 110067, India Kathleen R. Reitsma, U.S. Department of Agriculture, Agricultural Research Service, North Central Regional Plant Introduction Station, Iowa State University, Ames, Iowa 50011-1170, USA Dipak Santra, University of Nebraska–Lincoln, Panhandle Research and Extension Center, 4502 Avenue 1, Scottsbluff, Nebraska 69361, USA Senapathy Senthilvel, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru PO, Hyderabad 502324, AP, India Abhishek Sharma, Department of Vegetable Crops, Punjab Agricultural University, Ludhiana 141 004, India Jo~ ao Silva Dias, Technical University of Lisbon, Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal Praveen Kumar Singh, Indian Institute of Vegetable Research, Regional Station, Sargatia, Kushinagar 274 406, UP, India Hari Upadhyaya, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru PO, Hyderabad 502324, AP, India Xiao-Ming Wu, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan 430062, Peoples Republic of China

Deceased

Plate 2.1 Phenotypes of different varieties of B. oleracea: (a,b) var. acephala; (c) var. oleracea (wild type collected from Spain); (d) var. capitata; (e) var. botrytis; (f) var. italica; (g) var. caulorapa; (h) var. alboglabra.

Plate 2.2 IllustrationsofcolecropsandturnipfromtheJulianaAniciaCodex(512 CE)basedon Dioscorides: (a) Krambe hemeros ¼ cultivated krambe [Brassica oleracea]; (b) Krambe agria ¼ wild krambe [B. cretica]; (c) Gagguli [B. rapa]. (Source: Der Wiener Dioskurides 1998, 1999).

Plate 2.3 Phenotypes of vegetable and oilseed variants of Chinese B. rapa: (a) var. chinensis; (b) var. parachinensis; (c) var. narinosa; (d) var. purpurea; (e) var. chinensis; tai-tsa; (f) var. oleifera; (g, h) var. pekinensis; (i) var. parachinensis; (j) var. narinosa.

Plate 2.4 Phenotypes of Chinese vegetable and oil variants of B. juncea: (a, b, h, j) leaf mustard var. multiceps; (c, d) stem mustard var. tsatsai; (e, f, k) root mustard var. megarrhiza; (g, i) stalk mustard var. utilis; (l) oil mustard var. oleifera.

Plate 3.1 Representative fruits of nondessert melon landraces of India: (1) var. acidulus, (2) var. flexuosus, (3) var. chate, (4) var. momordica, (5) cv. Wanga (var. chate?), (6) semidomesticated and ‘‘wild’’ melons.

1 Dedication: Molly M. Jahn Plant Breeder and Geneticist I. L. Goldman Department of Horticulture University of Wisconsin—Madison 1575 Linden Drive Madison, Wisconsin 53706, USA I. BIOGRAPHICAL SKETCH II. RESEARCH PROGRAM III. TEACHING IV. ADMINISTRATION V. AWARDS AND RECOGNITION VI. THE WOMAN LITERATURE CITED SELECTED PUBLICATIONS OF MARGARET M. JAHN GERMPLASM RELEASES AND PATENTS

Volume 35 of Plant Breeding Reviews is dedicated to the illustrious career of Molly M. Jahn, Molly is a dynamic leader in plant breeding, and her career is an inspiration to a new generation of students entering this profession. I. BIOGRAPHICAL SKETCH Molly Jahn was born June 4, 1959, and raised near Detroit, Michigan. As a child, she was fascinated by nature and field biology. A serious illness that kept her hospitalized in Ann Arbor, Michigan, for a long period gave her time to think, and when she recovered, she was determined to pursue a career as a biologist.1 Avery bright student, Molly was selected as the Midwest Scholar at Swarthmore College. Her start as a geneticist was, however, a humbling one. After failing her first genetics test, her professor, John B. Jenkins, asked her if she had studied. When Plant Breeding Reviews, Volume 35, First Edition. Edited by Jules Janick. Ó 2012 Wiley-Blackwell. Published 2012 by John Wiley & Sons, Inc. 1

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she replied that she had studied diligently for the exam, he replied that students who did poorly either were not very bright or did not know how to study, hoping it was the latter. He allowed her to come to see him once each week with a list of questions, which she did faithfully. Soon he offered her a job doing lab preparations, and later he served as the mentor who encouraged her to apply for a National Science Foundation fellowship to attend graduate school and to apply to at least one graduate school. Numerous examples in Molly’s academic career show incredibly successful outcomes emerging from simple beginnings, and these are a testament to her drive, determination, and vision. It is rare to find a scientist like Molly Jahn who combines high intellect and a strong sense of purpose with such an intuitive sense of the future and its possibilities. Throughout this chapter, specific events and turning points in Molly Jahn’s career are identified from descriptions she provided in an interview for the book Democracy and Higher Education (Peters et al. 2010). Molly was awarded the NSF fellowship and was admitted to graduate study at the Massachusetts Institute of Technology (MIT), where she set off to pursue her interest in genetics, integrating the avalanche of molecular insights of the time in a system that would have some applied relevance. Several faculty members at MIT were extremely influential in helping her shape her scientific priorities and experimental approaches, notably Phil Sharp and Frank Solomon. But while visiting her parents back in Michigan, she picked up a book about her maternal greatgrandfather Saunders and his four brothers, each of whom had made major contributions in plant breeding and related agricultural sciences under the tutelage of their father, William Saunders. Her great-grandfather had a distinguished career as a physicist at Harvard, and the family still met regularly for large reunions at Hamilton College, where one of the brothers had been a chemist and a successful peony breeder. Another brother, Charles, together with his father bred ‘Marquis’ wheat, a shortseason cultivar that opened the western Canadian plains for settlement. When the Canadian Parliament committed to a federal agricultural research system in Canada, her great-great-grandfather served as the founding director of the experiment station in Ottawa, Canada, for agricultural research where her great-grandfather, the youngest son, was raised. Much as Molly was drawn to the rigor and excitement of molecular genetics at MIT, she was also drawn to the practical relevance of genetics toward crop improvement. In 1983, she made the difficult decision to shift graduate programs, bringing the background she had acquired to agriculture. So it was with a combination of heredity, aptitude, and good luck that Molly found her way to Cornell University in 1983 to begin graduate work in plant breeding.

1. DEDICATION: MOLLY M. JAHN PLANT BREEDER AND GENETICIST

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After moving to Ithaca, New York, Molly, following the example of one of her mentors at MIT, looked for a system where she could study mutations that conferred resistance to plant viral disease with the idea that these mutations likely would occur in genes of both fundamental and practical interest. She was particularly focused on genes or gene clusters that appeared to control resistance to sets of viruses, and all paths led to the largest family of plant viruses, the Potyviridae. Because of the role that vegetables play in the developing world and because of their genetic and botanical diversity, she was interested in finding an example of this phenomenon in a vegetable species and eventually settled on the I gene in Phaseolus vulgaris. To work on this problem, she became the student of Michael H. Dickson, a noted breeder of Phaseolus, Brassica, and carrots. From her very earliest days at Cornell, Henry Munger was a key mentor, co-chair of her graduate committee. Ultimately it was his position at Cornell that she filled, appointed in the spring of 1987 as an assistant professor in the Department of Plant Breeding. She received her Ph.D. from Cornell in early 1988 and was allowed to defer her faculty appointment to accept a prestigious postdoctoral fellowship, the Life Sciences Research Foundation fellowship, which she took to the laboratories of Drs. T. J. Morris and A. O. Jackson in the Department of Plant Pathology at the University of California—Berkeley and S. H. Howell at the Boyce Thompson in Ithaca, New York, prior to joining the Cornell faculty in 1991. Although Molly’s primary appointment was in the Department of Plant Breeding, eventually she also held a faculty appointment in the Department of Plant Biology. She assumed responsibility for the germplasm that had been developed by Henry Munger and was an outstanding steward of this important legacy in U.S. vegetable breeding, eventually releasing dozens of varieties, parents, and breeding lines with Munger. Molly and her students and staff worked primarily on Cucurbita, Cucumis, and Capsicum, conducting field-oriented plant breeding and basic laboratory research. Her research laboratory grew to include more than 30 scientists, staff, and students and was supported by a wide variety of funding sources, including federal agencies, contracts, and gifts from seed companies, royalties, private foundations, and significant gifts from individual donors with whom she developed close ties. Special among these were Paul H. Todd, a Cornell graduate and gifted chemist who was interested in her work on peppers, and Charles M. Werly. Molly’s research programs were widely recognized for their breadth and depth and were an ideal training ground for a large number of students, many of whom went on to careers in plant breeding in both the public and private sectors. Her work in plant breeding and plant genetics led to pioneering discoveries related to plant disease resistance and

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quality traits. Furthermore, germplasm releases from her program are now grown commercially on six continents under approximately 60 active commercial licenses. ‘Cornell’s Bush Delicata,’ for example, was named an All America Selection. This achievement was especially notable in that this open-pollinated cultivar combined the best characteristics of an heirloom on a compact, disease-tolerant, highly productive squash and was recognized because it outperformed the best hybrids on the market at the time for both yield and quality. More recently, a cucumber cultivar was noted with the MGA Green Thumb award, and Molly’s licenses now generate royalties that help support the breeding program now led by her student, Dr. Michael Mazourek, an assistant professor at Cornell University. Always committed to ensuring that her work benefited agriculture, Molly worked closely with Cooperative Extension where possible and filled this role herself where budget cuts had resulted in gaps. Because of this commitment to ensuring growers and seed companies had the full benefit of her work, she learned early how important detailed communication and strong partnerships were to her success. As she established herself in basic research, she consistently directed major grants in genomics towards outreach and impact. Molly’s long-term partnerships with George Moriarty, a Research Support Specialist in Plant Breeding and Genetics, and Henry Munger, Professor Emeritus, have been key to the development of so much useful germplasm in the Cornell program along with many key long term staff notably Mary Lyons Kreitinger. Under Henry Munger’s influence with strong support from long-time department chair Ronnie Coffman, Molly committed early to international engagement and has had active research relationships in many countries including Afghanistan, Argentina, Austria, Bangladesh, Brazil, Burkina Faso, Chile, China, Costa Rica, Egypt, Ethiopia, France, Ghana, Greece, Honduras, Hungary, India, Indonesia, Israel, Jordan, Kenya, Mali, Mexico, the Netherlands, Pakistan, Portugal, the Philippines, South Korea, Spain, Sweden, Thailand, Taiwan, Tunisia, Turkey, and South Africa. She welcomed international scholars and students and strongly encouraged U.S. students to acquire international experience. Many have continued their commitment to international engagement and agricultural research. These efforts have resulted in the transfer of many important traits from the Cornell program for use in national programs and seed companies all over the world and sparked ongoing interest in underinvested and indigenous types and species. Molly also worked with the McKnight Foundation for a decade as a charter member of the Oversight Committee of Collaborative Crops.


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II. RESEARCH PROGRAM Beginning with her dissertation research, Molly’s focus on genetics of disease resistance in plants resulted in many publications with two pronounced themes. First, she provided key evidence of the importance of the host translation factor, eIF4E, in virus resistance and provided the first example in plants of a bimolecular interaction whose outcome determined infectivity. This work began with the classical identification and revision of known genes for recessive disease resistance in pepper and concluded with isolation of a series of allelic variants at this gene that varied in the range of isolates controlled. This work was based on a murine model, further establishing the relevance of mammalian model systems for crop improvement. A key observation that defines another important first in plant genetics and plant virology was that eIF4E variants driven by a strong promoter could confer dominant negative disease resistance, despite the presence of a wild-type allele in the cell. A second theme was focused on the organization of disease-resistant genes in the genome. In contrast to prevailing ideas that suggested that the evolutionary pressure on disease resistance loci would lead to rapid diversification and scrambling of these regions of plant genomes, Molly’s laboratory showed conservation of these positions across genera in the Solanaceae, culminating in a definitive study published in 2009 that demonstrated that these loci are conserved across wide evolutionary distances while diversification of specificity occurs frequently even within narrowly defined germplasm pools. The significance of her fundamental research into both the structure and the function of plant disease-resistance genes earned her a berth on the Plant Cell Editorial Board in 2004 and service on the executive committee and as chair of the Plant-Microbe Subcommittee. Another area of fundamental inquiry and significant impact has been her efforts to identify the molecular basis for quality traits in Capsicum, notably color and pungency. She and colleagues used a candidate gene approach to efficiently identify genes with both qualitative and quantitative effects on fruit color in Capsicum, resulting in a widely cited publication. More notable, however, is the definitive work from her laboratory over a decade that defined and mapped loci responsible for both qualitative and quantitative variation in pungency including the C or Pun1 locus for presence/absence of pungency. Capsaicinoid biosynthesis in pepper represents an ideal model to study the appearance of unique and evolutionarily significant metabolic capacities in plants with significant implications for the culinary and pharmacological uses of a number of Capsicum spp.

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In addition to research, student training, and teaching, Molly pioneered a number of new models for public-private partnerships at land grant institutions. These models were based in part on her own experience at Cornell with the licensing of plant germplasm to commercial companies. Those companies became fewer and fewer by the late 1990s, as consolidation whittled the vegetable seed industry down to several major players in the global marketplace. Molly and her colleagues created the Public Seed Initiative (PSI), a mechanism for bringing smaller seed companies and growers together with public sector germplasm. The PSI was, in a sense, a traditional model of cooperation among growers and public sector researchers and allowed for both conventional and organic producers to source public seed. Through the efforts of the PSI, greater connections have been made between smallerscale growers and seed companies, facilitating the marketplace for specialized seed. This has been accomplished through farm-based trials of public sector cultivars and enhanced relationships with Extension educators, who then translate the information to growers in new ways. As part of this project, Molly became aware of an important market in the northeastern United States that was almost entirely underserved by the public sector research establishment, namely organic agriculture. She was a public sector pioneer in the area of breeding and selection in and for organically managed production systems. In 2004, she was awarded the largest federal grant of its kind at the time to establish the Organic Seed Partnership, an effort to integrate public and private sector research efforts with large participatory networks for selection in organically managed production environments and trialing. This effort involves hundreds of farmers across the country connected to public and private sector research programs with particular emphasis on smaller companies with limited research capacities. Molly and her colleagues at Cornell were also instrumental in creating and maintaining the Vegetable Breeding Institute (VBI). The VBI works to assure the continued development of improved vegetable breeding lines and varieties to meet future needs of the vegetable industry and the general public. Through the VBI, which includes faculty from Cornell and, more recently, the University of Wisconsin—Madison, vegetable breeding programs train graduate and undergraduate students to become capable vegetable breeders of the future. The VBI currently has more than two dozen member companies that help support these objectives and participate in yearly field days to exchange information with public sector breeders. Molly Jahn has been a strong advocate for funding and support of plant breeding activities in the public sector. While many public plant


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breeders have lamented the lack of funding for their work, Molly always challenged this notion by arguing that if the work is worth doing and can intersect with strong science and the private sector, resources should be available. Her advocacy helped foster support for plant breeding programs at Cornell and later at the University of Wisconsin—Madison as well as nationally through her work at the United States Department of Agriculture. Molly’s influence has also been to encourage plant breeders and other agricultural scientists to think about the societal impacts of their research and to consider the vitality of the rural economy when planning their work.

III. TEACHING Molly’s core teaching was focused around plant genetics, and the class she taught for many years in that subject at Cornell was a mainstay for graduate students in the field. Plant genetics has become a remarkably active field in the last several decades due to an infusion of insights gained from molecular biology and molecular genetics. Molly was able to incorporate these elements into her courses and bring the best of modern plant genetics to her students. Molly is widely known for thinking far ahead for solutions to problems, and she brought this perspective to her teaching. Characteristic of Molly’s approach was the integration of information gained from other fields, such as mammalian biology, physical sciences, and ecology, into her core subjects. This syncretic format had great benefits for her students, who broadened and deepened their learning and gained valuable insight into the pursuit of knowledge. Molly also taught sophomore plant genetics and many other courses in plant biology during her years at Cornell and is widely regarded as a challenging and beloved instructor by graduate students. She also mentored 14 postdoctoral scientists, 13 international visiting scientists, and served as the major advisor to 19 graduate students during her years at Cornell. As testament to her tireless efforts at mentoring, her former students and mentees characterize Molly’s fierce support of their learning and research projects as absolutely transformative in their education.

IV. ADMINISTRATION In 2006, Molly Jahn was recruited to be dean of the College of Agricultural and Life Sciences and director of the Wisconsin Agricultural Experiment Station at the University of Wisconsin—Madison. Her faculty

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affiliations at Wisconsin are with the Department of Genetics and the Department of Agronomy. Her work there has focused on revitalizing the partnership between the research powerhouse in the College of Agricultural and Life Sciences and the highly varied constituency the college serves. She has been incredibly effective at increasing the resource base of the college, presiding over a substantial increase in extramural funding during her deanship. She also was a driving force behind modernizing administrative and departmental structures and introducing new concepts aimed at improving the efficiency of the use of state resources. Molly is also very well known for serving as an advocate for production agriculture, forestry, the life sciences and higher education in the state. She developed new models for bringing rural students to study at Madison and has championed the cause of curricular reform to capitalize on efficiencies and natural alliances among the sciences. Toward this end, new models for biology instruction, a new major in environmental sciences, a simplified and streamlined degree structure, and reaccreditation of all college-accredited degree programs and animal research were secured. New initiatives in global health, internationalization of curriculum and pre-professional advising in health sciences were launched, and 50 new faculty members were hired, many of whom were recruited to fill new faculty roles. During her deanship, major capital commitments to update facilities and expand infrastructure were secured, including major renovation of Babcock Hall, new construction for the Wisconsin Energy Institute, and a plan for a new Meat and Muscle Biology Laboratory. Emphasizing responsiveness and a strong sense of the land grant mission, Molly has established herself as an important voice in the nearly $60 billion agricultural industry of the state of Wisconsin. Molly also played an instrumental role in developing the Great Lakes Bioenergy Research Center and the Wisconsin Bioenergy Initiative, which together represent close to $200 million in federal and state investment in bioenergy research and outreach. These efforts began in 2007 and are already paying large dividends for the state of Wisconsin and the nation as researchers investigate the potential for biomass-derived energy and the potential trade-offs and synergies, should relevant technologies be commercialized and implemented at scale. In late 2009, Molly took a leave of absence from the University of Wisconsin to serve on a formal loan to the federal government to provide interim leadership at U.S. Department of Agriculture in Washington, D.C., in the mission area of Research, Education, and Economics, initially as Deputy Under Secretary and, effective as of the departure of Dr. Rajiv Shah, subsequently as Acting Under Secretary for


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Research, Education, and Economics. Her work in Washington brought together many of her skills and talents to help advance the science agenda for agriculture, forestry, food safety, nutrition, and environmental sciences during the early phase of the Obama administration. Molly returned to the deanship in Madison on June 1, 2010, and continued through the end of December, 2010. In January, 2011, she took on a brandnew challenge as she transitioned from the deanship to Special Advisor to the Chancellor and Provost for Sustainability Sciences. In this role Molly returned to a more substantial focus on the science that will support decision making with respect to land management strategies, the deployment of innovations on landscapes and our food and energy future.

V. AWARDS AND RECOGNITION Molly Jahn has received numerous awards in her career, among them fellowship in the American Association for the Advancement of Science, the Vegetable Breeding Award of Excellence from the American Society for Horticultural Science, the Wisconsin Dairy Communicator of the Year from the Wisconsin Dairy Business Association, the Service to Industry Award from the Wisconsin State Cranberry Association, a major teaching award at Cornell University, the National Garden Bureau Gold Medal for the winter squash cultivar ‘Bush Delicata’, and the MGA Green Thumb award for her cucumber variety Salt and Pepper. She is widely recognized as a leader in the fields of vegetable breeding and sustainability science and is considered one of the country’s most important voices on the continued relevance of the land grant university in today’s world.

VI. THE WOMAN Molly Jahn is widely known as a visionary leader in the areas of plant breeding, sustainable agriculture and sustainability sciences, and international development, and has been a national and international presence in these fields for many years. She exhibits limitless energy, intellectual brilliance, and a vision for the future that set her apart from her peers. She has served as an inspiration to students, visiting scientists, and colleagues in both science and policy, and her opinions are sought by leaders across a wide spectrum of agriculture and agricultural science fields. Her advocacy for plant breeding education, improved quality and disease resistance in vegetable cultivars grown worldwide, advocacy for small-scale vegetable and seed production in the United States and

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abroad, her work in organic agriculture, and her creation of novel models for public-private partnerships place her among the most widely respected voices for the future U.S. agriculture as a diverse, highly productive, balanced system. She has long been an advocate for the role that vegetables in particular, and improved, stabilized yields of crops and livestock in general will play for human welfare around the world. LITERATURE CITED Peters, S.J., T.R. Alter, and N. Schwartzbach. Democracy and higher education: Traditions and stories of civic engagement. Profile of Molly Jahn. 2010. p. 75–98. Michigan State Univ. Press, East Lansing.

SELECTED PUBLICATIONS OF MOLLY M. JAHN Journal Papers Miller, M.D. [Jahn, M.M.], and F. Solomon. 1984. Kinetics and intermediates of marginal band reformation: Evidence for peripheral determinants of microtubule organization. J. Cell Biol. 99:70–75s. Kyle, M.M. [Jahn M.M.] and R. Provvidenti. 1987. Inheritance of resistance to potyviruses in Phaseolus vulgaris L. I. Two independent genes for resistance to watermelon mosaic virus-2. Theor. Appl. Genet. 74:595–600. Kyle, M.M. [Jahn, M.M.], and M.H. Dickson. 1988. Linkage of hypersensitivity to five potyviruses with the B locus for seed coat color in Phaseolus vulgaris L. J. Hered. 79:308–311. Valyasevi, R., M.M. Kyle [Jahn], P. Christie, and K. Steinkrauss. 1990. Plasmids of Bacillus popilliae Dutky. J. Invert. Pathol. 56:286–288. Kyle, M.M. [Jahn, M.M.], and R. Provvidenti. 1993. Inheritance of resistance to potyviruses in Phaseolus vulgaris L. II. Linkage relations and utility of a dominant gene for lethal necrotic response to soybean mosaic virus. Theor. Appl. Genet. 86:189–196. Gilbert, R.Z., M.M. Kyle [Jahn], H.M. Munger, and S.M. Gray. 1994. Inheritance of resistance to watermelon mosaic virus in Cucumis melo. HortScience 29:107–110. Murphy, J.F., and M.M. Kyle [Jahn]. 1994. Isolation of leaf mesophyll protoplasts from Capsicum species and inoculation with three pepper viruses. Plant Cell Rep. 13:397–400. Fisher, M.L., and M.M. Kyle [Jahn]. 1994. Inheritance of resistance to potyviruses in Phaseolus vulgaris L. III. Cosegregation of phenotypically similar dominant resistance to nine potyviruses. Theor. Appl. Genet. 89:818–823. Prince, J.P., V.K. Lackney, C. Angeles, J.R. Blauth, and M.M. Kyle [Jahn]. 1995. Genetic similarity among Capsicum genotypes as measured by restriction fragment length polymorphism and randomly amplified polymorphic DNA markers. Genome 38:224–231. Murphy, J.F., and M.M. Kyle [Jahn]. 1995. Alleviation of restricted systemic movement of pepper mottle potyvirus in Capsicum annuum cv. ‘Avelar’ by coinfection with a cucumovirus. Phytopathology 85:561–566.


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Munger, H.M., Y. Zhang, S.L. Fenton, and M.M. Kyle [Jahn]. 1995. Leaf blower adapted for large scale inoculation of plants with mechanically-transmitted viruses. HortScience 30:1266–1267. Fisher, M.L., and M.M. Kyle [Jahn]. 1996. Inheritance of resistance to potyviruses in Phaseolus vulgaris L. IV. Inheritance, linkage relations, and environmental effects of systemic resistance to four potyviruses. Theor. Appl. Genet. 92:204–208. Hoffmann, M.P., R.W. Robinson, M.M. Kyle [Jahn], and J.J. Kirkwyland. 1996. Defoliation and infestation of Cucurbita pepo genotypes by diabroticite beetles. HortScience 31:439–442. Valkonen, J.P. T., M.M. Kyle [Jahn], and S. Slack. 1996. Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses. Ann. Appl. Biol. 129:25–38. Collmer, C.W., M.F. Marston, S.M. Albert, S. Bajaj, H.A. Maville, S.E. Ruuska, E.J. Vesely, and M.M. Kyle [Jahn]. 1996. The nucleotide sequence of the coat protein gene and 30 untranslated region of azuki mosaic potyvirus, a member of the bean common mosaic subgroup. Mol. Plant-Microbe Int. 9:758–761. Zhang, Y., M.M. Kyle [Jahn], K. Anagnostou, and T.A. Zitter. 1997. Screening melon (Cucumis melo L.) for resistance to gummy stem blight caused by Didymella bryoniae in the greenhouse and field. HortScience 32:117–121. Prince, J.P., Y. Zhang, E.R. Radwanski, and M.M. Kyle [Jahn]. 1997. A high-yielding and versatile DNA extraction protocol for Capsicum. HortScience 32:937–939. Kyle, M.M. [Jahn], and A. Palloix. 1997. Proposed revision of nomenclature for potyvirus resistance genes in Capsicum. Euphytica 97:183–188. Murphy, J.F., J.R. Blauth, K.D. Livingstone, V.K. Lackney, and M.M. Jahn. 1998. Genetic mapping of the pvr1 locus in Capsicum and evidence that distinct potyvirus resistance loci control responses that differ at the cellular and whole plant level. Molec. Plant Microbe Interact. 11:943–951. Silberstein, L., I. Kovalski, R. Huang, K. Anagnostou, M.M. Jahn and R. Perl-Treves. 1999. Molecular variation in melon (Cucumis melo L.) as revealed by RFLP and RAPD markers. Scientia Hort. 79:101–111. Livingstone, K.D., V. Lackney, J.R. Blauth, R. Van Wijk, and M.M. Jahn. 1999. Genome mapping in Capsicum and the evolution of genome structure in the Solanaceae. Genetics 152:1183–1202. Zuniga, T., J.P. Jantz, T.A. Zitter, and M.M. Jahn. 1999. Monogenic dominant resistance to gummy stem blight in two melon (Cucumis melo L.) accessions. Plant Dis. 83:1105–1107. Grube, R.C., E.R. Radwanski, and M.M. Jahn. 2000. Comparative genetics of disease resistance within the Solanaceae. Genetics 155:873–887. Jahn, M.M., I. Paran, K. Hoffmann, E.R. Radwanski, K.D. Livingstone, R.C. Grube, E.Aftergroot,M.Lapidot,andJ.Moyer.2000.GeneticmappingoftheTswlocusforresistance to tomato spotted wilt tospovirus in Capsicum and its relationship to the Sw-5 allele for resistance to the same pathogen in tomato. Molec. Plant-Microbe Interact. 13:673–682. Grube, R.C., J.R. Blauth, M. Arnedo, C. Caranta, and M.M. Jahn. 2000. Identification and comparative mapping of a dominant potyvirus resistance gene cluster in Capsicum. Theor. Appl. Genet. 101:852–859. Grube, R.C., Y. Zhang, J.F. Murphy, F. Loaiza-Figueroa, R. Provvidenti, and M.M. Jahn. 2000. A new source of resistance to Cucumber mosaic virus in Capsicum frutescens. Plant Dis. 84:885–891. Anagnostou, K., M.M. Jahn, and R. Perl-Treves. 2000. Inheritance and linkage analysis of resistance to zucchini yellow mosaic virus, watermelon mosaic virus, papaya ringspot virus and powdery mildew resistance in Cucumis melo L. Euphytica 116:265–270.

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Collmer, C.W., M.F. Marston, and M.M. Jahn. 2000. The I gene of bean: A dosage-dependent allele conferring extreme resistance, hypersensitive resistance, or spreading vascular necrosis in response to Bean common mosaic virus. Molec. Plant-Microbe Interact. 13:1266–1270. Thorup, T.A., B. Tanyolac, K.D. Livingstone, S. Popovsky, I. Paran, and M.M. Jahn. 2000. Candidate gene analysis of organ pigmentation loci in the Solanaceae. Proc. Nat. Acad. Sci. (USA) 97:11192–11197. Livingstone, K.D., G. Churchill, and M.M. Jahn. 2000. Linkage mapping in populations with karyotypic rearrangements. J. Hered. 91:423–428. Ben Chaim, A., R.C. Grube, M. Lapidot, M.M. Jahn, and I. Paran. 2001. QTL mapping of resistance to cucumber mosaic virus in Capsicum annuum cv. Perennial. Theor. Appl. Genet. 102:1213–1220. Ben Chaim, A., I. Paran, R.C. Grube, M.M. Jahn, R. van Wijk, and J. Peleman. 2001. QTL mapping of fruit-related traits in pepper (Capsicum annuum). Theor. Appl. Genet. 102:1016–1028. Porch, T.G., and M.M. Jahn. 2001. Effects of high temperature stress on microsporogenesis in heat-sensitive and heat-tolerant genotypes of Phaseolus vulgaris. Plant Cell Environ. 24:723–731. Celebi-Toprak, FR., S.A. Slack, and M.M. Jahn. 2002. Nytbr, a new gene for dominant hypersensitivity to Potato virus Y maps to chromosome IV in potato. Theor. Appl. Genet. 104:669–674. Welsh, R., B. Hubbell, D.E. Erwin, and M.M. Jahn. 2002. GM crops and the pesticide paradigm. Nature Biotechnol. 20:548. Blum, E., K. Liu, M. Mazourek, E.-Y. Yoo, M.M. Jahn, and I. Paran. 2002. Molecular mapping of the C locus for presence of pungency in Capsicum. Genome 45:702–705. Brown, R.N., A. Bolanos, J. Myers, and M.M. Jahn. 2003. Inheritance of resistance to four cucurbit viruses in Cucurbita moschata. Euphytica 129:253–258. Chen, J.-F., X.D. Luo, J.E. Staub, M.M. Jahn, C.-T. Qian, F.-Y. Zhuang, and G. Ren. 2003. An allotriploid derived from an amphidiploid diploid mating in Cucumis. Euphytica 131:235–241. Lotfi, M., A.R. Alan, M.J. Henning, M.M. Jahn, and E.D. Earle. 2003. Production of haploid and doubled haploid plants of melon (Cucumis melo L.) for use in breeding for multiple virus resistance. Plant Cell Rep. 21:1121–1128. Aluru, M.R., M. Mazourek, L.G. Landry, J. Curry, M.M. Jahn, and M.A. O’Connell. 2003. Capsaicinoid biosynthesis: Characterization of genes for branched-chain fatty acid biosynthesis. J. Expt. Bot 54:1655–1664. Blum, E., M. Mazourek, M.A. O’Connell, J. Curry, T. Thorup, K. Liu, M.M. Jahn, and I. Paran. 2003. Molecular mapping of capsaicinoid biosynthesis genes and QTL analysis for capsaicinoid content in Capsicum. Theor. Appl. Genet. 108:79–86. Rose, J.K.C., S. Bashir, J.J. Giovannoni, M.M. Jahn, and R.S. Saravanan. 2004. Tackling the plant proteome: Practical approaches, hurdles and experimental tools. Plant J. 39:715–733. Porch, T.G., M.H. Dickson, M.C. Long, D.R. Viands, and M.M. Jahn. 2004. General combining ability effects for reproductive heat tolerance in snap bean. J. Agric. Univ. Puerto Rico 88(3–4):161–164. Porch, T.G., M.H. Dickson, M. Long, D.R. Viands, and M.M. Jahn. 2004. General combining ability effects for reproductive heat tolerance in snap bean. J. Agr. Univ. Puerto Rico 88(3–4):161–164. Nelson, R.J., R. Naylor, and M.M. Jahn. 2004. The role of genomics research in the improvement of orphan crops. Crop Sci. 44:1901–1904.


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Griffiths, P. D., M.M. Jahn, and M.H. Dickson. 2004. Cornell 501: A snap bean breeding line (Phaseolus vulgaris L.) tolerant to white mold. HortScience 39:1507–1508. Naylor, R.L., W.P. Falcon, R.M. Goodman, M.M. Jahn, T. Sengooba, H. Tefera, and R.J. Nelson. 2004. Integrating new genetic technologies into the improvement of orphan crops in least developed countries. Food Policy 29:15–44. Frantz, J.D., and M.M. Jahn. 2004. Five independent loci each control monogenic resistance to gummy stem blight in melon (Cucumis melo L.). Theor. Appl. Genet. 108:1033–1038. Frantz, J.D., J. Gardner, M.P. Hoffmann, and M.M. Jahn. 2004. Greenhouse screening of Capsicum accessions for resistance to European corn borer (Ostrinia nubilalis) HortScience 39:1336–1338. Frantz, J.D., J. Gardner, M.P. Hoffmann, and M.M. Jahn. 2004. Greenhouse screening of Capsicum accessions for resistance to green peach aphid (Myzus persicae) HortScience 39(6):1332–1335. Chen, J., X. Luo, C. Qian, M.M. Jahn, J.E. Staub, F. Zhuang, Q. Lou, and G. Ren. 2004. Cucumis monosomic alien addition lines: Morphological, cytological and RAPD analysis. Theor. Appl. Genet. 108:1343–1348. Alba, R., Z. Fei, P. Payton, Y. Liu, S.L. Moore, P. Debbie, J.S. Gordon, J.K.C. Rose, G. Martin, S.D. Tanksley, M. Bouzayen, M.M. Jahn, and J. Giovannoni. 2004. ESTs, cDNA microarrays and gene expression profiling: Tools for dissecting plant physiology and development. Plant J. 39:697–714. Paran, I., J. Rouppe van der Voort, V. Lefebvre, M.M. Jahn, L. Landry, R. van Wijk, H. Verbakel, B. Tanyolac, C. Caranta, A. Ben Chaim, K.D. Livingstone, A. Palloix, and J. Peleman. 2004. An integrated genetic map of pepper. Molec. Breed. 13:251–261. Quirin, E.A., E. Ogundiwin, J.P. Prince, M. Mazourek, M.O. Briggs, T.S. Chlanda, K.-T. Kim, M. Falise, B.C. Kang, and M.M. Jahn. 2005. PCR-based detection of Phyto.5.2, a major QTL controlling resistance to Phytophthora capsici in Capsicum. Theor. Appl. Genet. 110:605–612. Yeam, I., B.C. Kang, J.D. Frantz, and M.M. Jahn. 2005. Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum. Theor. Appl. Genet. 112:178–186. Stewart, C.S., B.C. Kang, K. Liu, M. Mazourek, E.Y. Yoo, S.L. Moore, B.D. Kim, I. Paran, and M.M. Jahn. 2005. The Pun1 gene in pepper encodes a putative acyltransferase. Plant J. 42:675–688. Qian, C.T., M.M. Jahn, J.E. Staub, X.-D. Luo, and J.F. Chen. 2005. Meiotic chromosome behavior in an allotriploid derived from an amphidiploid diploid mating in Cucumis. Plant Breed. 124:272–276. Liu, K., B.C. Kang, H. Jiang, S.L. Moore, C.B. Watkins, T.L. Setter, and M.M. Jahn. 2005. A GH3-like gene isolated from Capsicum chinense L. pepper fruit is regulated by auxin and ethylene. Plant Mol. Biol. 58(4):447–464. Liu, K., H. Jiang, S.L. Moore, C.B. Watkins, and M.M. Jahn. 2005. Identification of fruitspecific lipid transfer protein in Capsicum chinense. Planta 223:672–683. Kang, B.-C., I. Yeam, and M.M. Jahn. 2005. Genetics of resistance to plant viruses. Annu. Rev. Phytopath. 42:581–621. Kang, B.-C., I. Yeam, J.D. Frantz, J.F. Murphy, and M.M. Jahn. 2005. The pvr1 locus in pepper encodes a translation initiation factor eIF4E that interacts with Tobacco etch virus VPg. Plant J. 42:392–405. Henning, M. J, H.M. Munger, and M.M. Jahn. 2005. ‘PMR Delicious 51’: An improved open-pollinated melon with resistance to powdery mildew. HortScience 40(1): 261–262.

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Henning, M.J., H.M. Munger, and M.M. Jahn. 2005. ‘Hannah’s Choice F1’: A new muskmelon hybrid with resistance to powdery mildew, Fusarium race 2 and potyviruses. HortScience 40:492–493. Cadle-Davidson, M.M. and M.M. Jahn. 2005. Resistance conferred against bean common mosaic virus by the incompletely dominant I locus of Phaseolus vulgaris is active at the single cell level. Arch. Virol. 150:2601–2608. Perez, K., I. Yeam, M.M. Jahn, and B.C. Kang. 2006. Megaprimer-mediated domain swapping for construction of chimeric viruses. J. Virol. Methods 135(2):254–262. Luo, X.D., L.F. Dai, S.B. Wang, J.N. Wolukau, M.M. Jahn, and J.F. Chen. 2006. Male gamete development and early tapetal degeneration in cytoplasmic male-sterile pepper investigated by meiotic, anatomical and ultrastructural analyses. Plant Breed. 125:395–399. Lou, Q.F., J.F. Chen, L.Z. Chen, J.N. Wolokau, B.C. Kang, and M.M. Jahn. 2006. Identification of an AFLP marker linked to a locus controlling gynoecy in cucumber and its conversion to a SCAR marker useful in plant breeding. L. Acta Hort. Sinica 31(2):256–261. Liu, K., H. Jiang, S.L. Moore, C.B. Watkins, and M.M. Jahn. 2006. Identification of fruitspecific lipid transfer protein in Capsicum chinense. Planta 223:672–683. Chen, J.F., G. Ren, X.D. Luo, J. Staub, and M.M. Jahn. 2006. Inheritance of aspartate aminotransferase (AAT) in Cucumis species as revealed by interspecific hybridization. Can. J. Bot. 84:1503–1507. Cadle-Davidson, M.M., and M.M. Jahn. 2006. Patterns of accumulation of Bean common mosaic virus in Phaseolus vulgaris genotypes nearly isogenic for the I locus. Ann. Appl Biol. 148:179–185. Cadle-Davidson, M.M., and M.M. Jahn. 2006. Differential gene expression in Phaseolus vulgaris I locus NILs challenged with Bean common mosaic virus. Theor. Appl. Genet. 112:1452–1457. Brown, C.R., T.S. Kim, Z. Ganga, K. Haynes, D. DeJong, M.M. Jahn, I. Paran, and W.P. DeJong. 2006. Segregation of total carotenoid in high level potato germplasm and its relationship to beta-carotene hydroxylase polymorphism. Am. J. Pot. Res. 83:365–372. Ben Chaim, A., Y. Borovsky, M. Falise, M. Mazourek, B.C. Kang, I. Paran, I., and M.M. Jahn. 2006. QTL analysis for capsaicinoid content in Capsicum. Theor. Appl. Genet. 113:1481–1490. Stewart C., M. Mazourek, G. Stellari, M. O’Connell, and M.M. Jahn. 2007. Genetic control of pungency in C. chinense via the Pun1 locus. J. Expt. Bot. 58:979–991. Porch T.G., R. Bernsten, J.C. Rosas, and M.M. Jahn. 2007. Climate change and the potential economic benefits of heat tolerant bean varieties for farmers in Atlantida, Honduras. J. Agr. Univ. Puerto Rico 91(3–4):133–148. Kang, B.C., I. Yeam, H. Li, K.W. Perez, and M.M. Jahn. 2007. Ectopic expression of a recessive resistance gene generates dominant potyvirus resistance in plants. Plant Biotech. J. 5:526–36. Garces-Claver, A., S. Moore Fellman, R. Gil-Ortenga, M.M. Jahn, and M. Arnedo-Andres. 2007. Identification, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp. Theor. Appl. Genet. 115:907–916. Cavatorta, J., G. Moriarty, M. Henning, M. Glos, M. Kreitinger, H.M. Munger, and M.M. Jahn. 2007. Marketmore 97: A monoecious slicing cucumber inbred with multiple disease and insect resistances. HortScience 42:707–709. Yeam, I., J.R. Cavatorta, D.R. Ripoll, B.C. Kang, and M.M. Jahn. 2007. Functional dissection of highly conserved amino acid substitutions in the recessive potyvirus resistance genes encoding eIF4E. Plant Cell 19:1–16.


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Cavatorta, J.R., A.E. Savage, I. Yeam, S.M. Gray, and M.M. Jahn. 2008. Positive Darwinian selection at single amino acid sites conferring plant virus resistance. J. Mol. Evol. 67:551–559. Zhuang, Y., J.-F. Chen, and M.M. Jahn. 2008. Expression and sequence variation of the cucumber Por gene in the synthesized allotetraploid Cucumis hytivus. Mol. Biol. Rep. Online http://www.springerlink.com/content/l44q32385l431471/fulltext.pdf. Wu, F., N.T. Eannetta, Y. Xu, R. Durrett, M. Mazourek, M.M. Jahn, and S.D. Tanksley. 2009. A COSII genetic map of the pepper genome provides a detailed picture of synteny with tomato and new insights into recent chromosome evolution in the Genus Capsicum. Theor. Appl. Genet. 118:1279–1293. Mazourek, M., G. Moriarty, M. Glos, M. Fink, M. Kreitinger, E. Henderson, G. Palmer, A. Chikering, D. Rumore, D. Kean, J. Myers, J. Murphy, C. Krame, and M.M. Jahn. 2009. Peacework: A cucumber mosaic virus-resistant early red bell pepper for organic systems. HortScience 44:1464–1467. Mazourek, M., E.T. Cirulli, S.M. Collier, L.G. Landry, B.-C. Kang, E.A. Quirin, J.M. Bradeen, P. Moffett, and M. Jahn. 2009. The fractionated orthology of Bs2 and Rx/Gpa2 supports shared synteny of disease resistance in the Solanaceae. Genetics 182:1351–1364. Online www.genetics.org/cgi/rapidpdf/genetics.109.101022v1.pdf. Mazourek, M., A. Pujar, Y. Borovsky, I. Paran, L. Mueller, and M. Jahn. 2009. A dynamic interface for capsaicinoid systems biology. Plant Physiol. 150:1806–1821. Stellari, G.M., M. Mazourek, and M. Jahn. 2010. Contrasting modes for loss of pungency between cultivated and wild species of Capsicum. Heredity 104:460–471. Miller, J.K., E.M. Herman, M.M. Jahn, and K.J. Bradford. 2010. Strategic research, education and policy goals for seed science and crop improvement. Plant Sci. 179:645–652. Cavatorta, J., K.W. Perez, S.M. Gray, J. Van Eck, I. Yeam, and M.M. Jahn. 2011. Engineering resistance to plant viral disease using a modified potato gene. Plant Biotechnology Journal. In press.

Books Kyle, M.M. [Jahn, M.M.], ed. 1993. Resistance to viral diseases of vegetables: Genetics and breeding. Timber Press, Portland, OR. Popp, J., M. Matlock, and M.M. Jahn. 2011. Biotechnology and sustainability. Cambridge University Press, Cambridge, U.K.

Book Chapters Munger, H.M., M.M. Kyle [Jahn], and R.W. Robinson. 1992. Cucurbits. p. 42–56. In: Historical review of traditional crop breeding practices. Group of National Experts on Safety in Biotechnology Working Group. Directorate for Science Technology and Industry/Committee for Scientific and Technological Policy. Organization for Economic Cooperation and Development. Paris. Munger, H.M., M.M. Kyle [Jahn], and R.W. Robinson. 1992. Cucurbits. p. 42–56. In: Historical review of traditional crop breeding practices. Group of National Experts on Safety in Biotechnology Working Group. Directorate for Science Technology and Industry/Committee for Scientific and Technological Policy Organization for Economic Cooperation and Development, Paris:. Munger, H.M., M.M. Kyle [Jahn], and R.W. Robinson. 1992. Cucurbits. p. 42–56. In: Historical review of traditional crop breeding practices. Group of National Experts

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on Safety in Biotechnology Working Group. Directorate for Science Technology and Industry/Committee for Scientific and Technological Policy. Organization for Economic Cooperation and Development, Paris. Superak, T.H., B.T. Scully, M.M. Kyle [Jahn], and H.M. Munger. 1993. Interspecific transfer of viral resistance. p. 217–236. In: Resistance to viral diseases of vegetables: Genetics and breeding. M.M. Kyle [Jahn], ed. Timber Press, Portland OR. McCouch, S.M., P. Ronald, and M.M. Kyle [Jahn]. 1993. Biotechnology and crop improvement for sustainable agricultural systems. p. 157–191. In: M.B. Callaway and F. Forella (eds.), Crop improvement for sustainable agricultural systems. Univ. Nebraska Press, Lincoln, NE. Kyle [Jahn], M.M., and R. Provvidenti. 1993. Genetics of broad spectrum viral resistance in bean and pea. p. 153–166. In: M.M. Kyle [Jahn], (ed.), Resistance to viral diseases of vegetables: Genetics and breeding. Timber Press, Portland OR.

GERMPLASM RELEASES AND PATENTS Plant Variety Protection Certificates Bugle, powdery mildew–resistant butternut squash awarded September 2001. Molly Jahn and George Moriarty Cornell’s Bush Delicata, powdery mildew–resistant winter Cucurbita pepo awarded May 2002. Molly Jahn and George Moriarty

Cornell Open-Pollinated and Hybrid Squash Cultivars Cucurbita pepo Cornell’s Bush Delicata (2002) All America Selection Harlequin F1 (2002) Celebration F1 (2004) Success PM (2004) Romulus PM Zucchini (2005) Sweet REBA winter squash (acorn type) (2005) Three inbred PMR pumpkin parent lines used in three hybrid pumpkin varieties One parent of three commercial hybrid summer squash varieties

Cucurbita moschata Bugle (2002) Parents of two leading commercial hybrids Bright Eyes (NY07-140A) (2009) Little John NY-05-130 (2009) Oro Verde NY07-131C-N (2009) Honeynut (NY07-134A)

Cucumis melo Hannah’s Choice F1 (2005) PMR Delicious 51 (2005) Farmer’s Daughter (2009)


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Cucumis sativus Marketmore 97 with Henry Munger (1997) Poinsett 97 with Henry Munger (1997) Poinsett 2000 with Henry Munger (2000) Greenfinger NY 08-143 (2006) Platinum NY06-873 (2008) Salt and Pepper NY08-7107 (2009) Silver Slicer (2009)

Capsicum annuum Peacework (2007) CU Early NY06-368 (2008) King Crimson (2009)

Patents U.S. Patent Application No. 10/538,434, Pub. No. 2006–0294618 A1 ‘‘Recessive plant viral resistance results from mutations in translation initiation factor eIF4E (allowed Feb. 5, 2010). PCT/US2009/061675. ‘‘Mutated eIF4E sequences from potato which are useful in imparting virus resistance.’’ Publication number WO/2010/048398 (published 29 April 2010).

2 History, Evolution, and Domestication of Brassica Crops Shyam Prakash National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012, India Xiao-Ming Wu Oil Crops Research Institute of Chinese Academy of Agricultural Sciences Wuhan 430062, People’s Republic of China S. R. Bhat National Research Centre on Plant Biotechnology Indian Agricultural Research Institute New Delhi 110012, India

ABSTRACT Brassica crops are unique as various plant parts have been modified during domestication for use—for example, roots, leaves, stems, and inflorescences in various vegetables and seeds in edible oils and condiments. The genus Brassica comprises six crop species: B. nigra (2n ¼ 16), B. oleracea (2n ¼ 18), B. rapa (2n ¼ 20), B. carinata (2n ¼ 34), B. juncea (2n ¼ 36), and B. napus (2n ¼ 38). Of these, B. oleracea, B. rapa, and B. juncea (2n ¼ 36) are highly polymorphic, displaying a range of morphotypes. Cytogenetic evidence point to an archetype with a basic chromosome number of x ¼ 6. However, molecular markers strongly suggest the involvement of two evolutionary pathways: B. nigra in one direction and B. oleracea/B. rapa together in the other. Comparison of chromosome collinearity and comparative chromosome painting (CCP) suggested the paleopolyploid nature of the three basic genomes, which are composed of three variants of Plant Breeding Reviews, Volume 35, First Edition. Edited by Jules Janick. 2012 Wiley-Blackwell. Published 2012 by John Wiley & Sons, Inc. 19

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S. PRAKASH, X.-M. WU, AND S.R. BHAT

an ancestral genome originating through an ancient hexaploid event, now referred to as the triplication theory. The Brassicaceae paleoarchetype had the chromosome constitution 2n ¼ 8; following another cycle of genome duplication it produced a tetraploid genome of 2n ¼ 4x ¼ 16 referred to as the ancestral crucifer karyotype (ACK). Another cycle of genome duplication resulted into a hexaploid (2n ¼ 6x ¼ 24). This hexaploid ancestor gave rise to three diploid basic genomes following reduction in chromosome number. An alternative view proposes that a reduction in chromosome number in ACK resulted into a smaller Brassica genome, known as the ancestral Brasssicaceae karyotpe with 6 haploid chromosomes. This subsequently diverged into nigra and rapa/oleracea lineages 7.3 to 4 million years ago. Brassicas are believed to have originated in the countries surrounding the Mediterranean basin and further extension into southwest and central Asia encompassing mainly Mediterranean, Irano-Turanian, and SaharoSindian phytogeographical regions. It is now believed that B. rapa was the first species to be domesticated followed by B. nigra and B. juncea; B. oleracea entered into cultivation later. The history of the domestication of B. carinata and B. napus is relatively recent. Ancient Indian, Chinese, Greek, and Roman literature is extremely rich in detailing information concerning brassica crops. Based on information from these sources and genetical and molecular evidence, possible domestication centers have been constructed. Brassicas first entered into domestication as vegetables and later as edible oil crop. KEYWORDS: Brassica carinata; Brassica juncea; Brassica napus; Brassica nigra; Brassica oleraceae; Brassica rapa ABBREVIATIONS I. INTRODUCTION A. Crop Species B. Origin of the Word Brassica II. ARCHETYPES AND EVOLUTION OF BASIC GENOMES AND DERIVED ALLOPOLYPLOIDS A. Basic Karyotypes B. Diploid Genomes C. Allopolyploid Genomes III. ETHNOBOTANY, ORIGIN, AND DOMESTICATION A. Origin of Diploid Species B. Brassica nigra C. Brassica oleracea 1. Taxonomy and Origins 2. Cabbage 3. Cauliflower and Broccoli 4. Chinese Kale (B. alboglabra) D. Brassica rapa 1. Taxonomy and Origin 2. European Forms 3. Indian Forms 4. Chinese Forms 5. Turnip

2. HISTORY, EVOLUTION, AND DOMESTICATION OF BRASSICA CROPS

21

E. Brassica carinata F. Brassica juncea 1. Taxonomy 2. Origin and Domestication 3. Indian Forms 4. Chinese Forms G. Brassica napus IV. CONCLUDING REMARKS ACKNOWLEDGMENTS LITERATURE CITED

ABBREVIATIONS ACK cp FISH ITS Mya RAPD RFLP rDNA rRNA

Ancestral crucifer karyotype Chloroplast Fluorescence in situ hybridization Internal transcribed spacers of nuclear ribosomal DNA and 5.8S rRNA gene Million years ago Randomly amplified polymorphic DNA Restriction fragment length polymorphism Ribosomal DNA genes Ribosomal RNA

I. INTRODUCTION A. Crop Species The origin and domestication of Brassica crops of the Brassicaceae is a fascinating story. Literatures from several ancient civilizations (Indian, Chinese, Greek, and Roman) have frequent references of these crops. The earliest mention of these can be traced back to a Chinese almanac (ca. 3000 BCE), Assyrian cuneiform documents (ca. 1800 BCE), and Indian Aryan literature (ca. 1500 BCE). Gautama Buddha in India (6th century BCE) told a parable concerning mustard seeds. Ancient Greek and Roman writers make frequent references to Brassica crops. Among the references are Theophrastus (370–285 BCE) in Historia de Plantis (Inquiry into Plants); Marcus Porcius Cato (234–149 BCE) in De Agri Cultura (On Farming); Pedanius Dioscorides (20–70 CE) in Peri Ylis Iatrikis, Latinized

22


as De Materia Medica (On Medical Matters); Lucius Junius Moderatus Columella (4–70 CE) in De Re Rustica (On Agriculture); and Gaius Plinius Secundus—known to us as Pliny the Elder—(23–79 CE) in Historia Naturalis (Natural History). Plant parts, particularly seeds, have been excavated from several ancient sites (Allchin 1969; Hyams 1971; Ghosh et al. 2006; Wu et al. 2009). In the botanical and agricultural books of the Renaissance, descriptions of various crops along with their usage, often accompanied by illustrations, are found in the herbals of Fuchs (1542), Tragus (1552), Mattioli (1571), Dodonaeus (1578), de Lobel (1581), Durante (1585), Dalechamps (1587), Gerard(e) (1597, 1633), Bauhin (1623), and Bauhin and Cherler (1651). Comprehensive taxonomy, geographical distribution, cytogenetical and biochemical evidence, and, in recent years, use of molecular markers have greatly helped in reconstructing the past events about crops’ origin, evolution and domestication. However, the origin of any crop involves two separate aspects: (1) the evolutionary processes that led to the origin of the wild species prior to its domestication, and (2) the history of domestication and superdomestication resulting from human intervention. The brassicas are important components of the cuisine of many cultures. These represent a valuable source of vitamin C, dietary fiber, and anticancer compounds (Fahey et al. 1997). In the majority of crop plants, domestication usually has enhanced a single plant part for use by humans, such as seeds, fruits, or roots. However, the Brassica crops are unique in that practically every plant part has been selected and elaborated to yield different crop plants.They provide edible oils, condiments (seeds), and vegetables (roots, leaves, stems, and inflorescences). The Brassica crops complex comprises six species (Table 2.1). Brassica oleracea, B. rapa, and B. juncea are highly polymorphic, displaying a range of morphotypes, although B. nigra is cultivated exclusively as condiment mustard. The cultivated B. oleracea forms exhibit enormous morphological variability in leaf, stem, and inflorescence and are collectively referred to as cole crops—a term given in 1901 by L. H. Bailey, the American botanist and horticulturist (Bailey 1922). Various forms of B. oleracea are popular vegetables worldwide. Forms of B. rapa are variously referred to as turnip rape (oilseed forms of Europe and Canada), sarson (oil seed forms of the Indian subcontinent), and leafy vegetables (China and other southeast Asian countries). B. carinata, the Ethiopian mustard, has a range of uses: for example, vegetables, edible and industrial oils, a condiment, and medicinal. Its cultivation is restricted primarily to Ethiopia but also extends to Kenya. Brassica juncea (Indian or brown mustard) is a major source of edible oil on the Indian subcontinent, northern China, and eastern European countries, as root and leaf


Table 2.1.

23

Cultivated species of the genus Brassica and their variations.

Botanical name

2n

Common name

Usage

B. nigra B. oleracea var. acephala var. alboglabra var. botrytis var. capitata var. fruticosa var. gemmifera var. gongylodes var. italica var. sabauda B. rapa spp. oleifera var. brown sarson var. toria var. yellow sarson ssp. rapifera ssp. chinensis ssp. pekinensis ssp. nipposinica ssp. parachinensis B. carinata B. juncea B. napus spp. oleifera spp. rapifera

16 18

Black mustard

Condiment (seed)

Kale Chinese kale Cauliflower Cabbage Branchingbush kale Brussels sprouts Kohlrabi Broccoli Savoy cabbage

Vegetable, fodder (leaves) Vegetable (stem, leaves) Vegetable (inflorescence) Vegetable (head) Fodder (leaves) Vegetable (head) Vegetable, fodder (stem) Vegetable (inflorescence) Vegetable (terminal buds)

Turnip rape Brown sarson Toria Yellow sarson Turnip Pak-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

Oilseed Fodder

20

34 36 38

Source: Prakash et al. 2009.

vegetables in China, and as hot mustard condiment used in mayonnaise, salad dressing, and sauces in Europe, Canada, and America (Skrypetz 2003). Brassica napus is a major edible oilseed crop widely grown in Europe, Canada, China, and Australia. One of its root variants, rutabaga or swede (ssp. rapifera), is grown for fodder, particularly in Scandinavian countries and England. Following the determination of chromosome numbers in the early 1920s, the Japanese scientist Morinaga carried out pioneering cytogenetical investigations involving hybridizations and the study of chromosome pairing behavior. Morinaga (1934) interpreted 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

24


Fig. 2.1. Cytogenetic relationships of crop brassicas (U, 1935). Solid and broken lines in the allopolyploids represent female and male parents, respectively. (Source: U 1935; Prakash et al. 2009).

(n ¼ 19) that have evolved through convergent allopolyploid evolution between any two of the diploid species. Another Japanese scientist U (1935) represented these cytogenetical relationships diagrammatically, now famously referred to as the triangle of U (Fig. 2.1). Various lines of evidence such as cytogenetics, molecular cytogenetics and molecular markers gathered during last several decades have substantiated these relationships (Prakash et al. 2009). A journey into the past through evolutionary events of origin and domestication of these crops makes a fascinating story, which we have attempted to synthesize in this review. Useful information dealing with some of these aspectes are given in recently published books, such as Biology of Brassica Coenospecies (Go´mez-Campo 1999) and Vegetable Brassicas and Related Crucifers (Dixon 2007). B. Origin of the Word Brassica Several views have been proposed concerning the origin of the word Brassica, and its etymology has been discussed since 1727 (see Prakash and Hinata 1980; Dixon 2007; Maggioni et al. 2010). Henslow (1908) quoted Hermann Boerhaave (1727) that the term Brassica originated from the Greek a ´ po´@úbsáxein [apooibsazein], Lat. vorare (to devour). Hegi (1919) believed that it originated from the Celtic word for cabbage, Bresic or Bresych, and a contraction of praesecare (to cut off early), as the leaves were removed from the stem for cattle fodder according to the Roman scholar Marcus Terentius Varro (116–27 BCE) in his De Lingua


25

Latina. Another possible derivation is from the Greek word ßoá ´ o´sein (crackle), as the leaves produce a crackling noise on being broken off (Gates 1953). The Greek word bráskh (braske) has also been favored for its derivation, which was a local name used by Greeks in southern Italy (Maggioni et al. 2010). A word in the Phoenician dialect of the Punic language Burutzim is also considered a possible progenitor of the word Brassica (Herv e 2003). The written word Brassica first appeared, in the 3rd-century BCE Latin literature by the playwright Plautus. II. ARCHETYPES AND EVOLUTION OF BASIC GENOMES AND DERIVED ALLOPOLYPLOIDS A. Basic Karyotypes There has been continuing debate and conflicting views on the origin and evolution of basic karyotypes in Brassica. In the initial stages, these views were based on classical cytological studies. During last 25 years, use of DNA markers has unraveled facts for interprating the phylogenetic relationships and origin of basic genomes (Prakash and Hinata 1980; Go´mez-Campo 1999). In the early 1940s, it was widely believed that the three basic genomes originated from one archetype, sharing common ancestry, making them secondary balanced polyploids. Based on several cytological surveys, a scenerio emerged suggesting that this archetype was a smaller genome. However, variable basic chromosome numbers of the archetype ranging from 3 to 9 were proposed. For example, Catcheside (1934, 1937) suggested x ¼ 6 as the basic number, his conclusion based on secondary chromosome associations in B. napus and B. oleracea. He also proposed the primitive haploid number in Brassicaceae as x ¼ 7 and assummed that x ¼ 6 arose from it by fusion of two chromosomes. Alam (1936) and Haga (1938) agreed with Catcheside by observing secondary chromosome pairing in the three basic diploid species. Sikka (1940) carried out detailed cytogenetical analysis in several Brassica and related species and proposed a basic number of x ¼ 5 from observing 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. He suggested that the evolution in Brassica occurred toward tetraploidy, citing in evidence the chromosome series 2n ¼ 30, 60, 90, and 120 in the genus Crambe, all multiples of x ¼ 5. However, pachytene chromosome analysis of the basic genomes (R€ obbelen 1960) provided compelling evidence in support of x ¼ 6 as the constitution of basic archetype and a monophyletic origin of the

26


diploid species. Meiotic chromosome pairing in the haploids of B. oleracea (2n ¼ 8, 2II þ 4I, Thompson 1956), B. nigra (2n ¼ 8, 2II þ 4I) (Prakash 1974a), and a related species B. tournefortii (2n ¼ 10, 1III þ 2 II þ 3I) (Prakash 1974b) also lent support to that proposal. However, investigations in the last 20 years using molecular markers firmly disprove the theory of monophyletic origin. The first indication came from nuclear DNA restriction fragment length polymorphisms (RFLPs) by Song et al. (1988a), which was further corroborated by investigations from nuclear, chloroplast, and mitochondrial DNA RFLPs. These studies strongly suggested that two evolutionary pathways are involved in the origin of diploid species: B. nigra evolved in one direction and B. rapa/ B. oleracea together in the other. Also these investigations clearly revealed a vertical division of the subtribe Brassicinae into two lineages, referred to as Nigra and Oleracea/Rapa (Warwick and Black 1991; Pradhan et al. 1992). Earlier, cytogenetical investigations predicted this divergence between B. nigra and B. oleracea/B. rapa lineages based on chromosome pairing in hybrids (Mizushima 1950; Prakash and Hinata 1980). Arabidopsis, a member of the Brassicacea, is regarded as a model species in plant molecular and genomic researches. Its genome has completely been sequenced (Arabidopsis Genome Initiative 2000), and this information is widely used for comparative mapping using Arabidopsis-derived probes to understand the architecture and organization of Brassica genomes and deciphering their evolution. Two strategies have been followed to unravel the constitution of ancestral karyotype and reconstruct the evolutionary events leading to the origin of current karyotypes. These are: (1) comparative genetic mapping (i.e., the comparison of chromosome collinearity) and (2) comparative chromosome painting. Availability of virtually repeat-free A. thaliana bacterial artificial chromosome clones has considerably helped in comparative chromosome painting in several Brassicaceae species. Many duplicated regions have been discovered that could not have been identified through classical genetical analysis and provided additional insight into karyotype evolution. Arabidopsis-genome sequence information indicated a large number of duplications, both intra- and interchromosomal, suggesting that this genome is a relic of an ancient whole genome duplication. Comparative RFLP mapping of the three basic genomes and Arabidopsis also confirmed the paleopolyploid nature of the three basic species (B. nigra, B. oleracea and B. rapa), suggested that the existing diploid genomes are paleopolyploids, and, for the first time, indicated that these are composed of three variants of an ancestral genome evolved probably by an ancient hexaploid event (Lagercrantz and Lydiate 1996; Lagercrantz 1998; Babula et al. 2003). This hypothesis


27

is now referred to as the triplication theory. It envisages that a common ancestral genome of Arabidopsis and Brassica underwent genome duplication events twice before their split while the third duplication event was a genome triplication after this divergence and was confined to the Brassiceae lineage only. There is evidence that this tribe represents a monophyletic lineage and that all the taxa are derivatives of this hexaploid genome (Lysak et al. 2005). This triplication model finds support from the earlier studies involving classical cytogenetics and ribosomal DNA (rDNA) markers, such as those by Chen and Heneen (1991) and Cheng and Heneen (1995), who observed three pairs of satellited chromosomes with active nucleolus organizer regions in B. nigra and S. arvensis and occurrence of 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) and related species S. alba (Schrader et al. 2000) and S. arvensis (Ali et al. 2005). It is inferred that the number of three chromosome pairs carrying the 25S rDNA gene is basic for the Brassicaceae (Ali et al. 2005). The Brassicaceae paleoarchetype had in all probability a genome comprising 8 somatic chromosomes (n ¼ 4) and following another cycle of genome duplication produced a tetraploid genome having 2n ¼ 4x ¼ 16, an event that occurred 24 to 40 million years ago (Mya) (Henry et al. 2006). This ancestral crucifer karyotype (ACK) has been put forward as the common ancestor of family Brassicaceae. It had a genome possessing 8 haploid chromosomes (AK1–AK8) and some 24 conserved genome blocks (Lysak et al. 2006; Schranz et al. 2006). This genome was similar to the present-day A. lyrata and Capsella rubella (2n ¼ 16, 230 Mbp) from which derived the genomes of existing Brassica species and A. thaliana (Schranz et al. 2006). The similar karyotypes of Arabidopsis and Capsella lineages further substantiated that the ancestral Crucifer karyotype had resemblance with Arabidopsis–Capsella karyotypes and possessed 16 somatic chromosomes (Lysak et al. 2006). The evidence in support of this n ¼ 8 ACK karyotype is derived as follows: (1) x ¼ 8 is the most common base number in the Brassicaceae (Warwick and Al-Shehbaz 2006), and (2) 8 chromosomes of A. lyrata and Capsella rubella possess nearly identical linkage groups (Bolvin et al. 2004; Kuittinen et al. 2004; Koch and Kiefer 2005; Lysak et al. 2006, 2007). The ACK gave rise to the Arabidopsis genome (2n ¼ 10) having 157 Mbp of DNA (Johnston et al. 2005; Schranz et al. 2006) around 14.5 to 20.4 Mya (Yang et al. 1999) in one pathway due to chromosomal rearrangements, mainly fusions and gene loss, while members of the tribe Brassiceae originated in the other pathway.

28


Several investigations based on molecular marker maps (Lagercrantz 1998; O’Neill and Bancroft 2000; Rana et al. 2004; Park et al. 2005; Parkin et al. 2005) and fluorescence in situ hybridization (FISH) maps (Lysak et al. 2005; Ziolkowski et al. 2006) suggested that diploid Brassica genomes evolved from this tetraploid following another cycle of genome duplication. The resulting genome was a hexaploid with chromosome constitution of 2n ¼ 6x ¼ 24. Ziolkowski et al. (2006) also proposed that the final event of genome triplication was allopolyploidization (Fig. 2.2) involving hybridization between an Arabidopsis-like diploid and a tetraploid genome. This allopolyploidization was responsible for, in part, increase in DNA content from 230 Mbp to 529–696 Mbp of diploid Brassica species (Johnston et al. 2005). However, the mechanism of reduction in chromsome numbers from 24 to 16–20 is not properly explained. Mand akov a and Lysak (2008) extended chromosome painting investigations to eight species with x ¼ 7 (2n ¼ 14, 28) comprising six X

X

Brassicaceae common ancestor (diploid)

X′

Polyploidization Brassicaceae common XX′ ancestor (allotetraploid?) Chromosomal diploidization

Y

Scenario 2 translocation

Scenario 1 translocation

Y′

Polyploidization

Arabidopsis ancestor (diploid)

ancestor I Y′Y′ Brassica (tetraploid)

Y′′′

Chromosome number reduction

Y′′ Brassica ancestor II (diploid)

A Present-day A thaliana (diploid)

Brassica ancestor I (diploid)

Polyploidization

Y′Y′Y′′ Brassica ancestor III (hexaploid) Chromosomal diploidization

B Present-day B. oleracea (diploid)

Fig. 2.2. Model of diploid Brassica genome evolution via hexaploidization. (Source: Ziolkowski et al. 2006).


29

Brassicaceae tribes. They proposed that a karyotype with n ¼ 7 chromosomes, referred to as Proto-Calepineae karyotype (PCK, n ¼ 7), was derived from the ACK due to the loss of one chromosome. They further suggested that this prototype was the progenitor of the tribe Brassiceae prior to undergoing whole-genome triplication and was followed by diploidization. Thus, the original triplication pattern was lost. However, some genome blocks were retained as single or duplicate copies, others in three copies, while a few were increased further due to homologous recombination. These authors did not rule out the possibility of further reduction in chromosome number to n ¼ 6 as originally proposed by cytogenetical research. Although the mapping sequence data suggest triplication of the ancestral genome involving three events of polyploidy, it is not readily accepted as it fails to explain the origin of extant chromosome numbers. This ancestral hexploid genome (2n ¼ 24) would have required extensive chromosome number reduction to yield 2n ¼ 16, 18, and 20 karyotypes and would require genome downsizing, which is not supported by genome size data. A possible model is proposed by Qiu et al. (2009) to resolve this ambiguity. Since the monogenomic diploids have more or less identical or similar chromosome numbers as the putative tetraploid ancestral genome (ACK, 2n ¼ 16), these diploids might have evolved from it after insertion of transposable elements, segmental duplications, and chromosomal rearrangements rather than undergoing another round of genome duplication followed by chromosome number reduction. Thus, x ¼ 8 can be considered the most likely ancestral chromosome number of the family. Qui et al. (2009) emphasized that transposable elements have played a major role in genome evolution of Brassica species, a view also suggested earlier by Lim et al. (2007) and Alix et al. (2008). For example, transposable elements account for 20% (139 Mbp) of the total DNA content (696 Mbp) of B. oleracea. Brassica genomes also have lower gene density than Arabidopsis, which is associated with larger introns and spacers and extensive gene arrangements. Hence, it appears to be a reasonable explanation that reconciles triplication of sequences and chromosome number changes. However, Mun et al. (2009) although agreeing with the triplication theory, believed that large-scale deletion of duplicated genes in the triplicated genome, along with less accumulation of transposons, resulted in smaller-size Brassica genomes. Considering all these different hypotheses on evolution of diploid karyotypes, an alternative view envisages a reduction in chromosome number in ACK (n ¼ 8) or PCK (n ¼ 7) resulted in a smaller genome. It had six haploid chromosomes (ABCDEFG) and can be regarded as the

30


progenitor of tribe Brassiceae. We refer to it as ancestral Brassiceae karyotype. Several mechanisms—reciprocal translocations, pericentric inversions leading to generation of acrocentric chromosomes, and elimination of unstable minichromosomes at meiosis—are reported to operate for reducing the chromosome number in Brassicaceae (Lysak et al. 2006; Mand akov a and Lysak. 2008). This prototype (ancestral Brassiceae karyotype) subsequently diverged into Nigra and Rapa/ Oleracea lineages 7.3 to 4 Mya (Wroblewski et al. 2000) or about 7.9 Mya (Lysak et al. 2005) (Fig. 2.3). Thus, x ¼ 6 is most likely the basic chromosome number of the tribe Brassiceae and the genus Brassica.

Fig. 2.3. Proposed model of the origin of basic and alloploid Brassica species (solid and broken lines in allopolyploids represent female and male parents, respectively).


31

Cytogenetical evidence also unequivocally suggests that the Brassicae ancestral karyotype had a constitution of x ¼ 6. R€ obbelen (1960), for the first time, following pachytene analysis, recognized six basic types of chromosomes in each genome based on absolute length, symmetry of arms, and shape of heterochromatic centromeric region, and proposed that selective chromosome duplication in the archetype resulted in the evolution of basic genomes. According to his scheme in B. rapa, two chromosomes, A and D, are duplicated, and one chromosome, F, is triplicated, with the constitution AABCDDEFFF. B. nigra has the constitution ABCDDEFF and is tetrasomic for chromosomes D and F, and B. oleracea is a triple tetrasomic for three chromosome types B, C, and E with the constitution ABBCCDEEF. Truco et al. (1996) also proposed a model of genome evolution based on the conservation of marker arrangement. The model envisages that these basic genomes were derived from six ancestral chromosomes (W1–W6) (Fig. 2.4), which underwent several duplications and rearran-

A10

A5

C5

A4

B6

C1

B3

A1

B5 W3

C4 A6

C7

W5

W4

Cx W6

C6

C3

W1 W2

A6

A7

Bx

C8 B1

A8 C9

B8 B7

B4 B2

A9 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: Truco et al. 1996).

32


gements. 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 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. The two chromosomes B7 and C9 do not share homology with any other group. Panjabi et al. (2008) used intron polymorphism markers selected from Arabidopsis single-copy genes to construct a detailed molecular map of B. juncea. A comparative study of B. juncea, A. thaliana, and three diploid Brassica genome maps revealed a high degree of colinearity. They also proposed the evolutionary events that might have contributed to karyotype variations in the three basic genomes. Significant similarity between the five linkage groups of A and B genomes was observed. Chromosome rearrangements, mostly translocations, were thought to be responsible for karyotype diversification. Ancestral blocks that remained unaltered since their inception were identified based on map information of the B genome. It was observed that three linkage groups of the B genome (B4, B5, and B6) are similar to the A genome. The remaining five chromosomes—B1, B2, B3, B7, and B8—appear to have originated through (1) rearrangements without any loss of chromosome (B1, B2, B3, and B8 consisting of four linkage groups of A/C genomes: A1–C1, A2–C2, A3–C3, and A10, respectively), and (2) rearrangements with variations in chromosome number (chromosome B7 by fusion of two linkage groups A7–C7/A8–C8). Both A and C genomes display a high degree of collinearity and show only minor changes. However, it is difficult to predict whether these changes occurred in one of the lineages after their divergence or independently in both the lineages. No structural changes were observed in the A1–C1, A2–C2, A3–C3, and A7–C7. The major changes in the evolution of A and C genomes involve rearrangements: mainly the translocations in the C4, C5, and C6, which are specific to the C genome and probably occurred after the diversification of A and C genomes; and C8 and C9 have rearranged blocks. C9 was derived by fusion of half of A9 and entire A10 while C8 is constituted of the other half of A9 and the entire A8. B. Diploid Genomes In spite of their origin from two different lineages, these three genomes possess similar genetic information with many duplications (Slocum et al. 1990; Chyi et al. 1992; Jackson et al. 2000; Parkin et al. 2003).


33

However, the gene organization and distribution on chromosomes is different (Truco et al. 1996). Following their evolution, these genomes underwent genetic diploidization and regulation of pairing forming a strictly bivalent regime. Chromosome differentiation and repatterning occurred mainly through duplications and translocations (Hosaka et al. 1990; McGrath et al. 1990; Truco and Quiros 1994; Quiros 1999) as well as deletions (Hu and Quiros 1991). Because of their secondary balanced nature, these changes were tolerated and adjusted (Kianian and Quiros 1992a). The B genome is separared from A/C genomes by a large number of rearrangements and also possesses cytoplasm distinct from A/C types of B. rapa/B. oleracea but still retains some homology (Palmer et al. 1983; Yanagino et al. 1987; Warwick and Black 1991; Pradhan et al. 1992). In comparison, A and C genomes are less differentiated (Lagercrantz 1998) and also cytogenetically very close, as expressed in high degree of chromosome pairing in hybrids between them (Mizushima 1950; Olsson 1960b; Wen et al. 2008). Supporting evidence is provided by: (1) FISH mapping of two families of repetitive DNA, which are confined to pericentromeric regions of most chromosomes of A and C genomes but absent in the B genome (Harrison and Heslop-Harrison 1995); (2) structural analysis of rDNA intergenic spacers (Bhatia et al. 1996); (3) collinearity between these two genomes as revealed by comparative analysis (Scheffler et al. 1997; Panjabi et al. 2008); and (4) extent of homologous pairing detected by genomic in situ hybridization (Snowdon et al. 1997a; Ge and Li 2007), FISH, and molecular markers (Nicolas et al. 2007). RFLP analysis also indicated that C genome is more conserved than A or B genomes as detected by microsatellites (Bornet and Blanchard 2004). These basic species are characterized by very small genomes (Table 2.2). Johnston et al. (2005), using internal transcribed spacers of nuclear ribosomal DNA and 5.8S rRNA gene (ITS), attempted to present an evolutionary overview of family genome size and reported an ancestral Table 2.2.

1c nuclear DNA content and genome size in Brassica species.

Species B. carinata B. juncea B. napus B. nigra B. oleracea B. rapa Source: Johnston et al. 2005.

1c nuclear DNA content (pg SE)

Genome size (1x) (Mbp)

1.308 0.018 1.092 0.001 1.154 0.006 0.647 0.009 0.710 0.002 0.539 0.018

642 534 566 632 696 529

34


genome size of approximately 0.20 pg. However, a reconstruction by Lysak et al. (2009) from five data sets found a mean value of 0.50 pg. Thus, there is only a slight increase in genome size. It is also estimated that B. rapa and B. oleracea separated from each other 7.3 4 Mya (Wroblewski et al. 2000; Inaba and Nishio 2002) and incorporation of transposons played a major role in their divergence (Alix et al. 2008). C. Allopolyploid Genomes It is now well established that the three high-chromosome species— B. carinata, B. juncea, and B. napus—originated in nature following alloploid evolution involving different combinations of the diploid species, as proposed for the first time by Morinaga (1934) and subsequently confirmed by U (1935). Several sets of evidence, such as taxonomy, artificial syntheses, molecular analysis, and chromosome mapping and painting, have unequivocally established allotetraploid origin of these three species (see Prakash et al. 2009 for references). It is also conclusively established from the information from Fraction-1 protein (Uchimiya and Wildman 1978) and chloroplast and mitochondrial DNA restriction patterns (Erickson et al. 1983; Ichikawa and Hirai 1983; Palmer et al. 1983; Yanagino et al. 1987; Warwick and Black 1991; Pradhan et al. 1992; Cunha et al. 2004) that B. nigra and B. rapa were the cytoplasm donors of B. carinata and B. juncea, respectively. The maternal parent of B. napus is not yet clearly established. Based on RFLP patterns of the plastid genomes, Erickson et al. (1983) suggested B. oleracea as the C genome donor of B. napus. Subsequent study by Song and Osborn (1992) indicated B. montana, a related wild species of B. oleracea, as the most likely C genome donor of B. napus. However, plastid simple sequence repeats analysis did not support B. montana as the maternal parent. On the contrary, it identified B. rapa as the most likely plastid donor (Flannery et al. 2006). Recently, Allender and King (2010) made a detalied investigation involving a lage number of accessions of B. napus, B. montana, B. rapa, B. oleracea, B. carinata, B. nigra, and B. juncea and employed a combination of chloroplast and nuclear genetic markers to resolve this issue. Their study also ruled out the possibility of any B. oleracea or B. montana having participated in the origin of B. napus and supported polyphyletic origin of B. napus. This is in agreement with the earlier study by Palmer et al. (1983), which suggested that chloroplast genomes in B. carinata and B. juncea are highly conserved since their origin while the chloroplast genome of B. napus has gone through evolutionary alteration. Also, both mitochondrial and chloroplast genomes in all the three species have


35

been coinherited over generations (Palmer 1988). Considerable cytoplasmic influence on the evolution of nuclear genomes of alloploid species is also reported. The alloploid genomes are highly plastic, and allopolyploidy is accompanied by an increase in DNA amount, but eventually some DNA is eliminated due to diploidization (Lukens et al. 2004, 2006). Narayan (1996) reported the amount of DNA in the allopolyploid species of B. napus, B. juncea, and B. carinata to be less than the sum of their putative parent species by 0.095, 0.094, and 0.049 pg less, respectively, an overall reduction of >6% (Table 2.2). Compelling evidence suggest rapid and extensive structural rearrangements of chromosomes in these alloploid species since their evolution (Slocum 1989; Slocum et al. 1990; Kianian and Quiros 1992a,b; Song et al. 1993, 1995; Poulsen et al. 1993; Harrison and Heslop-Harrison 1995). These chromosomal rearrangements arising as a result of homologous recombination also induce morphological variants (Sharpe et al. 1995; Parkin and Lydiate 1997; Osborn et al. 2003; Pires et al. 2004; Udall et al. 2005; Nicolas et al. 2007). In fact, such morphological and physiological variants of nonhomologous origin have been obtained in synthetic B. juncea population (Prakash 1973b). These events are regarded as progressive steps in evolution. However, Song et al. (1995) disputed this view. Their investigations employing RFLP markers in natural and synthetic B. juncea indicated that the nuclear genomes in B. juncea have not undergone drastic changes and have remained largely unchanged since its origin, a view also supported by Axelsson et al. (2000) and Panjabi et al. (2008). In spite of considerable homolology between chromosomes of the partaking genomes, these present-day allopolyploid species exhibit true diploid-like meiosis devoid of any higher-order chromosome associations. Several mechanisms have been proposed for such bivalent-forming regimes. When the two genetically distinct genomes come together, they should adjust in a common nucleus by regulating gene expression and chromosome pairing. It is suggested by several researchers that this diploid-like meiosis is genetically regulated in Brassica and its related genera (Prakash 1974c; Attia and R€ obbelen 1986; Eber et al. 1994; Sharpe et al. 1995; Jenczewski et al. 2003). The other factors are point mutations, gene conversion, and DNA methylation (Szadkowski et al. 2010). The role of ribosomal RNA (rRNA) genes has also been highlighted as a contributing factor. It is observed in a number of allopolyploids that rRNA genes from only one parent are transcribed while the transcription of such genes of the other parent are suppressed, a phenomenon referred to as nucleolar dominance. A hierarchy of nucleolar dominance—namely, B. nigra > B. rapa > B. oleracea—has been

36


demonstrated in allotetraploids (Chen and Pickard 1997; Pickard 2000; Ge and Li 2007). These results suggest that nucleolar dominance may contribute decisively in preferential stabilization of chromosomes from the parent exerting nucleolar dominance. All these factors lead to stabilization of newly evolved allopolyploids and their successful colonization (Prakash et al. 2009). III. ETHNOBOTANY, ORIGIN, AND DOMESTICATION A. Origin of Diploid Species A hypothetical model for the origin of Brassica has been proposed by Song et al. (1990) and is depicted in Fig. 2.5. According to this scheme, Brassica or related species with n ¼ 7 and n ¼ 8 evolved from a common ancestor through two pathways: one group comprising Hirschfeldia incana (n ¼ 7) or a closely realated species as the primary ancestor of B. nigra, B. fruticulosa, and others in Nigra lineage. Diplotaxis erucoides (n ¼ 7) or a close relative was the primary ancestor for the other group, which gave rise to B. oleracea, B. rapa, and other species in Rapa/Oleracea lineage. In support, D. erucoides has been shown to be very close to B. rapa/ B. oleracea by Harbinder and Laksmikumaran (1990) based on analysis of repeat sequence (satellite DNA) and nucleotide sequences of the S-locus related gene, SLR1 (Inaba and Nishio 2002). This closeness is also reflected in their nuclear DNA (Song et al. 1990) and chloroplast (cp) DNA (Warwick and Black 1991; Pradhan et al. 1992) and high chromosome pairing in their hybrids (Vyas et al. 1995). This ancestral D. erucoides evolved into a common ancestor having n ¼ 9, which in turn gave rise to B. oleracea and B. rapa. Since B. oleracea is considered to be an older species (Prakash and Hinata 1980; Song et al. 1990), it evolved first. Primitive forms of B. rapa were subsequently derived from one of the wild or very primitive cultivated B. oleracea form. Brassicas are believed to have originated in countries surrounding the Mediterranean basin and further extended into southwest and central Asia encompassing mainly the Mediterranean, Irano-Turanian, and Saharo-Sindian phytogeographical regions. This whole region can be regarded as the cradle of development for this group (Fig. 2.6). This area represents the world’s most traveled corridor of prehistoric times with the migrating people carrying a variety of seeds with them, thus making their diffusion possible. Go´mez-Campo and Prakash (1999) believed that, chronologically, B. rapa (turnip rape) was the first diploid species to be domesticated several millenia ago as a multipurpose crop (e.g., roots in turnip, leaves in Chinese vegetable forms, seed in turnip


37

Common ancestor, n=7

common ancestor -1, n=7

common ancestor -2, n=7

Diplotaxis erucoides or related n=7 species with A/C type cytoplasm

Hirschfeldia incana or related n=7 species with B type cytoplasm

B. fruticulosa, n=8

bridge species, n=8

B. nigra

common ancestor, n =9

Sinapis arvensis, n=9 primitive B. oleracea,n=9

wild B. oleracea and

primitive B. rapa, n=10

turnip, turnip rape

pak-choi

B. alboglabra, both n=9

primitive cultivated B. oleracea-kales

Cabbage group

sarson group

Chinese cabbage and other leafy forms

Broccoli and cauliflower

Fig. 2.5. Hypothetical model for genetic evolution of Brassica diploid species. (Source: Adapted from Song et al. 1990).

rape and sarson) that was widely adopted by all the civilizations in the regions of domestication. Brassica nigra is also an ancient species. B. oleracea entered into cultivation later as its natural area (Atlantic coast) was too far from major centers of its domestication. Although we do not have any precise information about B. carinata, it seems its domestication history is not too old, similar to B. napus, which entered into cultivation only 400 years ago. These three species (B. oleracea, B. rapa, and B. juncea) are highly polymorphic with a range of morphotypes. B. rapa and B. oleracea with close genomic homologies exhibit

38


B.napus B.oleracea B.rapa B.juncea

B.carinata

Fig. 2.6. Geographic distribution and probable places of origin of different Brassica species. (Source: Dixon 2007).

parallel series of differentiation in Asia and Europe, respectively, while B. rapa and A genome containing B. juncea exhibit such parallelism in Asia (Table 2.3). These represent the classical examples of parallel structural evolution in plants. Parallelism is apparent also in nuclear DNA variations (Song et al. 1988b). One of the striking features of these species is that while in India, B. rapa and B. juncea are grown for reproductive products (i.e., seeds), in East Asian countries they are cultivated for vegetative growth. Another interesting point is that European B. rapa variation resembles broccoli group of B. oleracea, and the variation in East Asian group of B. rapa is similar to cabbage group of B. oleracea. Nishi (1980) pointed out that this might not be due to ecological adaptations of the plants but rather to cooking habits of the regions concerned and their wider geographical distribution. B. Brassica nigra This species was collected from the wild and subsequently cultivated for its medicinal uses since antiquity. Hippocrates (480 BCE) and Pliny

39

Latin English/local Distribution








Bush

Elongated stem

Heading

Kohlrabi type

Loose head

Savoy

Stalking type

Turnip

Source: Prakash and Hinata 1980.

var. acephala Kale Worldwide

Latin English Distribution

Basic

var. botrytis, B. alboglabra Broccoli, kailaan Worldwide

var. sabauda Savoy cabbage Worldwide

var. acephala Portuguese kale Atlantic islands

var. gongylodes Kohlrabi Worldwide

var. capitata Cabbage Worldwide

var. acephala Marrow stem kale Europe

var. acephala Thousand headed borecole Europe

B. oleracea

B. rapa ssp. rapifera Turnip Worldwide

Central and S. China, Japan

ssp. chinensis

ssp. pekinensis Hakusai Southeast Asia

ssp. pekinensis Chinese cabbage Southeast Asia

ssp. pekinensis Chinese cabbage Southeast Asia

Japan

ssp. japonica

B. rapa Spinach mustard Japan, China

B. rapa

Nomenclature of parallel morphological variations in Brassica oleracea, B. rapa, and B. juncea.

Type

Table 2.3.

Turnip mustard North China

Kigarashi Japan, China

Chirimen takana Japan, China

Katsuo-na Japan, China

Pickling mustard Central China

Heading mustard South China

Tashin-chetsai Taiwan

Shelifong China

B. juncea Leaf mustard Japan, China

B. juncea

40


referred to its medicinal value. Greeks believed that it had been made known to mankind by Aesculapius, the god of medicine, and Ceres, the goddess of seeds (Hedrick 1919). The New Testament also mentions it (sıńapi, sinapi). Its natural distribution is in Mediterranean area extending into Middle East and central Asia. It probably originated in central and south Europe (Bailey 1922; Zeven and Zhhukovsky 1975) and was available for domestication to Mediterranean civilizations (Mesopotamia, Egyptian, Greek, Roman), where it was an important condiment. Several herbalists mentioned B. nigra under the name Sinapis. We find S. primum in Mattioli (1571) and Dodonaeus (1578), S. sativum Erucae aus Rapifolis in de Lobel (1581), and Sinapi sativum in Gerarde (1597, 1633). ‘‘It has great virtues because it is hot, dilates the passages and destroys the humours’’ wrote de Glanville (1518) in a French book. The word mustard is believed to be a derivative of Latin mustus ardens because of the pungent seeds. Its Middle English/Old French word is mustarde and was originally an occupational name for a person dealing in hot spices. People in Rhodes consume its infloresecence boiled and seasoned with salt, lemon juice, and olive oil. In Sicily, it is cultivated for medicinal uses while in Turkey, it is used as a spice for flavoring sausage. It is called sanafitch in Ethiopia and is grown for seed oil, spice, as a leaf vegetable, and for medicinal use (Tsunoda 1980). RFLP investigations suggest that B. nigra has diverged much less from the wild ancestor since its origin (Song et al. 1988a). It still shows wild or semidomesticated characters (e.g., tall plant stature, enormous vegetative growth, small-size pods with reduced numbers of small seeds). A related wild species S. arvensis has traditionally been considered very close to B. nigra, which is reflected in high levels of chromosome pairing in their hybrids (BSa, 2n ¼ 17, up to 8 II) (Mizushima 1950). Several investigations substantiate the closeness of B. nigra and S. arvensis, including research on seed proteins (Vaughan and Denford 1968), fraction 1 protein (Uchimiya and Wildman 1978), nuclear DNA RFLPs (Song et al. 1988a; Poulsen et al. 1994), 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 (Tsukamoto et al. 1993; Simonsen and Heneen 1995), cytology (Cheng and Hennen 1995), karyotypes (Yuan et al. 1995), randomly amplified polymorphic DNA (RAPD) patterns (Wu et al. 1996), nuclear sequence of S-locus related gene SLR1 (Inaba and Nishio 2002), and ITS/trnL sequence data (Warwick and Sauder 2005). A previous concept was that B. nigra evolved from


41

S. arvensis, but now it is well setteled that B. nigra preceded S. arvensis (Song et al. 1990). Both these species overlap in geographical distribution and still have not developed a strong reproductive isolation barrier. C. Brassica oleracea 1. Taxonomy and Origins. B. oleracea is a highly polymorphic species with extensive variation in leaf, stem, and inflorescence morphology. It represents a classical example of structural evolution in plants (Plate 2.1). The variants provide a range of vegetable forms and are considered important sources of vitamins and fibers (Lorenz and Maynard 1988; Rubatzky and Yamaguchi 1997) and anticarcinogenic compounds (Rosa et al. 1997). Bailey studied many cultivated forms of B. oleracea and applied for the first time the general term ‘‘cole crops’’ in 1901 in his book Principles of Vegetable-Gardening (Bailey 1922). This B. oleracea complex includes at least six well-defined groups designated as varieties. Snogerup (1980) and Dixon (2007) described the major cultivated groups: 1. Kales (var. acephala), which develop a strong main stem bearing edible foliage and include marrow stem kale, collards, and green and dwarf Siberian kales. Landraces of these kales are widely scattered. 2. Cabbages (var. capitata) characterized by formation of heads consisting of tightly packed leaves and include headed cabbages, Brussels sprouts, and savoy cabbage. 3. Kohlrabi (var. gongylodes) grown for its thickened stout edible stem particularly in China and Vietnam. 4. Inflorescence kales (var. botrytis, var. italica), which are cultivated for thickened edible inflorescences and include cauliflower, broccoli, and sprouting broccoli. Almost all cauliflowers are white- or cream-curded but occasionally forms with colored curds also occur. 5. Branching bush kales (var. fruticosa) used to be grown for edible foliage. These are very popular in European supermarkets as ‘‘fresh leaves.’’ 6. Chinese kale (B. alboglabra) widely cultivated in southeast Asian countries where the flower bud, flower stalk, and young leaves are consumed. It has close morphological resemblance with related wild species B. cretica ssp. nivea. Vast information is available about the antiquity of these cole crops. Maggioni et al. (2010) in a recent and informative article documented in

42


detail the antiquity and domestication of these crops considering the literary, linguistic, and historical sources and presented the terminolgy used by the Greeks and Romans. Krámbh (krambe) was the main word for B. oleracea in ancient Greek (before ca. 330 BCE). Theophrastus described several different forms of cole crops and refers to branching bush kales and stem kales, sometimes with curled leaves. Dioscorides also referred to cole crops, but the original manuscript was not illustrated. The first illustration of various brassicas is found in the Dioscoridean recension known as the Juliana Anicia Codex dated 512 (Plate 2.2). The Romans Cato and Pliny mention several forms of coles including stem kales and headed cabbages. Cato devotes a chapter (De brassica) to cole crops in De Agricultura. Based on their description, it appears that quite a large number of forms were cultivated by Romans. Farmers collected their own seeds and as a result of cross-pollinations because of self-incompatible nature, new variations arose that led to the development of local cultivars through selection. Subsequently, stabilization gave rise to modern-day types. Early Greek and Roman authors used the word caulis, which means ‘‘stem,’’ to define the entire cole plant. Names of cole crops in modern European languages are derived from it: Kohl in German; cole, collard, and kale in English; kal in Scandinavian; cal in Gaelic; cole in Spanish; chou in French; cavolo in Italian; and couve in Portuguese. Medieval information about cole crops can be found in the Capitulare de Villis imperialibus, which describes plants occurring in the garden of Charlemagne (ca. 800 CE). Cauli, probably kale or cabbage, and ravacauli, kohlrabi, are mentioned. Several Ibero-Arabic treatises describe brassicas in detail. The best-known ones, by Ibn-al-Awam (12th century) and Ibn-al-Baithar (13th century), draw heavily from Greek and Roman writings. Kale is described as caules onati in the 14th-century Tacuinum Sanitatis, the illustrated manuscripts based on an 11th-century Arabic manuscript Taqwim al-Sihha bi al-Ashab al-Sitta (Rectifying Health by Six Causes) written by Sa’dun Ibn Butlan of Baghdad (Dauney et al. 2009). Cultivated B. oleracea forms have been well depicted in paintings produced in Flanders and Holland from the 16th to 19th century (Zeven and Brandenburg 1986). Pieter Aertsen (1509–1575), his nephew Joachim Beuckelaer (1535–1575), and Floris Gerritsz van Schooten (1590–1655) painted cauliflower and cabbage. Cauliflowers are of normal size and the curd is well formed. Floris van Dijck (1575–1651) and Frans Snijders (1579–1657), however, painted cauliflower with loose curds. A painting by Adriaen van Utrecht (1599–1652) shows cauliflower with both long and short stems. Cabbages are more


43

commonly painted than cauliflower. Gerard Dou (1613–1675), Joachim Beuckelaer, and Peter Aertsen frequently depict cabbages in their paintings. Red cabbage is painted by many artists and must have been quite common at that time. Lucas van Valckenburg (1540–1597) illustrates a red cabbage with greenish leaves and reddish ribs. Although kale was common during this period, it is not found in paintings; Brussels sprouts are not found in this period. Almost all the 16th- and 17th-century illustrated herbals have extensive descriptions of cole crops. One of the earliest was by Macer Floridus in De Herbarum viribu (1506), where the plant appears to be wild and is called caulis (Henslow 1908). Dodonaeus (1583) described several forms; one of these, Flourie Coleivort (Brassica Cypria), appears to refer to cauliflower and is described in this way: ‘‘the small stems grow together in the center, thick set and fast throng together’’ (Giles 1941). Gerarde (1597) in The Herball or Generall Historie of Plants devotes an entire chapter to cabbages and gave 15 examples of coleworts or forms of B. oleracea (Fig. 2.7). Brussels sprouts are referred to as Brassica capitata polycephalos in Dalechamps (1587), B. ex.capitibus pluribus Bauhin 1623), and B. polycephalos (de Lobel 1581). Cabbage is referred to as B.quartum genus (Fuchsius 1542), Caulis capitularis (Tragus 1552), B. capitata (Mattioli 1571), B. capitata albida (Dalechamps 1587), B. alba sessilis glomerata, aut capitata Taclucae habitu (de Lobel 1581), B. capitata alba (Bauhin and Cherler 1651), and B. capuccia (Durante 1585). Kohlrabi is named B. caule rapum gerens (Dodonaeus 1583), Rapa Br. peregrine, caule rapum gerens (de Lobel 1581), B. raposa (Durante 1585; Dalechamps 1587), B. gongylodes (Mattioli 1571), Caulorapum rotundum (Gerarde 1597, 1633), and B. caulorapa (Bauhin and Cherler 1651). Precise information about the progenitors of these brassicas is unavailable although several theories have been proposed. It was earlier believed that all the cole crop forms evolved through mutations, human selection, and adaptation from the primitive kales growing wild along the Mediterranean coast from Greece to England, where it might have been cultivated by Celts. Kale traveled to the eastern Mediterranean region between first and second millennia BCE, where it became fully domesticated with explosive diversification giving rise to a range of cultivated forms. However, this concept is disputed at present, and the Mediterranean kales are considered as mere escapes from early cultivation. These species occur in cliffs and in rocky islets, in more or less small isolated places with distinct ecogeographical centers and mainly confined to Mediterranean region (Fig. 2.8). These wild forms are closely related to cultivated forms and might have participated in their origin and domestication. Snogerup (1980) has grouped them in this way:

44


Fig. 2.7.

Illustrations of various cole crops and mustard. (Source: Gerard 1633).


45

Distribution of wild B. oleracea

B. oleracea

B. montana

B. rupestris-Incana complex

B. hilarionsis

B. insularis

B. cretica

©EnchantedLearning.com

Fig. 2.8. Distribution of wild species of Brassica oleracea complex. (Source: Snogerup 1980).

B. cretica grows in the Aegean area, southen Greece, and southwestern Turkey. It is a woody and much-branched perennial plant. Ssp. nivea occurs in Paloponnesos and Kriti while ssp. cretica grows in maritime cliffs. B. rupestris-incana complex. A number of regional variants in this complex occur in Sicily and south-central Italy. The different variants include B. incana, B. villosa, B. rupestris, and B. drapenensis. However, their ranks are uncertain. These are characterized by tall main stem and large petiolated leaves. B. insularis occurs in Corsica, Sardinia, and Tunisia. It is characterized by stiff, fleshy, glabrous leaves and large fragrant flowers. B. macrocarpa is endemic in west Sicily with very thick fruits containing seeds in two rows in each locule. B. montana occurs in northeastern coastal area of Spain, southern France, and northern Italy. It is a shrubby perennial plant. B. oleracea occurs on coasts of northern Spain, western France, and southwestern Britain. It is a stout perennial plant with a strong vegetative stock.

46


B. hilarionis with a very narrow endemism in the Kyrenia Mountains of Cyprus, very much resembles B. macrocarpa except for large pink flowers. These wild species, all 2n ¼ 18, are easily crossable with oleracea forms and produce fertile or semifertile hybrids (Kianian and Quiros 1992c; Go´mez-Campo 1999). Harberd (1972) assigned them to the same cytodeme or crossing group in a cytotaxonomical investigation while Gladis and Hammer (1992) suggested subspecies status to them. Bailey (1922, 1930) described a number of cultivated forms, created some new species, and suggested wild B. oleracea as the possible progenitor. Neutrofal (1927) and Snogerup (1980) believed B. montana to be the progenitor of cabbage and kales and B. rupestris of kohlrabi, while Schiemann (1932) suggested different Mediterranean wild species as the progenitors. Schulz (1936) specifically mentions the role of B. cretica in the origin of cauliflower and broccoli. Helm (1963) in a detailed scheme of evolution of different forms (Fig. 2.9), proposed three different pathways for the origin of different forms from wild B. oleracea but did not specify precisely any species. Accordingly, in one direction, the oldest form var. ramosa (thousand-headed kale) gave rise to var. gemmifera probably in Belgium from which developed monstrosities such as var. dalechampi. In another direction, the wild cabbage was the progenitor of var. costata (Portuguese kale), which originated in the Iberian peninsula. Cabbage (var. capitata) and savoy cabbage (var. sabauda) both derived from it in Italy. The kales and collards (vars. acephala, sabellica, selensia, and palmifolia) gradually developed from one stock that also gave rise to the forage crop marrow stem kale (var. medullosa), which also gave rise kohlrabi (var. gongylodes). Sprouting broccoli (var. italica), which initially developed from wild var. sylvestris, gave rise to cauliflower, with both the forms originating in the eastern Mediterranean. However, in recent years, these concepts have changed; these Mediterranean kales are regarded as mere escapes from early cultivation. At present, a polyphyletic origin is proposed from several wild Mediterranean B. oleracea (Gustafsson 1979; Snogerup 1980; Mithen et al. 1987). Several wild species are mentioned in this context, although the molecular investigations did not support this concept (Hosaka et al. 1990; Song et al. 1990). The low chloroplast diversity does not indicate multiple domestication events (Allender et al. 2007). Gates (1953), following a study of different wild species and cultivated forms, came to the conclusion that cabbages, Brussels sprouts, and kale originated from B. oleracea var. sylvestris in western Europe, cauliflower and broccoli in the eastern Mediterranean, and kohlrabi in the middle


47

Fig. 2.9. Schematic representation of the origin of various Brassica oleracea cultivars. (Source: Adapted from Helm 1963).

Mediterranean. In recent years, the theory of origins again reverted to the old concept of implicating wild Atlantic B. oleracea as the progenitor with subtantial introgressions from different wild species, which had been responsible for increasing the varaibility and adaptability of cultivated forms. Song et al. (1988b, 1990) carried out studies on cultivated and wild forms within B.oleracea group using nuclear DNA RFLPs, divided them into three groups (kales, cabbages and broccoli), and suggested a monophyletic origin. Louarn et al. (2007), based on microsatellite marker investigations, agreed with this grouping. Song et al. (1988b) believed that ancient wild progenitor was similar to wild B. oleracea and B. alboglabra, which must be the closest ancestors of cultivated forms. The earliest cultivated form was probably a leafy kale from

48


which originated a variety of kales due to natural hybridization, gene introgression, and selection along the Mediterranean coast and north Atlantic from Greece to Wales. However, the linguistic, literary, and historical sources favor the origin of B. oleracea in the north-central and northeastern Mediterranean extending from southern Italy to Greece and the west coast of Turkey (Maggioni et al. 2010). This whole region also overlaps with the distribution of related wild species of B. oleracea. Many studies in recent years have been carried out to investigate the evolutionary relationships between these wild taxa and between them and crop types to assess their possible role in the evolution of crop forms using both nuclear and organelle-based molecular markers (Lazaro and Aguinagalde 1996, 1998a,b; Lann er 1998; Panda et al. 2003; Allender et al. 2007; Louarn et al. 2007; Mei et al. 2010). Although the results from nuclear and cp genomes are not consistent, these sudies broadly suggested the possibility of co-ancestry and gene flow between wild and crop types. There is a close association of cultivated types and B. incana, B. montana, B.cretica, and B. hilarionis (Mei et al. 2010). At the same time, the relative lack of cp diversity in these species (Panda et al. 2003; Allender et al. 2007) indicates a single rather than multiple centers of domestication of cole crops. 2. Cabbage. Neolithic farmers collected cabbage for food before it entered into cultivation. The word cabbage is a derivative of Latin caboche or caputium, meaning ‘‘head.’’ It was an important ingredient of Greek and Roman cuisine. However, the early forms did not form a compact head as found in modern cultivars. De Candolle (1824) believed that it was independently domesticated by the Basques and also at different places in Europe. In evidence, he cited several names for cabbage: kap or kab, caul or kohl in Latin, German, and Celtic. However, the idea of domestication by Celts in western and northwestern Europe is not very convincing since all the Celtic names are derived either from Greek (krambai and kaulos) or Latin (brassica, caput, olus and caulis). Most likely, the invading Celts in 6–8th-century BCE found it already domesticated by the native Ligurians and Iberians and adopted it along with its name. Another point of contention is how a wild plant from the Atlantic coast reached Egypt and Mesopotamia. Go´mez-Campo and Gustaffson (1991) believed that cabbages migrated along the sea tin route linking the British ‘‘Casiteride’’ Islands with the eastern Mediterranean. The Tartessians from southwest Spain were exploring the tin mines of Cornualles and traded tin with the seafaring Phoenicians. The Greek word krambe for ‘‘cabbage’’ is most likely of Phoenician origin. A word shaw’t in Papyrus Harris, a document written in 1166 BCE on the death of


49

Ramses III comprising a list of offerings to the god Amon, has been interpreted as ‘‘cabbage.’’ However, its authencity is questionable (Aufrere 1987; Nunn 2002) as the Egyptians used the word gramb for cole crops, which is a possible derivative of Greek krambe. The Greek Theophrastus and the Romans Cato and Pliny mentioned stem cabbage kales and headed cabbages. Theophrastus mentions three and Dioscorides mentions six types of cabbage, indicating a diversity of forms at that time. Ch evre et al. (1998) describe the myriad uses of cabbage in 16thcentury French books. According to Mizauld (1578): ‘‘The ancient Egyptians, who dearly loved wine, used to eat cooked cabbage at their meals before eating anything else; and at their banquets and feasts the first dish used to be cabbage so that the wine should not harm them. The juice of raw cabbage sipped with wine serves as a remedy against vipers’ bites; and as a plaster with fenugreek flour is a sovereign remedy against gout and other diseases of the joints.’’ Brussels sprouts are supposed to have been developed in 14th century near Brussels in Belgium. Gerarde (1597, 1633) described a similar form of kale with finely dissected leaves and numerous buds as ‘Parseley cabbage’ (Henslow 1908). It is reported that these sprouts were served at the royal wedding feast of Alcande de Bredrode in 1481 at Brussels (Hyams 1971). 3. Cauliflower and Broccoli. Cauliflower and broccoli produce large dense structures made up of modified infloresences. In cauliflower, the undifferentiated mass of inflorescence meristems form a structure called curd. About 10% to 15% of the meristems develop into normal flowers with the rest aborting. The word cauliflower is dervied from Latin caulis (stem) and floris (flower) or Greek kaylo´z. It is believed that both these forms evolved in eastern Mediterranean (Hyams 1971; Snogerup 1980). Although early Greeks and Romans mentioned sprouting forms of cabbage, probably a distinction was not apparent between broccoli and cauliflower. Possibly both were considered as variants of the same form. Yahya ibn Muhammad Ibn-al-Awam, a Moor living in Spain in the 12th century, was the first to make a clear distinction between heading and sprouting forms. He compiled a Spanish-Arabic treatise Kitab-al-Felahah (Book of Agriculture, ca. 1140) wherein he devoted a full chapter to cauliflower, described three types, and called it Syrian or Mosul cabbage or quarnabit, the current Arabic word for ‘‘cauliflower.’’ Ibn-al-Baithar, in the 13th century, also mentioned it. Hyams (1971) is of the opinion, based on Ibn-Al-Awam’s observations, that cauliflower was, noticed, selected, and propagated in Syria. Dodonaeus (1583), while describing various forms of cole group, referred to it as B. cypria, indicating its possible origin in Cyprus. It is now generally regarded that cauliflower originated from

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broccoli (Giles 1944; Crisp 1982; Gray 1982). This is also supported by Smith and King (2000), based on the distribution of alleles for curd development in B. oleracea. In fact, this close relationship between these two forms is further substantiated by the presence of a nonsense mutation in a floral gene (Purugganan et al. 2000). Crisp (1982) hybridized cauliflower and broccoli, which led him to conclude that a single major gene mutation in broccoli produced cauliflower. In recent years, use of various molecular markers has unraveled the molecular genetic basis of domestication in several crops including cauliflower. Several floral homeotic genes controlling flower traits have been identified in Arabidopsis thaliana. Mutations in these genes cause floral deformities. These genes belong to MADS-box regulatory gene family (Yanofsky 1995; Riechman and Meyerowtz 1997) and include APETALA 1 (AP1) responsible for floral meristem identity and correct specification of sepals and petal organs (Mandel et al. 1992; Gustafson et al. 1994). The other gene CAULIFLOWER (CAL) also specifies the floral meristem identity (Kempin et al. 1995). Mutations in both these genes arrest the development at the inflorescence meristem stage and result in a dense mass of inflorescence meristems as in cauliflower (Kempin et al. 1995). These results suggest the involvement of B. oleracea orthologues referred to as BoCAL (Kempin et al. 1995) and BoAPL (Anthony et al. 1993, 1996; Carr and Irish 1997) in curd formation. Further studies by Lowman and Purugganan (1999) revealed that BoAPL is present in two copies in B. oleracea genomes and are referred to as BoAPL-A and BoAPL-B. Mutant genes have a reduced ability to specify floral meristem identity. BoAPL-B is transcriptionally silenced in both cauliflower and broccoli and appears to be a pseudogene. Genetic studies by Crisp and Tapsell (1993) suggested the involvement of several loci in curd formation with at least one major and several modifier genes. Lowman and Purugganan (1999) believed that variation in at least two loci is responsible for curd formation: a nonsense mutation in BoCAL and a 9 bp insertion in exon 4 at BoAPL-B. Brassica oleracea ssp. botrytis still possesses a functional BoAPL-A gene as the flowers that are produced from the curd are normal. Smith and King (2000) reconstructed the possible events leading to domestication of cauliflower based on genetic, phenotypic, and molecular information, together with ecogeographic distribution of alleles, and proposed a two-step process. They considered Sicilian Purple types as a primitive curding type that was selected following introduction of a mutant BoCAL-a or BoAP1-a allele into the gene pool of basic heading plants. Follwing the introduction of a mutant copy of the second locus, either BoCAL-a or BoAP1-a, present-day cauliflower with fully developed curd evolved. This specific


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genetic architecture with curd formation to develop normal flowers and capability to produce seeds was selected by farmers so that the mutant could be preserved (Purugganan et al. 2000). Broccoli includes several heading forms with a single large terminal inflorescence made up of a mass of fully differentiated flower buds of which only few abort before flowering. A related form is sprouting broccoli, where the inflorescence is branched. The word broccoli is derived from the Italian brocco and the Latin brachium, which means ‘‘arm’’ or ‘‘branch’’ (Bosewell 1949). Cultivation of broccoli dates back to an earler period than cauliflower (Vilmorin 1885). It is believed that the raw forms were introduced to Italy from the eastern Mediterranean during the fourth to sixth century BCE, where diversification took place and many forms similar to modern types, including heading and sprouting forms, originated (Schery 1972). Ancient Romans referred to broccoli as cyma. The first documented description of it was given by herbalist Dalechamps (1587) in Historia Generalis Plantarum; it is called ‘‘sprout cauliflower’’ or ‘‘Italian asparagus’’ in Miller’s Gardeners’ Dictionary of 1724 (Hedrick 1919). 4. Chinese Kale (B. alboglabra). Although originated and cultivated in initial stages in Mediterranean, Chinese kale was domesticated in southern China (Guangdong Province). It represents an ancient domesticate without any history of wild progenitors, although possible similarities to B. cretica ssp. nivea are apparent (Dixon 2007). It does not require vernalization and it flowers early. Su shi (1037–1101), a famous poet and gastronomist in the Song dynasty, praised it as delicious as mushroom in a poem. A number of more primitive varieties of B. oleracea that were once under cultivation have either disappeared or are grown only in a very limited area. These include branching or thousand-headed kales and true kales and collards (var. ramosa and var. viridis) and palm cabbage—var. palmifolia, which had a wider distribution in Italy (Hamelt 1998; Farnham et al. 2008). Nonheading forms of cabbage became popular in the British Isles, Portugal, and Spain and were known as coleworts by the English, a term that became ‘‘collards’’ in the Americas during the 1700s (Zohary and Hopf 1993). Collards are considered the oldest forms of cultivated B. oleracea. D. Brassica rapa 1. Taxonomy and Origin. The cultivated forms of Brassica rapa, a highly polyomorphic species, exhibit distinct infraspecific types and comprise three different groups:

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1. Oleiferous. Grown in Canada, Australia, and Europe, where it is known as turnip rape. The forms cultivated in the Indian subcontinent are called sarson. 2. Leafy forms. Extensively cultivated in southeast Asian countries as vegetables. These are also used as oilseeds in central and southern China. 3. Turnip or root-forming type. The root-forming types are grown for vegetable and fodder with a wide global distribution. The antiquity of oilseed B. rapa is not well documented, although references to oleiferous forms are found in ancient Indian Aryan literature (ca. 1500 BCE). B. rapa seeds were found in the stomach of Tollund man (a mummified corpse of the fourth-century BCE found in Scandinavia) (Renfrew 1973). Wild forms of B. rapa grow from the western Mediterranean region through Europe to central Asia and the Near East (Sinskaia 1928; Vavilov 1949; Mizushima and Tsunoda 1967; Zeven and Zhukovsky 1975; Tsunoda 1980). The Fertile Cresent region comprising present-day Iran-Iraq-Turkey is most likely the place of its origin. It probably was first domesticated at some of these places perhaps several millennia ago. B. rapa in one way or other has been widely used by all the civilizations in this region. Burkill (1930) suggested Europe as the place of its origin and also believed that it was originally biennial but, through selection and domestication, annual forms were developed. Sinskaia (1928) and Vavilov (1949) strongly considered central Asia, Afghanistan, and the adjoining northwest part of the Indian subcontinent as one of the independent centers of its origin. Based on comparative morphology, Sun (1946) proposed the existence of two races, the Western race comprising oilseed forms and turnip, and the Eastern race comprising vegetable forms. Protein analysis (Denford 1975), isozyme distribution patterns (Denford and Vaughan 1977), nuclear RFLP (Song et al. 1988b), RAPD (Chen et al. 2000; He et al. 2003) and Amplified fragment length polymorphism (AFLP) studies (Guo et al. 2002; Zhao et al. 2005; Takuno et al. 2007) substantiate the existence of two races. The most likely explanation is that these groups represent two independent centers of origin with Europe being the primary center for oleiferous forms. Turnip later traveled further eastward through the Middle East. Once the primitive or semidifferentiated types entered India and China, they developed toward oilseed forms in India and toward leafy forms in China, primarilysouth China. China is also the center of origin of a unique form of ssp. oleifera (oilseed form) (Li 1981). 2. European Forms. European and North American oleiferous forms are quite distinct from Indian forms. It is believed that Europe took to its


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cultivation in the early Middle Ages. The first commercial plantings of B. rapa occurred in the Netherlands in the 16th century; its oil was first used for lamps and as lubricants in steam engines. Since World War II, its cultivation was taken up on a large scale in Europe and Canada, where at present ‘‘double-low’’ cultivars dominate. European and Canadian oilseed forms, spring-type cool-season crops, possess better cold tolerance. For this reason, they have become the predominant crop in the more northern regions of Europe and in some parts of Scandinavia. In Canada, it is often referred to as Polish rape or summer turnip rape. 3. Indian Forms. The Indian forms comprise three ecotypes: brown sarson, yellow sarson, and toria. Brown sarson is believed to be the oldest, and has been identified with the Sanskrit word sarshap. The modern Hindi word sarson appears to be a derivative of sarshap. Two views exist regarding its origin: (1) it evolved independently from the original stock in the northwest of India in the foothills of Himalayas, or (2) it reached nortwestern India in a subdifferentiated state through Iran. Hinata and Prakash (1984) are of the view that only primitive forms entered India with migrating people and from these the brown sarson was the first to evolve in northwestern India and later spread eastward. Two forms of brown sarson are cultivated in India, the self-incompatible lotni and the self-compatible tora. Since all the cultivated and wild forms of B. rapa are self-incompatible, it is assumed that lotni is older and tora is a derivative form. The tora form is very similar to yellow sarson in inflorescence shape, flower morphology, introse anthers, and growth rate. The only difference is in the brown seed color. In view of these similarities and its cultivation in the traditional yellow sarson area, Hinata and Prakash (1984) proposed that the tora form arose through hybridization with yellow sarson while lotni brown sarson arose somewhere in eastern India. In fact, these tora forms can be produced easily through such hybridization. Cultivation of yellow sarson is confined to a very limited area in eastern parts of India. In all probability it evolved from brown sarson as a mutant. Yellow sarson is characterized by yellow seeds and self-compatibility. Another important feature is the occurrence of 2-, 3-, and 4-valved siliquae. Yellow sarson has flowers of higher growth rate after the initiation than self-incompatible brown sarson. Its self-compatibility trait is controlled by a modifying gene (m) that is independently inherited from the S alleles. This recessive epistatic gene m suppresses the action of S allele on the stigma side but not on the pollen side. It was selected by farmers because of its attractive yellow seed color and a deceptive bold seed size. It was mentioned as siddhartha in Sanskrit literature of 1000 BCE. Hinata and Prakash (1984)

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suggested the tentative dates and place for its origin as ca. 1200 BCE in northwestern India. Toria perhaps evolved as a mutant form of the brown sarson population, differing in being dwarf with a shorter life cycle. Lotni brown sarson, being self-incompatible, is allogamous and hence can allow perpetuation of mutant alleles in its population. Most of the variability in toria is met within types from submountainous tracts of the Himalayas where toria-like plants were selected from brown sarson populations and thrived in relatively warmer climates. In spite of numerous references to brassicas in Sanskrit literature, the descriptions are not precise enough to identify the species. Several names referring to these crops are mentioned, such as rajika, swesarshapa, and siddartha. Hinata and Prakash (1984) proposed that sarshap connotes only brown sarson and siddhartha yellow sarson. 4. Chinese Forms. The vegetable forms of B. rapa (Plate 2.3) are extensively cultivated in China, Korea, Malaysia, Vietnam, Taiwan, Indonesia, and Japan. These are eaten fresh as salad, boiled, or salt-pickled. Because of foliar diversity, much confusion arose in naming various forms. Linnaeus (1753) described B. chinensis, for the first time, using plants raised from the seeds collected in China by Osbeck in 1751. Subsequently, many investigators described them under various names (Prakash and Hinata 1980). Bailey (1922, 1930) assigned species ranks to these varieties as B. parachinensis, B. pekinensis, B. narinosa, B. chinensis, and B. nipposinica. At present, these have been designated as subspecies by most researchers and include: ssp. chinensis Pak choi with large thick leaves and broad thick white petioles and does not form a head ssp. narinosa Resembles ssp. chinensis but differs from typical pak choi types by its flat appearance and many dark green leaves ssp. japonica Pot herb mustard. Forms a large stump with many narrow thin leaves ssp. pekinensis Chinese cabbage. Cabbage-like heads of different shapes formed by tightly overlapping light green large leaves with a wrinkled surface that have large white midribs It is now widely accepted that these forms originated from oilseed forms after B. rapa it was introduced into China, probably in the first century CE, through western Asia or Mongolia (Burkill 1930; Nishi 1980). There is supporting evidence in the Yun T’ai in Ch’i-min-yao-shu, a Chinese agriculture treatise of the fifth or sixth century (Li 1969). Li (1982) has discussed the origin of various forms as represented in Fig. 2.10.


Fig. 2.10.

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Origin of various leafy forms of Brassica rapa. (Source: Adapted from Li 1982).

Pak-choi (ssp. chinensis) with a narrow or wide green-white petiole was the first to evolve in central China and is most closely related to the primitive European forms (Song et al. 1988b). Its antiquity is suggested by a vast range of morphological varations (Li 1982) and high level of DNA polymorphism (Figdore et al. 1988; Song et al. 1988b). Among the East Asian forms, this is the most primitive type from which originated ssp. parachinensis in central China. The Chinese cabbage (ssp. pekinensis) has a well-documented history and origin (Li 1982). It first appeared as a loose-leaved form as a result of hybridization between pakchoi and turnip in the 10th century in the city of Young-Chow. A book Ben-Cao-Tou-Jing (The Classics of Illustrated Medical Herbs) by Su Song has recorded this information. Its hybrid origin is also supported by nuclear RFLPs (Song et al. 1988b). This primitive form is still grown in southern China. The appearence of a heading form with thick petioles is documented in the 12th century for the first time. Semi-heading and solid compact head forms subsequently evolved. These two forms are

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mentioned in the 14th-century treatice, Shua-Pu-Tsa-Su (Miscellanea of Gardening). Selection by farmers for better types resulted in heads with fluffy tops or fully solid heads, which were described in ShuinTian-Fu-Tse (Local Records of Shuin Tian Fu), a compiliation of the 17th century. These vegetable forms are also extensively grown in Japan and are believed to have entered through China or Siberia in the Meji era in the 19th century (Aoba 2000). During cultivation, a unique vegetable form, ssp. japonica, was selected by farmers. It possesses many basal branches and leaves. Because of its close similarities to B. juncea, it is suggested that natural hybridization with B. juncea might have played a role in its evolution (Nishi 1980). There are two prominent forms: Mizuna with deeply dissected bipinnate leaves and Mibuna with slender entire leaves. 5. Turnip. Turnip is of European origin, and its seeds have been excavated from Neolithic sites in Switzerland, 8000 BCE (Hyams 1971). The domestication history is ancient, as is evident from the Assyrian word laptu, ca. 1800 BCE (Oppenheim et al. 1973; Vogl et al. 2007). Turnip is mentioned during the period of Ashur-Nasir-Apli in the ninth century BCE (Leach 1982). The earliest record of its cultivation is found in a list of plants grown in the garden of Merodachbaladan (722–711 BCE) in Babylonia (Zohary and Hopf 1993). It is also referred to in the Jewish Mishna composed in the first century. De Candolle (1886) proposed its cultivation in Europe around 2500–2000 BCE and its spread to Asia after 1000 BCE. Many Semitic, Greek, and Slavic words, such as meip, erifinen, rapa, and rippa, indicate the antiquity of its domestication (Reiner et al. 1995). Turnip formed an important component of the cuisine of ancient Greek and Roman civilizations. There are charred remains from the ancient site of Sparta in Greece (Hather et al. 1992). It appears that biennial forms of turnip were selected for food value of the swollen hypocotyl. A Chinese book Shih Ching (Chinese Book of Poetry) comprising poems that were composed between 1000–500 BCE and said to have been edited by Confucius (551–479 BCE) refers to turnip as feng (Keng 1974). Theophrastus mentioned it as gongylis, and this name is used in the Juliana Anicia Codex of Dioscorides from 512 CE that contains an image of turnip (Plate 2.2). Both Columella and Pliny described different types of turnip. Pliny used the word napus and mentioned five types: Cornithian, Cleonaceum, Liothasium, Boeoticum, and Green. Columella provides significant information about turnips and described Long Roman, Round from Spain, the Syrian, the White, and the Egyptian. During the


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Middle Ages, turnip was an important food plant in Europe. It is referred to as napi in Capitulare de Villis of Charlemagne (ca. 800); as ruba by Hildegard von Bingen (1098–1179), and as napo and rapa by Albertus Magnus (1193–1280) (Vogl et al. 2007). It is also mentioned and illustrated in the late 14th-century handbook Tacuinum Sanitatis (Daunay et al. 2009). The word turnip is derived from the Middle English word nep from napus, which together with turn (made round) became turnip (Bosewell 1949), but this word appeared only after 1400. In the early 16th century, rape and turnep were still in use to indicate this plant. Many herbalists frequently mentioned turnip of two types, the common flat and the long ones. Hedrick (1919) lists the many names used by herbalists: Rapum (Mattioli 1571), Rapum vulgare (Dodonaeus 1578), and Rapum majus (Gerarde 1597, 1633) for the flat turnips; Rapum tereti, rotunda oblangaque radici (de Lobel 1581), Rapum oblongius (Dodonaeus 1578), Rapum rediceoblonga (Gerarde 1597, 1633) and Rapum sativum rotundum et onlongum (Bauhin and Cherler 1651) for the long ones. It is a very well known vegetable in the entire Middle East (Arab: lift, Persian: salgham). It probably arrived with the invading armies and from there spread eastward to India, where it also is known by the Persian word salgham. Turnip now has a bad image as a poor man’s crop. E. Brassica carinata Ethiopian mustard is cultivated on the East African Plateau, particularly in Ethiopia and in parts of the east and west coasts of the African continent. No information is available regarding its origin. It is considered to have evolved in the highlands of Ethiopia and adjoining portion of East Africa and the Mediterranean coast (Go´mez-Campo and Prakash 1999). Brassica nigra, which grows wild in this region, and a kale-like form that has been in cultivation since ancient times are the possible ancestors (Mizushima and Tsunoda 1967), and which underwent natural hybridization in the remote past (Prakash and Hinata 1980; Song et al. 1988a), a point demonstrated by observing wide genetic diversity based on RAPDs (Teklewold and Becker 2006). In Ethiopia, resource-poor small-holder farmers produce the crop for several uses. Young tender leaves and stem tips are eaten raw in salads while older leaves and stouter stem portions are cooked and eaten like collards. Flowering stalks may be cooked and eaten like broccoli. The seeds are used for a mustard preparation and as a source of edible oil. Ethiopian mustard has also been reported as a useful fodder crop. Because of its better drought tolerance and resistance to fungal

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pathogens, it is gaining area in semiarid areas of southern Europe, western Canada, Australia, and India (Malik 1990; Rakow and Getinet 1998; Cardonea et al. 2003). F. Brassica juncea 1. Taxonomy. The history of mustard utilization and domestication dates back to 6000–7000 years ago. Carbonized mustard seeds have been excavated from Banpo in China (ca. 4800 BCE), which contains the remains of several well-organized Neolithic settlements (Institute of Archaeology Report, 1963) as well as from a site in the Indus Valley (ca. 2500 BCE) in the northwest part of the Indian subcontinent (Allchin 1969). There are references in ancient Indian and Chinese literature as early as 2500 BCE (Prakash and Hinata 1980; Hinata and Prakash 1984; Go´mez-Campo and Prakash 1999; Manohar et al. 2009). The earliest Chinese literature record of brassicas being used as a vegetable appeared in Xiaxiaozhen (Ancient Almanac) in the Xia dynasty (about 3000 BCE) and Shijin-Gufeng (A Collection of Poems) in the Zhou dynasty (1122–247 BCE). The species entered Europe during the Middle Ages as a medicinal crop and was later grown as a vegetable, condiment, and oil crop (Hemingway 1976). Because of its highly polymorphic nature and importance for human nutrition, B. juncea has long attracted the attention of botanists, taxonomists, and plant breeders. In ancient times, Greeks used the word aıń while Romans referred to it as sinapi. Pliny described it as thlaspi. Herbalists mentioned its use for medicinal purposes and described it as Sinapi alterum (Mattioli 1571) and S. sativum alterum (Dodonaeus 1583; Gerarde 1597, 1633). Since the 17th century, many expeditions were carried out to collect different accessions of B. juncea in several countries; they resulted in a vast accumulation of synonmy, which was described either under Sinapis or Brassica (Prakash and Hinata 1980). The botanists Cosson of France (1859) and Czernjaew of Russia (1859) finally transferred it to Brassica with the species name juncea. Hooker and Thomson (1861), for the first time, reported polymorphism in B. juncea particularly the large variations in leaf shape. Chiefly due to this character, it was accorded the rank of subspecies and occasionally species. Bailey (1922), in a comprehensive investigation on various forms of B. juncea primarily collected from China and Japan but also from India and Europe, confined his observations mainly to variations in leaf form. He created several botanical varieties: crispifolia, having large crisped curled and fringed leaves; japonica with pinnatifid basal leaves; and multisecta with multifid leaves.


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Vaughan et al. (1963), who reviewed the taxonomy and vast synonymy of B. juncea in detail, considered morphological variations, volatile oils, and seed protein characters to place all the variations into four well-defined groups: B. juncea var. sareptana with lyrate lobed basal leaves and includes S. ramosa Roxb., B. juncea Hook. Fil. & Thomson (Prain), B. besseriana Andrz., B. juncea Coss. (Bailey), and B. juncea var. sareptana Sinsk. B. juncea var. integrifolia with entire or small lobed basal leaves, including B. rugosa var. cuneifolia Prain, S. patens Roxb., B. juncea var. foliosa Bailey, B. integrifolia Rupr., B. juncea var. subintegrifolia Sinsk., and B. juncea var. integrifolia Sinsk. B. juncea var. japonica with dissected basal leaves, including B. juncea var. longidens Bailey, B. juncea var. japonica Bailey, and B. juncea var. multisecta Bailey. B. juncea var. crispifolia with dissected and crisped lower leaves including B. juncea var. crispifolia Bailey, B. juncea var. subcrispifolia Sinsk, and B. juncea var. crispifolia Sinsk. In a recent publication, Dixon (2007) distinguished seven groups: 1. Hakarishina (B. juncea). Oilseed form of Indian subcontinent, central Asia and Europe 2. Nekarashina (var. napiformis) with enlarged roots 3. Hsueh li hung (var. foliosa) and Nagan sz kaai (var. japonica) with dissected leaves 4. Azanina (var. crispifolia) with dissected leaves and a general appearance of curly kale used for salads and as ornamentals in the United States 5. var. integrifolia with entire succulent leaves 6. var. rugosa, a large-size plant with leaves having flat entire midribs 7. Ta hsin tsai (var. bulbifolia) with plants having succulent stems Chinese researchers (Fu et al. 2006; Qi et al. 2007, 2008; Wu et al. 2009) describe variants following Gladis and Hammer (1992): leaf mustard (B. juncea var. multiceps), stem mustard (B. juncea var. tsatsai), root mustard (B. juncea var. megarrhiza), oilseed mustard (B. juncea var. juncea), and seed stalk mustard (B. juncea var. utilis) are the major representatives of this diversified crop (Plate 2.4). These forms are widely grown in most Southeast Asian countries; China is the leading country in terms of crop area. These forms are consumed as pickled

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leaves, stem and root, seed oil, and seed mustard. At present, oil forms are simply referred to as B. juncea Czern. while the Chinese variations are given the varietal rank as mentioned. 2. Origin and Domestication. Prain (1898), for the first time, proposed that B. juncea evolved in China and subsequently spread to India through a northeastern route. He substantiated his views by observing that a wild form of B. juncea, which he referred to as Sinapis patens, is common along this route. This view proved to be incorrect as S. patens is now identified as Nasturtium indicum (Hooker and Anderson 1872; Schulz 1919). Prain was later supported by Sinskaia (1928). She believed that East European B. juncea is also of Chinese origin from where it migrated naturally from the Kirgiz steppes, where it grows wild. She further speculated that most primitive forms have lyrate-pinnatisect leaves from which evolution occurred in three directions: (1) the East Asian forms with bipinnate leaves dissected into threadlike segments, (2) the Chinese forms with crisp leaves, and (3) nondivided leaves comprising the central Asian and Indian forms. Burkill (1930) and Sun (1970) did not agree with a Chinese origin for B. juncea; both suggested the Middle East as the place of origin. Sun (1970) argued that since parent species do not occur naturally in China, B. juncea had to be introduced from outside. Russian botanist Vavilov (1949) proposed Afghanistan and adjoining regions as its primary center of origin and central and western China, eastern India, and Asia Minor through Iran as the secondary centers. Cytogenetical, biochemical, and in recent years molecular evidence has suggested the polyphyletic origin at many places with a sympatric distribution of parental species (Olsson 1960a; Prakash 1973a; Vaughan 1977; Prakash and Hinata 1980; Song et al. 1988a) The region of Middle East (Fig. 2.6) has been favored strongly as the place of its origin by many researchers as both parental species grow wild in this region (Olsson 1960a; Mizushima and Tsunoda 1967). Wild forms of B. juncea can still be seen in the plateau of Asia Minor and adjoining southern Iran (Tsunoda and Nishi 1968; Tsunoda 1980). Regions of northwest India and southwest China constitute two important secondary centers for the domestication of B. juncea as they exhibit enormous variation. Sun et al. (2004), while studying variations in B. juncea, also suggested central China, mainly the Gansu and adjoining region, as the major center of diversity. Biochemical evidence for the existence of two different races was provided by studies of Vaughan et al. (1963) and Vaughan and Gordon (1973). The seeds of Chinese forms have a marked mucilagenous epidermis and produce allyl isothiocynate, while the


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Indian forms produce 3-butenyl isothiocynate. RFLP studies (Song et al. 1988a) provide support to this concept of two main centers of origin: the Middle East Indian region where mainly oil forms evolved and China where leafy types developed. 3. Indian Forms. The earliest record of B. juncea from India is the excavated seeds from the ancient sites of the Indus Valley civilization (ca. 2300–1500 BCE) in the present-day Sindh and Punjab states of Pakistan. Its inhabitants were mostly meat eaters who used mustard oil for cooking, food preservation, and body massage (Prakash 1961). When migrating Aryans came to northwest India around 1800 BCE, they learned to use B. juncea oil from the original inhabitants. Ancient Sanskrit literature of Aryans is replete with references of Brassica species. Rigveda, Atharvaveda, Chandogya Upanishad, and Brahmanas used several names, such as siddhartha, rajika, baja, sarshap, and svet sharshap (Hinata and Prakash 1984; Watt 1989). However, the use of oil was not very popular with the Aryans (Atharvaveda). In the period around 800–300 BCE, mustard seeds were used for food seasoning and mustard stalks were consumed as vegetables, as mentioned often in early Buddhist and Jain works, such as Jataka and Acharanga Sutra. In the same period, the Buddhist texts Bodhyan Grhya Sutra, Sankhyan Grhya Sutra, and Sankhyan Sautra Sutra mentioned the use of mustard oil for cooking. It is also mentioned in Buddhist canonical literature Dhammapada, Suttanipita, and Samyutta Nikaya. During 300 BCE to 75 CE, known as the Maurya and Sunga period, Kautilya (ca. 300 BCE), the author of famous Arthashastra, mentioned three types of mustard. Mustard seeds were used to add pungency (Kashyap Samhita, ca. 200 BCE). The medical treatise Charak Samhita (3rd century CE) mentioned the use of mustard oil in food preparation and also of mustard stalks as vegetables. The period from 300 to 750 was an era of great prosperity in India. During this time three famous Chinese travelers, Fahi-an, HuenTsang, and Itsing, came to India and provided an excellent record of Indian food habits. Mustard oil was in common use as a frying medium (Itsing in India 671–689; Huen Tsang in India 632–643). Itsing also mentioned two types of mustard—white and black— which were produced in large quantities, and their oil was used for cooking (Takakusu 1896). A Sanskrit book Sukraniti (1100) referred to mustard oil as a common medium for frying and cooking and mustard stalks as winter vegetables. It is evident that in a period of over 3,500 years, mustard came to occupy an important place in the Indian diet. The only seed remains of Brassica species excavated are of B. juncea. In spite of numerous references to brassicas in Sanskrit literature, the

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descriptions are not precise enough to identify the species. Watt (1889) interpreted rajika, Siddhartha, and svet sarshapa as referring to blackand brown-seeded B. juncea and white-seeded Sinapis alba, respectively. Similarly sarshap was used both for brown sarson and B. nigra (Watt 1889). Hinata and Prakash (1984) are of the view that sarshap denoted only brown sarson and siddhartha unmistakably indicated yellow sarson. The present-day Hindi word sarson is clearly a derivative from sarshap. Vedic Aryans used rajika for B. juncea and were unaware of B. nigra and Sinapis alba. All the available evidence indicates that B. juncea was under cultivation in the Indus Valley around 3000 BCE. The art of extracting oil was known to this civilization, with the oil being used for massage. Seeds of B. juncea have been excavated from Chanhu-daro, a site of this civilization (Allchin 1969). With the arrival of Aryans in northwest India around 1500 BCE, its oil was adopted first as a preservative and later for cooking and massage purposes. Subsequently around 1000 BCE, it spread eastward with the stream of migrating people. Its carbonized seeds have been recovered from Damoder River valley site in eastern India, ca. 1000 BCE (Ghosh et al. 2006) and, by ca. 700 CE, it became firmly established as an oil crop in the Indo-Gangetic plain of north India, as evident from the reports of Chinese travelers Huen Tsang (ca. 640) and Itsing (ca. 690). Conflicting views have been expressed regarding the route of its entry into India. Hinata and Prakash (1984) strongly believe that it entered into northwest India from the Middle East, the place of its origin, through Afghanistan between 4500–2300 BCE but not before that date. This is supported by the fact that an ancient Neolithic site of agriculture at Mehrgarh (7000–4700 BCE), in the area of Indus Valley civilization and on a historical route connecting it to west Asia through the Iranian Plateau, did not yield seeds of any Brassica species (Jarrige and Meadow 1980). Natural hybridizations between the parental species with a sympatric distribution in Aghanistan and adjoining region also originated new genetic stocks. However, Indian forms exclusively developed in the direction of oilseed types with lyrate pinnatisect leaves. Several factors, such as introgressive hybridization leading to incorporation of adaptive gene complexes, played vital roles in the further adaptation of Brassica species. A leafy form of B. juncea with large leaves and thick white fleshy stalks is cultivated as a vegetable in the foothills of the Himalayas from Kashmir in the west to Assam and Sikkim in the east. Roxburgh (1832) referred to it as Sinapis rugosa and S. cuneifolia. It is very similar to the Chinese leafy form and might have been brought by Buddhist monks who since ancient times frequently traveled the Himalayas and subsequently established this species in cooler climates.


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4. Chinese Forms. China has a long history of B. juncea cultivation (Wen 1980; Chen 1982). The southwest region exhibits enormous polymorphism in vegetable forms, as described earlier. The history of mustard utilization and domestication dates back 6000–7000 years (Liu 1996). Carbonized mustard seeds stored in a gallipot were excavated from the Banpo site in China in 1963, and carbon dating indicated that the seeds belonged to the New Stone Age, ca. 4800 BCE (Institute of Archaeology Report 1963). The earliest Chinese literature record of brassicas being used as vegetable appeared in Xiaxiaozhen (Ancient Almanac) in the Xia dynasty (ca. 3000 BCE) and Shijin-Gufeng (A Collection of Poems) in the Zhou dynasty literature (1122–247 BCE) (Wu et al. 2009). During the West Han dynasty (206 BCE–24 CE), its use had been recorded as a flavoring agent. Dai in his work Liji (The Book of Rites) referred to a ‘‘sliced jam of fish with mustard.’’ It was a popular crop in the first century CE, as frequent references are available in Tu-Bin-Jin-Cao (Illustated Book of Medicinal Herbs) by Su (10–61 CE). Chia-Ssu-hsieh’s book Ch’i-min-yao-shu of the late 5th or early 6th century described various uses of leaves and seeds (Li 1969). Wang (1576–1588) mentioned a root form in his work Gua Guo Shi (Explanations of Cucurbits and Vegetable Crops). During the Ming dynasty, a famous work by Li (1578) described many forms for their leaves and shoots. Wen (1980) presented a fascinating account of the origin of variations (Fig. 2.11). Based on ancient literature from the fifth century, he proposed that the primitive type was an annual plant with poor leaf growth and was cultivated for its pungent seeds. Subsequently, variants in leaf shape and heading forms were evolved and selected due to human intervention. During the Tang dynasty (607–907), broad-leaved forms were developed and used as greens in temperate and humid south China. A form with deeply dissected leaves adapted to arid regions was developed in northern China. It subsequently produced tillering forms that were more productive, branched early during vegetative growth, and were used for pickles. The Qing dynasty (1644–1911) witnessed the origin of types having leaves with broad, thick midribs and petioles. Later, headed forms with leaves with fleshy midribs and petioles evolved simultaneouly with forms having swollen stems. Fleshy root forms evolved independently from broad-leaved forms, most probably after the 12th century. In a recent study on these vegetable forms comprising accessions of all the diverse variations, Fu et al. (2006) employing RAPDs observed that these various accessions are not a homogenous group. Further, using sequence variations of ITS1, 5.8s rRNA, and ITS2, Qi et al. (2007) proposed their division into two clades, one having accessions close to Nigra lineage and other accessions closer to Rapa/Oleracea lineage.

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Fig. 2.11. Evolution of Chinese mustard crops: (a and b) primitive types; (c) var. oleifera; (d) var. rugosa; (e) var. napiformis; (f and g) var. crispifolia; (h and i) var. capitata, K1 and K2 var. tsa-tsai. (Source: Wen 1982).

These observations are in disagreement with earlier observations from cp, mitochondria, and nuclear DNA RFLPs (Wu et al. 2009). Another observation is that morphological and molecular classifications are incongruent as forms with similar phenotypes do not necessarily have closer relationships. These authors (Fu et al. 2006; Qi et al. 2007, 2008; Wu et al. 2009) also inferred that Chinese forms evolved separately from Indian forms, consistent with the earlier views of the existence of two races. Once some seed stocks were established, introgression from parental species B. nigra and B. rapa further differentiated these types mediated by human selection. There may be two possibilities for the origin of these variations: (1) different B. rapa leafy morphotypes hybridized with B. nigra, as amply demonstrated by obtaining B. juncea forms closely resembling the natural ones following artificial synthesis using various Chinese B. rapa parents (Prakash 1973a), and (2) mutations


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and/or nonhomologous recombination between A and B genome chromosomes. Such morphological variants closely resembling the natural forms were produced following the selfing of F1 interspecific hybrids B. rapa B. nigra (Prakash 1973b). G. Brassica napus This crop came into cultivation only in the 15th century. Two forms are now in cultivation: (1) oilseed rape, widely grown for its edible oil in Canada, Europe, China and Australia, and (2) root-forming swede or rutabaga for food and fodder. Theophrastus and Dioscorides mentioned a root form as bounias, which was wrongly identified as swede in the 16th century (Henslow 1908). Zwinger (1696) described a form steckruebenkohl, which in all probability represents an early form of rutabaga. The rutabagas include two forms, one with white flesh and the other with yellow. The first description of the white one is by Bauhin in Prodromus (1620), and it is named again in his Pinax (1623), where it is called napobrassica. Tournefort (1700) mentioned it as Brassica radice napiformi, or chou-navet, in France while De Candolle (1821) described it as navet jaune, navet de Suede, chou de Laponie, and chou de Suede. Its earliest reference in Germany as Kohlrabi unter der Erden appeared in 1748. Subsequently until the early 20th century, it was recognized in several languages as the underground form of B. oleracea var. gongyloides. The Linnean concept of B. napus was a turnip with an elongated root. Usage of Brassica napus, originally named as Brassica napus rapifera Metzger (Metzger 1833), for rutabaga created enormous confusion and also an erroneous idea that it is an ancient species (Ahokas 2007). It became popular as fodder in Scandinavia and later in the 18th century spread to England (McNaughton and Thow 1972). An important staple food plant earlier, particularly during World War I, it is now mostly grown for forage. It must have originated as a consequece of hybridization between turnip and some forms of B. oleracea in farmers’ fields. Such root-forming morphotypes were obtained from crosses between turnips and different B. oleracea forms (Olsson et al. 1955; Kato et al. 1968). The first reference of oil forms occurs in the Cruydt Boek of Dodonaeus (1554), where slooren is mentioned as being grown for seed oil (Toxopeus 1979). It was most likely to be an early form of winter rape and came into cultivation in the early 17th century (Baur 1944). Its oil, known as raepolie, was first used for illumination and later as an edible oil and for soap making. Spring forms were developed in the late 17th or early 18th century as a selection from winter forms suitable for locales

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where winter is not too severe. At present, in some European countries and in China, winter forms are cultivated. In Canada, northern Europe, and Australia, only spring forms are grown. The oil form of B. napus does not occur in the wild. Sinskaia (1928) and Schiemann (1932) proposed the southwest Mediterranean region as the place of origin where the constituent parents overlap in natural distribution. However, this overlapping probably did not exist (Tsunoda 1980). As Go´mez-Campo and Prakash (1999) observed, wild B. rapa is very poorly represented in Spain. Thus, it is difficult to envisage its arrival at Atlantic maritime cliffs where B. oleracea grows wild. These authors believed that it originated outside the Mediterranean region. Several researchers suggested its multiple origins in an agricultural environment (Olsson 1960b; Prakash and Hinata 1980). This has been substantiated by investigations based on organelle and nuclear RFLP analyses (Song and Osborn 1992). A study by Song et al. (1988a) identified B. rapa ‘‘spring broccoli raab’’ as the closest extant relative of maternal genome of B. napus.However, as stated earlier, the maternal donor of B. napus has not been unequivocably established. The plastid genome of B. napus shows considerable variability, suggesting its polyphyletic origin (Allender and King 2010). An introgression of genetic information from B. rapa, one of its constituent parents, has also played a major role in developing an array of cultivars (Aru´s et al. 1987; Aguinagalde 1988). Large-scale natural hybridizations between both the constituent parents have been recorded in fields; such hybridizations have contributed to the increase in B. napus variability (Bing et al. 1996). One of the most striking aspects of oilseed B. napus is that in a brief span of 400 years since its origin, its distribution, cultivation and production has far exceeded other oilseed brassicas. This change started in early 1980s. Busch et al. (1994) have documented an account of its introduction into Canada and development of canola-quality rapeseed. Spring forms of B. napus were introduced before World War II; the oil was first used as a marine lubricant during the war. Later in the 1950s, attention was paid to use it for human consumption. However, the presence of erucic acid (cis-13-docosenoic acid, 22:1, n-9) in oil and glucosinolates in meal prevented its adoption. Low–erucic acid cultivars were soon developed by Downey and Harvey (1963), followed by the world’s first zero-erucic and low-glucosinolate cultivar ‘Tower’ (Bell 1982). During the last 25 years, many high-yielding canola-quality conventional and hybrid cultivars have been bred and released in Canada, Australia, Europe, and China. The term canola is a special one and was coined and defined as seed, oil, and meal that contain 0.3% w/w WIN6 developed to second instar compared to 93% on control uninfected leaves. Goggin et al. (2006) showed that transformation of the susceptible ‘Moneymaker’ tomato with the Mi-1.2 gene (NB-LRR class) resulted in resistance to nematodes and aphids. The Mi-1.2 locus confers resistance against root-knot nematodes (Meloidogyne spp.), the potato aphid (Macrosiphum euphorbiae), and the sweet potato whitefly (Bemisia tabaci). A proteinaceous aspartic proteinase inhibitor, designated as tomato chymotrypsin inhibitor 21 (TCI21), was also expressed in tomato and found to increase mortality and delay growth of Egyptian cotton worm (Spodoptera littoralis) (Lison et al. 2006). More recently, Chen et al. (2007) indicated that the root-knot nematode resistance gene CaMi from hot pepper (Capsicum annuum) confers inheritable host plant resistance to this nematode in transgenic tomato. 4. Fungal Resistance. Thomzik et al. (1997) transformed tomato with two stilbene synthase genes from grapevine (Vitis vinifera). They characterized the transgenic plants for stable integration and expression of the transgene and host plant resistance in the transgenic tomato to downy mildew (Phytophthora infestans). Upon fungal inoculation, transgenic plants accumulated the phytoalexin transresveratrol, the product of stilbene synthase, and exhibited increased resistance to P. infestans. Inoculation of transgenic tomato with Botrytis cinerea (botrytis blight or gray mold) and Alternaria solani (early blight of tomatoes) also caused accumulation of resveratrol, but plants did not show significant resistance to these fungi. Tabaeizadeh et al. (1999) observed the effect of constitutive expresion of an acidic endochitinase gene, pcht28, from Lycopersicon chilense for resistance against Verticillium dahliae (verticillium wilt). The R1 plants were tested in the greenhouse for tolerance to V. dahliae race 1, 2 whereas R2 plants tested against race 2 showed a significantly higher level of resistance to the fungi than the nontransgenic plants. The resistance was confirmed by foliar disease symptoms and vascular discoloration index. Likewise, Kesarwani et al. (2000) were able to overexpress oxalate decarboxylase

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from Collybia velutipes in transgenic tobacco and tomato, both of which developed resistance to fungal infection. These transgenic tobacco and tomato plants showed remarkable resistance to phytopathogenic fungus Sclerotinia sclerotiorum (sclerotinia stem rot or white mold), which utilizes oxalic acid during infestation. Lin et al. (2004) introduced the Arabidopsis NPR1 (nonexpresser of PR genes) gene into a tomato cultivar, which possesses heat tolerance and resistance to Tomato mosaic virus (ToMV). The transgenic lines expressing NPR1 showed normal morphology and horticultural traits for at least four generations. Host plant resistance screening against eight important tropical pathogens revealed that, in addition to the innate ToMV resistance, the tested transgenic lines conferred significant levels of enhanced resistance to bacterial wilt (Ralstonia solanacearum) and fusarium wilt (Fusarium oxysporum), and moderate degree of enhanced resistance to gray leaf spot (Stemphylium botryosum f. sp. lycopersici) and bacterial spot (Xanthomonas campestris pv. vesicatoria). Transgenic lines that accumulated higher levels of NPR1 proteins exhibited higher levels and a broader spectrum of enhanced resistance to these pathogens, and their enhanced host plant resistance was stably inherited. The spectrum and degree of these NPR1-transgenic lines are more significant than in the transgenic tomatoes bred to date. Hence, these transgenic lines may be further used as tomato stocks, aiming at building up host plant resistance to a broader spectrum of pathogens by transferring these characteristics into tomato cultivars with good agronomic and organoleptic characteristics. The Arabidopsis thionin (Thi2.1) gene was also used by Chan et al. (2005) to genetically engineer enhanced resistance to various pathogens in tomato. A construct was created in which the fruit-inactive promoter RB7 was used to control the expression of the Thi2.1 gene. In transgenic lines containing RB7/Thi2.1, constitutive Thi2.1 expression was detected in roots and a little in leaves, but not in fruits. Host plant resistance assays revealed that the transgenic lines tested showed enhanced resistance to bacterial wilt and fusarium wilt. It was found that progression of bacterial wilt in transgenic lines was delayed by a systemic suppression of bacterial multiplication. Schaefer et al. (2005) introduced genes coding for an iris ribosomal-inactivating protein (I-RIP), a maize b-glucanase (M-GLU), and a Mirabilis jalapa antimicrobial peptide (Mj-AMP1) into tomato. Selected transgenic lines were inoculated with a suspension containing 2–3 104 conidial spores/ml of the fungal pathogen Alternaria solani. Two transgenic lines carrying either M-GLU or Mj-AMP1 transgenes had enhanced resistance to Alternaria solani vis- a-vis the parental control. None of the four lines carrying the I-RIP transgene showed resistance to this pathogen.

4. TRANSGENIC VEGETABLE CROPS

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5. Bacterial Resistance. The tomato resistance gene Pto encodes a serine/threonine protein kinase that is postulated to be activated by a physical interaction with the AvrPto protein. In this regard, Tang et al. (1999) showed that transgenic tomato plants exhibited significant host plant resistance to Pseudomonas syringae (bacterial speck) without avrPto and reduced bacterial growth compared to nontransgenic lines. These transgenic plants also showed more resistance to Xanthomonas campestris pv. vesicatoria (bacterial spot) and Cladosporium fulvum (leaf mold of tomato). Similarly, Lin et al. (2004) transformed tomato with the Arabidopsis NPR1 gene. They found that transgenic lines had significant levels of enhanced resistance to bacterial wilt and moderate resistance to bacterial spot (BS). Seong et al. (2007) isolated a pathogenesis-induced factor (CaPIF1) from pepper leaves after infection with the soybean pathogen Xanthomonas axonopodis pv. glycines 8ra. The overexpression of CaPIF1, which encodes a Cys-2/His-2 zinc finger transcription factor, resulted in major transcriptional modulation without exhibiting any visual morphological abnormality. The transgenic plants exhibited tolerance to cold stress and to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, whose tolerance was correlated with the expression levels of CaPIF1. The CaPIF1, a plant-specific unique Cys2/His-2 transcription factor, seems to regulate directly and indirectly gene expression, thereby leading to enhanced resistance to biotic and abiotic stresses. Huang et al. (2007) demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic tomato plants can enhance resistance to bacterial pathogens that infect leaf tissue. In their study, PFLP was applied to protect tomato cv. Cherry Cln1558a from the root-infecting pathogen Ralstonia solanacearum. Independent R. solanacearum resistant T1 lines were selected and bred to produce homozygous T2 generations. Selected T2 transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to R. solanacearum. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by Erwinia carotovora subsp. carotovora, but it was less apparent in transgenic line 26- 2-1a. The cationic lytic peptide cecropin B (CB), isolated from giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. Jan et al. (2009) investigated the effect of chemically synthesized CB on plant pathogens. The S50 (peptide concentration causing 50% survival rate of a pathogenic bacterium) of CB against two major pathogens of tomato R. solanacearum and X. campestris pv. vesicatoria were

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529.6 mg/ml and 0.29 mg/ml, respectively. The CB gene was then fused to the secretory signal peptide sequence (sp.) from the barley a-amylase gene, and the new construct, pBI121-spCB, was used for the transformations of tomato. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato (0.05 mg in 50 mg leaf) are far smaller than the S50 determined in vitro. CB transgenic tomato could therefore be a new mode of bioprotection against these two tomato pathogens. 6. Viral Resistance. Substantial efforts also have been made to engineer virus resistance in tomato plants. Whitefly-transmitted geminiviruses are widely distributed in tropical and subtropical regions around the world, causing yield losses in tomato and in other important vegetable crops, such as beans, cassava, and pepper. Motoyoshi and Ugaki (1993) reported the transformation (with a chimeric Tobacco mosaic virus [TMV] coat protein gene under the control of the CaMV 35S promoter) of an F1 hybrid between tomato and its wild relative Solanum peruvianum. Transgenic line 8804-150 accumulated 2.5 mg coat protein per gram fresh weight in fully developed fresh leaves and exhibited the strongest resistance to ToMV among the plants examined. The transgenic plants did not show any morphologic or physiologic differences vis-a-vis their nontransgenic control plants. An interspecific F1 tomato hybrid derived from Solanum pennellii that was sensitive to the Tomato yellow leaf curl virus (TYLCV) was also successfully genetically transformed with a TYLCV transgene-encoding capsid protein (V1). The R1 plants inoculated with TYLCV using whiteflies showed delayed disease symptoms and increased recovery from the disease (Kunik et al. 1994). McGarvey et al. (1994) indicated that transgenic R1 tomato plants expressing CMV satellite RNA fused to the GUS gene showed mild disease symptoms in the first two weeks after inoculation with virions of RNA preparations of CMV or Tomato aspermy virus (TAV), which was followed by a decrease in symptoms. Field trials of transgenic tomato plants expressing an ameliorative satellite RNA of CMV exhibited mild or no CMV symptoms and low viral titers relative to nontransformed plants. When infected with CMV, the transgenic lines showed 40% to 84% greater total marketable yield than parent lines. A significant negative correlation between satellite RNA levels and disease severity was found in transgenic lines (Stommel et al. 1998). High levels of resistance to Tomato spotted wilt virus (TSWV) were


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obtained in an inbred tomato line that was transformed with a DNA construct comprising the TSWV nucleoprotein (NP) gene (Hann et al. 1996). The high levels of resistance were maintained in hybrids derived from the parental transgenic tomato lines. Moreover, the transgenic-derived hybrids remained completely free of TSWV symptoms in field trials under high virus pressure. Xue et al. (1994) also successfully developed transgenic tomato plants expressing a high level of resistance to Cucumber mosaic virus (CMV) strains of subgroup I and subgroup II. These transgenic tomatoes that are resistant to isolates from both subgroups of CMV have practical significance for controlling this serious virus. Transgenic tomato plants exhibiting a broad commercial resistance to CMV infection have been developed by expressing coat protein (CP) genes from the CMV-D strain and two Italian isolates CMV-22 of subgroup I and CMV-PG of subgroup II (Kaniewski et al. 1999). Transgenic plants generated using CP from any of the strains showed broad resistance against CMV strains from both subgroups I and II. These transgenic lines were field-tested to assess the level of resistance and agronomic performance (Tomassoli et al. 1999). The target virus spread naturally by the indigenous aphid populations in multilocation trials in Italy. These trials showed, however, that CMV resistance of the transgenic tomatoes was less effective in the field than what was observed in growth chamber experiments. Transgenic tomato resistant to CMV—through expression of the CP gene—have been released in China (Chen et al. 2003), though limited information is available on their adoption rate. Nunome et al. (2002) also transformed tomato plants with a truncate replicase gene encoded by RNA 2 of CMV strain GT, subgroup II. The truncate replicase gene does not retain a C-terminal region of the gene that contains the GDD amino acid motif and the NTP binding motif. These motifs are considered to correspond to active or recognition domains of RNA-dependent RNA polymerase. Upon transformation with Agrobacterium tumefaciens, 137 individual transgenic lines were obtained. Each transgenic line was evaluated for resistance to CMV strain Ta-8. About 10% of the transgenic lines were highly resistant, and the remaining 90% showed a moderate resistance or were susceptible. The 15 lines were selected as resistant lines. Chenopodium amaranticolor was used to analyze the multiplication of CMV in the symptomless plants. Among the selected lines, three lines did not appear to show any multiplication of CMV in both inoculated and noninoculated leaves. The T1 progeny of the selected lines harbored the transgene according to transgene amplification by PCR and the kanamycin resistance assay. Several resistant lines of the

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T1 generation were resistant to viral inoculation. These resistant lines become suitable breeding lines for resistance to CMV. There is a project on CMV-resistant transgenic tomato in Indonesia and the Philippines (ABSPII 2009) because to date conventional breeding has not been able to develop resistant germplasm. Resource-poor tomato growers in the Philippines will gain significantly if transgenic tomato with resistance to CMV and TYLCV can become available to and adopted by them (Mamaril 2009). In Indonesia, a study has shown that the adoption of a transgenic tomato with resistant to CMV and ToLCV (Tomato leaf curl virus) will have a significant potential economic impact that would increase economic welfare (Ameriana 2009). Transgenic tomato with multiple virus resistance will also reduce the use of insecticides, thereby significantly contributing to maintaining environmental quality and minimizing pesticide residues in tomato products (Ameriana, 2009). Brunetti et al. (1997) had shown that high expression of a truncated version of the Cl gene of TYLCV, which encodes the first 210 amino acids of the multifunctional rep protein, confers resistance to TYLCV in transgenic tomato plants. N genes possess a putative nucleotide binding site and leucine-rich repeats and confer a gene-for-gene resistance against TMV and most other members of the tobamovirus family. Whitham et al. (1996) also genetically transformed tomato with the N gene from tobacco. Tomato transgenics expressing the N gene exhibit a hypersensitive response and effectively restricts TMV to the sites of inoculation, as in tobacco. Transgenic tomato plants expressing the CP gene of Physalis mottle tymovirus (PhMV) showed delay in symptom development indicating partial resistance to the virus (Vidya et al. 2000). 7. Plant Stress. Plants have developed responses to environmental extremes such as drought, salinity, extreme temperatures and hypoxia, which in turn may impact their growth, productivity, and quality. Water availability is expected to be highly sensitive to climate change, and severe water stress conditions will affect crop productivity, particularly that of vegetables (De la Penã and Hughes 2007). Water significantly influences fruit yield and quality of tomato. Drought therefore drastically reduces tomato productivity, and the magnitude of the yield loss will depend on its timing, intensity, and duration. About one-fifth of irrigated agricultural land is impacted by salinity, and it is rapidly expanding due to the increased use of underground water. Tomato, like most other crop plants, is sensitive to salinity (Foolad 2004, 2007). Our limited understanding of the molecular basis regulating salt tolerance in plants has hampered progress in developing


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salt-tolerant crops. Nonetheless, several genes with a potential role in imparting tolerance to salinity stress have been identified. Plants also vary in their responses to temperature extremes. Nonfreezing low temperatures also cause chilling injury while high temperatures impair crop productivity and quality in tomato. Cold damage in tomato plants occurs under temperatures below 6 C. Freezing tolerance of tomato occurs down to to 2 to 3 C whereas extended storage at temperatures below 12 C results in severe chilling injury. Temperatures over 30 C inhibit the synthesis of lycopene and affect the quality of the fruit (Almeida 2006). Furthermore, anaerobiosis occurs during waterlogging, flooding, poor drainage, or even irrigation and causes oxygen deficiency, anoxia with bare minimum oxygen availability, and hypoxia with only some oxygen availability in the rooting zone. Tomato and other plants respond to anaerobiosis in a variety of ways. There has been research on tomato responses to all these abiotic stresses. Plants show an elaborate signaling network that perceives signals from abiotic stresses and modulates the expression of select genes. Significant research advances have been made in understanding these pathways using Arabidopsis thaliana as a model system. Table 4.3 provides some highlights regarding progress on developing tolerance to abiotic stresses in tomato (e.g., to drought, salt, extreme temperatures, and hypoxia). Breeding for tolerance to drought and salt stresses could increase the productivity of tomato in many regions of the world and would help regain more arable land (Table 4.3). For example, the expression of AVP1—a vacuolar H þ pyrophosphatase from A. thaliana—in transgenic tomato led to enhanced performance under soil water deficit due to a strong and large root system allowing better use of limiting water (Park et al. 2005). Hsieh et al. (2002a,b) used CBF1 (C-repeat/dehydrationresponsive element-binding factor 1)/DREB1 genes driven by a strong constitutive 35S Cauliflower mosaic virus promoter to successfully engineer tolerance to chilling, drought, and salt stress in tomato plants showing dwarfism and low fruit and seed set. When the same gene (CBF1) was expressed using an abcisic acid (ABA)/stress-inducible promoter from barley HAV22 gene, the transgenic tomato plants showed enhanced tolerance to chilling, water deficit, and salt stress as compared to untransformed plants (Lee et al. 2003). The use of an inducible promoter eliminated the deleterious effects of the ectopic expression of CBF1 on plant growth and yield. Park et al. (2005) constitutively overexpressed the vacuolar H þ -pyrophosphatase in commercial tomato cultivars. The resulting transgenic plants exhibited greater pyrophosphate-driven cation transport into root vacuolar fractions, increased root

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Arabidopsis

Arabidopsis

Yeast

CBF1

Hþpyrophosphatase

Trehalose-6phosphate synthase

NHX1, vacuolar Na þ /H þ antiporter

Arabidopsis

Yeast

Arabidopsis

Drought CBF1

Salt HAL1 (K þ transport regulation) gene

Gene source

:NHX1 overexpressed

CaMV 35S:HAL1

CaMV 35S:TPS1

CaMV35S:AVP1D

ABRC1:CBF1

CaMV 35S:CBF1

Promoter:gene

Tolerance to high levels of salt; maintains high levels of K þ and low levels of intracellular Na þ Salt tolerance up to 200 mM NaCl; high Na þ concentrations in leaves but very low levels in fruits

Tolerance to cold, drought, and salt stress but dwarf phenotype and reduction in fruit set and seed number per fruit Tolerance to chilling, drought, and salt stress with normal growth and yield Greater pyrophosphate-driven cation transport into root vacuolar fractions, increased root biomass, and enhanced survivability during waterdeficit stress Improved tolerance to drought, salt, and oxidative stress

Phenotype

GM tomato for tolerance to abiotic stresses such as drought, salt, extreme temperatures, and hypoxia.

Gene product

Table 4.3.

Zhang and Blumwald 2001

Gisbert et al. 2000

Cortina and CulianezMacia 2005

Park et al. 2005

Lee et al. 2003

Hsieh et al. 2002a,b

References

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Hypoxia ACC deaminase

Cys-2/His-2 zinc finger protein-TF

sHSP (mitochondrial) CAPX (cDNA)

Extreme temperatures Heat shock factor, hsfA1b Choline oxidase

Betaine aldehyde dehydrogenase

CaMV 35S:MT-sHSP :cAPX

Tomato

Bacteria

CAMV, rolD, and PRB-1b: ACC deaminase

CaMV 35S:CaPIF1 overexpression

CaMV 35S:CodA

Arthrobacter globiformis I Tomato

Pepper (Capsicum annuum)

CaMV 35S:AtHsfA1b

CaMV 35S:BADH (2 genes)

Arabidopsis

Atriplex hortensis

Tolerance to flooding stress, rolD promoter protects to greater extent

Enhanced resistance to heat (40 C) and UV-B stress in tomato Zhongshu No. 5 compared to wild-type plants Tolerance to cold stress and to the bacterial pathogen P. syringae

Heat shock–induced chilling tolerance Improved chilling and oxidative stress tolerance Thermotolerance

Enhanced tolerance to salt stress

Grichko and Glick, 2001

Seong et al. 2007

Wang et al. 2006

Nautiyal et al. 2005

Park et al. 2004

Li et al., 2003

Jia et al. 2002

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biomass, and enhanced survivability under soil water deficit stress. Transgenic tomato plants transformed with the yeast trehalose-6-phosphate synthase (TPS1) gene exhibited improved tolerance to drought, salt, and oxidative stress, indicating a great potential of this gene to impart stress tolerance to plants (Cortina and Culianez-Macia 2005). Salt-tolerant transgenic tomatoes also ensued by increasing expression of the A. thaliana tonoplast membrane Na þ /H þ antiporter, AtNHX1, under a strong constitutive promoter (Zhang and Blumwald 2001). They were able to grow, flower, and set fruit at 200 mM NaCl. Although their leaves accumulated high concentrations of Na, the seeds showed low concentrations of this element. The tomato hairy root lines transformed with BADH-1 gene using root-inducing plasmid (pRi) plasmid accumulated betaine (Jia et al. 2003). Constitutive expression of Atriplex hortensis BADH gene in tomato led to enhanced tolerance to salt stress. Overexpression of yeast HAL1 in transgenic tomatoes, which is involved in the regulation of K þ transport, also imparted tolerance to high levels of salt (Gisbert et al. 2000). Similar to yeast, these transgenic tomatoes were able to maintain higher levels of K þ and decreased levels of intracellular Na þ than the control. Mishra et al. (2002) showed that heat stress transcription factor (HsfA1) underexpressing plants as well as their fruits exhibited extreme sensitivity to elevated temperatures. Cosilencing of HsfA1 by its transgenes resulted in reduced heat stress–induced accumulation of chaperones and heat shock factors, suggesting a unique function of HsfA1 as a master regulator for inducing thermotolerance. The effect of heat shock factor on chilling tolerance has also been studied by expressing AtHsA1b and gusA under CaMV 35S promoter. The transformed tomato showed more accumulation of heat shock–induced gene transcripts and enzymes, including a twofold increase in the specific activity of soluble isoforms of ascorbate peroxidase (Li et al. 2003). Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide, and hydroxyl radicals, are byproducts of biological redox reactions. ROS can denature enzymes and damage important cellular components. Plants develop antioxidant enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX), to scavenge ROS and detoxify them. Wang et al. (2006) studied the effect of increased cytosolic ascorbate peroxidase (cAPX) on heat and UV-B stress tolerance using transformed tomato cv. ‘Zhongshu No. 5’ plants. This research demonstrated in laboratory or field tests the potential to enhance tolerance to heat, UV-B, and sunscald stress by genetic engineering. Overexpression of cAPX in transgenic tomato enhanced resistance to heat (40 C) and UV-B stress compared to wildtype plants. When leaf disks were placed at 40 C for 13 hours, the


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electrolyte leakage of disks from wild-type was 93%, whereas two tested transgenic lines (A9, A16) exhibited 24% and 52% leakage respectively. When fruits of wild-type and transgenic plants were exposed to UV-B (2.5mW cm-2) for 5 days, the extent of browning was 95%, 33%, and 37%, respectively. In field tests, the detached fruits from field-grown transgenic plants showed more resistance to exposure to direct sunlight than fruits from wild-type plants. APX activity in leaves of cAPX transgenic plants was several times higher than in leaves of wild-type plants when exposed to heat, UV-B, and drought stresses. Nautiyal et al. (2005) developed transgenic tomato lines overexpressing tomato MT-sHSP and showed that vegetative tissues of T0 and T1 lines exhibited enhanced thermotolerance, whereas Zhaoa et al. (2007) tested the function of endoplasmic reticulum (ER)–located small heat shock proteins (ER-sHSPs) in ER stress by overexpressing LeHSP21.5 in tomato plants. ER stress is basically an imbalance between the cellular demand for protein synthesis and the capacity of the ER to promote protein maturation and transport, which leads to an accumulation of unfolded or malfolded proteins in the ER lumen. Gene expression in the transgenic lines greatly attenuated the lethal effect of tunicamycin (a potent inducer of ER stress) on tomato seedlings. Moreover, tunicamycin treatment led to lower levels of the chaperone-binding protein, protein disulfide isomerase, and the chaperone calnexin transcripts in transgenic tomato plants than in the nontransgenic tomato plants. These results suggest that the HSP LeHSP21.5 can alleviate the tunicamycin-induced ER stress by promoting proper protein folding. Transformation of ‘Moneymaker’ tomato with a chloroplast targeted codA gene of Arthrobacter globiformis encodes choline oxidase that catalyzes the conversion of choline to glycine betaine and improves chilling and oxidative stress tolerance of transgenic plants (Park et al. 2004). These authors saw that transgenic tomato plants accumulated up to 1.2 mM of glycine betaine per gram of fresh weight with the chloroplasts containing up to 86% of total leaf glycine betaine. These transgenic tomato lines produced 10% to 30% more fruit compared to untransformed plants. The overexpression of CaPIF1 enhanced tolerance to cold stress, which was correlated with CaPIF1 expression levels in transgenic plants (Seong et al. 2007). Grichko and Glick (2001) showed that a bacterial ACC deaminase under the transcriptional control of double CaMV 35S, the rolD promoter from Agrobacterium rhizogenes (root-specific expression), produced tomato plants with increased tolerance to flooding stress and lesser deleterious effects of root hypoxia on plant growth than the nontransformed plants.

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8. Vaccines. Plant delivery of oral vaccines has attracted much attention because this strategy offers several advantages over vaccine delivery by injection (Langridge 2000; Pascual 2007; Sunil Kumar et al. 2007). Oral vaccines also offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs worldwide. Tomato has been tested for expression of vaccines that can address human health issues of the developing world. Transgenic tomato plants potentially can bring several positive effects and improve human health. McGarvey et al. (1994) engineered tomato plants of cv. ‘UC82b’ to express a gene encoding a glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the environmental risk assessment strain of rabies virus. The G-protein was expressed in leaves and fruit of the transgenic plants, and it was found localized in Golgi bodies, vesicles, plasmalemma, and cell walls of vascular parenchyma cells. Ma et al. (2003) overexpressed hepatitis E virus (HEV) open reading frame 2 partial gene in tomato plants, to investigate its expression in transformants, the immunoactivity of expressed products, and explore the feasibility of developing a new type of plant-derived HEV oral vaccine. The recombinant protein was produced at 61.22 ng g1 fresh weight in fruits and 6.37–47.9 ng g1 fresh weight in the leaves of the transformants. It was concluded that the HEV-E2 gene was correctly expressed in transgenic tomatoes and that the recombinant antigen derived had normal immunoactivity. These transgenic tomato plants are a valuable tool for the development of edible oral vaccines. Chen et al. (2006) developed an effective antiviral agent against enterovirus 71 (EV71), that causes seasonal epidemics of hand, foot, and mouth disease associated with fatal neurological complications in young children, by transforming the gene for VP1 protein—a previously defined epitope and also a coat protein of EV71—in tomato plant. VP1 protein was first fused with sorting signals to enable it to be retained in the endoplasmic reticulum of tomato plant, and its expression level increased to 27 mg g1 in fresh tomato fruit. Transgenic tomato fruit expressing VP1 protein was then used as an oral vaccine, and the development of VP1specific fecal IgA and serum IgG were observed in BALB/c mice. Additionally, serum from mice fed transgenic tomato could neutralize the infection of EV71 to rhabdomyosarcoma cells, indicating that tomato fruit expressing VP1 was successful in orally immunizing mice. Moreover, the proliferation of spleen cells from orally immunized mice was stimulated by VP1 protein and provided further evidence of both humoral and cellular immunity. Results of this study not only demonstrated the feasibility of using transgenic tomato as an oral vaccine to generate


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protective immunity in mice against EV71 but also the probability of enterovirus vaccine development. The Gram-negative bacterium Yersinia pestis causes plague, which has affected human health since ancient times. It is still endemic in Africa, Asia, and the American continent. There is the urgent need for a safe and cheap vaccine due to the increasing reports of the incidence of antibiotic-resistant strains and concern with the use of Y. pestis as an agent of biological warfare. Out of all the Y. pestis antigens tested, only F1 and V induce a good protective immune response against a challenge with the bacterium (Benner et al. 1999). Alvarez et al. (2006) reported the expression in tomato of the Y. pestis F1-V antigen fusion protein. The immunogenicity of the F1-V transgenic tomatoes was confirmed in mice that were injected subcutaneously with bacterially produced F1-V fusion protein and boosted orally with transgenic tomato fruit. Expression of the plague antigens in the tomato fruit allowed producing an oral vaccine candidate without protein purification and with minimal processing technology, offering a good system for a largescale vaccination programs in developing countries. The future of edible plant-based vaccines through transgenic approaches will depend on producing them safely on sufficient amounts. B. Eggplant The eggplant, known as aubergine in Europe and brinjal in South Asia, is a popular vegetable crop grown in many countries throughout the subtropics, tropics, and Mediterranean area, since it requires a relatively long season of warm weather to give good yields. It was produced on 2 million ha in 2007 (Choudhary and Gaur 2009), with a global harvest of 32 million t. Asia contributed 91.5% of the world production (FAO 2009). India, the second largest producer in the world after China, produces 8 to 9 million t, a quarter of global production. A total of 1.4 million small marginal and resource-poor growers grow eggplant in all eight vegetable growing zones of India. The crop is often considered a poor person’s vegetable and is cultivated mainly on small family farms. It is an important source of nutrition and cash income for many resource-poor growers, since they transplant it from nurseries at different times of the year to produce two or three crops, each of 150 to 180 days’ duration. Growers start harvesting fruits at about 60 days after planting and continue the harvest for 90 to 120 days, thereby providing a steady supply of food for the family and a stable income. Eggplant was one of the first vegetable crops adopted by growers in India to be used as hybrids. Hybrids now occupy more than 50% of the eggplant-planted area.

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1. Insect Resistance. Eggplant is attacked by a number of insects including thrips, cotton leafhopper, jassids, and aphids. The most damaging pest is the eggplant fruit and shoot borer (FSB), Leucinodes orbonalis (Palada et al. 2006). Infestation is caused by adults migrating from neighboring fields, from eggplant seedlings, or from previously grown eggplants in the same planting area. Damage from L. orbonalis starts at the nursery stage and continues after crop transplanting until harvest. Losses have been estimated to be between 54% and 70% in India and Bangladesh and up to 50% in the Philippines (Choudhary and Gaur 2009). Recommended insect pest management practices include the prompt manual removal of wilted shoots, trapping male moths using pheromones to prevent mating, ensuring regular crop rotation, and using nylon net barriers. These methods, however, are not widely adopted by growers because of time and resource constraints or lack of awareness. There are no known eggplant cultivars resistant to the FSB, so the use of insecticide sprays continues to be the most common control method used by growers. The borers are vulnerable to sprays only for a few hours before they bore into the plant. Therefore, growers in India spray insecticides as many as 40 to 80 times over a 7-month cropping season (AVRDC 2001; Choudhary and Gaur 2009). Growers may even spray every other day, particularly during the fruiting stage, which contributes to the presence of pesticide residues. But despite the application of many insecticides, the eggplant fruits sold in the Indian market are still of inferior quality, infested with larvae from the borer (Choudhary and Gaur 2009). A survey of pesticide use in Central Luzon in the Philippines indicates that growers there spray up to 56 times with insecticides during a crop season to protect their eggplant crops against the borer (Palada et al. 2006). The decision of growers to spray is influenced more by subjective assessment of visual presence of the insect rather than methodology based on threshold levels. This reliance on visual assessment leads to gross overspraying with insecticides, higher insecticide residues, and unnecessary increase in growers’ exposure to insecticides. On average, 4.6 kg of active ingredient of insecticide per hectare per season is applied on eggplant (Choudhary and Gaur 2009). Such pesticide use, besides being detrimental to the environment and human health, also increases the cost of production, making this humble vegetable expensive for poor consumers. In Asia, chemical spraying for this insect accounts for 24% of the total cost of production (Choudhary and Gaur 2009). Intensive use of improperly applied insecticides raises serious concerns for environmental and human health. A study conducted in the Jessore district of Bangladesh found that 98% of farmers felt sickness and more than 3%


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were hospitalized due to various problems related to pesticide use (AVRDC 2003). FSB-resistant Bt eggplant was genetically engineered by Mahyco under a collaborative agreement with Monsanto , and the first Bt transgenic eggplant with resistance to FSB was produced in 2000. This GM eggplant incorporates the cry1Ac gene expressing insecticidal protein to confer resistance against FSB. This Bt-eggplant was effective against FSB, with 98% insect mortality in Bt-eggplant shoots and 100% in fruits compared to less than 30% mortality in non-Bt counterparts (ISAAA 2008). Similar to Bt-sweet corn, it is expected that Bt-eggplant cultivars will reduce pesticide applications and contribute to poverty reduction and overcoming food insecurity. Krishna and Qaim (2008) indicated that simulations using farm-survey data suggest the aggregate economic surplus gains of Bt-eggplant hybrids could be around U.S. $ 108 million per year in India. Eggplant consumers will capture a large share of these gains, but farmers and the seed company will also benefit. By sharing this technology with the public sector, Bt openpollinated eggplant cultivars eventually may become available (Krishna and Qaim 2007; Kolady and Lesser 2008), thereby making this technology more accessible for resource-poor farmers. The genetic engineering approach for eggplant improvement seems promising since it might also incorporate resistance or tolerance to other insects, nematodes, diseases, and abiotic stress as well as incorporating parthenocarpy. Recently, a gene-encoding oryzacystatin was introduced in eggplant, and the effect on Myzus persicae and Macrosiphum euphorbiae was examined (Ribeiro et al. 2006). The transgenic eggplant reduced the net reproductive rate, the instantaneous rate of population increase, and the finite rate of population increase of both aphid species compared with a control eggplant line. Age-specific mortality rates of M. persicae and M. euphorbiae were higher on transgenic plants. These results indicate that expression of oryzacystatin in eggplant has a negative impact on population growth and mortality rates of M. persicae and M. euphorbiae and could be a source of plant resistance for pest management of these aphids. Expression of Mi-1 gene isolated from tomato in eggplant cv. HP 83 conferred resistance to root knot nematode Meloidogyne incognita (Goggin et al. 2006). 2. Fungal Resistance. Attempts have also been made to engineer eggplant for fungal resistance. Overexpression of a yeast dD-9 desaturase gene in eggplant has resulted in higher concentrations of 16 : 1, 18 : 1, and 16 : 3 fatty acids, and such transgenics exhibited increased resistance to Verticillium wilt (Xing and Chin 2000). Transgenic plants challenged by

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Verticillium could also result in a marked increase in the content of 16:1 and 16:3 fatty acids. They have shown that cis-D9 16 : 1 fatty acid was inhibitory to Verticillium growth. Transgenic eggplants resistant to Verticillum and Fusarium wilts by overexpression of pathogenesisrelated genes, such as glucanase, chitinase, and thaumatin (singly and in combination), have been also produced (Rajam and Kumar 2007). 3. Plant Stress. Tolerance against osmotic stress induced by salt, drought, and chilling stress was achieved in eggplants expressing the bacterial mannitol-1-phosphodehydrogenase (mtlD) gene, which is involved in mannitol synthesis (Prabhavathi et al. 2002). Interestingly, these transgenic plants also showed enhanced resistance to fungal wilts caused by V. dahliae and F. oxysporum. Further, various transgenic eggplants overexpressing different genes (namely, arginine decarboxylase, ornithine decarboxylase, S-adenosylmethione decarboxylase, and spermidine synthase) encoding enzymes in the polyamine metabolic pathway have also been generated. These transgenic plants showed increased tolerance to multiple abiotic (salinity, drought, extreme temperature, and heavy metals) as well as biotic (fungal pathogens) stresses. More recently Prabhavati and Rajam (2007) showed that such transgenics expressing the mtlD gene with mannitol accumulation exhibit increased host plant resistance against three fungal wilts caused by F. oxysporum, V. dahliae, and Rhizoctonia solani under both in vitro and in vivo growth conditions. Mannitol levels could not be detected in the wild-type plants, but the presence of mannitol in the transgenics could be positively correlated with the disease resistance. These results and the previous data suggest that the mtlD gene can be used for engineering crop plants for both biotic and abiotic stress tolerance. 4. Parthenocarpy. Transgenic eggplants with parthenocarpic fruits were also developed by manipulating the auxin levels during fruit development through the introduction of iaaM gene from Pseudomonas syringae pv. savastanoi under the control of the ovule-specific promoter DefH9 from Antirhinum majus (Rotino et al. 1997). These transgenic eggaplants produced seedless parthenocarpic fruit in the absence of pollination without the external application of plant hormones, even at low temperatures, which normally prohibit fruit production in untransformed lines. Furthermore, these transgenic eggplants have exhibited significantly higher winter yields than the untransformed plants and a commercial hybrid under an unheated glasshouse trial (Donzella et al. 2000). Trials in open field for summer production and greenhouse for


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early spring production confirmed that transgenic parthenocarpic eggplant F1 hybrids gave a higher production coupled with an improved fruit quality with respect to the untrasformed controls (Acciarri et al. 2002). C. Potato Potato ranks as the third most important food crop after wheat and rice. The total 2007 potato harvest worldwide was about 323.5 million t in a total area of approximately 19 million ha. The top-ranking potato producers were China (approximately 65 million t in 4.4 million ha), Russia (37 million t in 2.9 million ha), and India (29 million t in 1.7 million ha); the highest national yields were recorded in New Zealand (50.3 t ha1), Belgium (46.9 t ha1), and France (45.4 t ha1). Potatoes yield on average more food energy per hectare than cereals as well as more edible protein and energy on a per-hectare and a per-day basis than either cereals or cassava (Horton 1988). Moreover, the biological value of potato protein is the best among vegetable sources and comparable to cow’s milk. The lysine content of potato complements cereal-based diets, which are deficient in this amino acid. 1. Insect Resistance. After the first Bt tomato was reported with partial resistance to lepidopteran insects, improvements through truncation of the Bt gene and codon modification were made to optimize protein expression in plants, and Bt potatoes expressing cry3A gene were developed. Bt-potato cultivars expressing resistance to Colorado potato beetle (Leptinotarsa decemlineata)—the most destructive insect pest of potato in North America—and aphids associated with Potato virus Y and Potato leafroll virus were approved for sale in the United States in 1995. On average, reported profits in United States were U.S. $ 55 ha1 for Bt-potato (Gianesi and Carpenter 1999), and ex-ante analysis suggested an average profit of U.S. $ 117 ha1 for virus-resistant potato in Mexico (Qaim 1998). These cultivars were marketed and sold under the trade names NewLeaf , NewLeafY, and NewLeafPlus by NatureMark , a subsidiary of Monsanto (Thomas et al. 1997). When NewLeaf cultivars were introduced in 1995, about 600 ha were grown commercially. As seed stocks increased, the commercial acreage reached 20,000 ha. Market success of the NewLeaf , NewLeafY, and NewLeafPlus potatoes could be attributed to the difficulty in controlling L. decemlineata and also high pest populations of aphids and associated virus problems due to mild winters in the Pacific Northwest (Thornton 2003). The added virus resistance benefited seed producers,

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while commercial growers benefited from higher yields and reduced need for insecticides (Thornton 2003). The processing industry and consumers benefited from improved quality. Potatoes were one of the first foods from a GM crop that was commonly served in restaurants. NewLeaf potato cultivars were the fastest cultivar adoption in the history of the U.S. potato industry (Thornton 2003), until potato processors, concerned about antibiotech organizations, consumer resistance, and loss of market share in Europe and Japan, suspended contracts for Bt-potatoes with growers in 2000, and they were taken off the market (Grafius and Douches 2008). Likewise, the crop area of GM insect- and virus-resistant potatoes did not increase at the same rate as in GM maize or GM cotton—irrespective of being highly effective and growers increasingly using them. This market growth for GM potatoes was not as rapid as the owners of the proprietary transgenic technology would have liked for four reasons: (1) growers’ ability to save potato tubers for future plantings; (2) the registration of a new insecticide (imidacloprid) that gave excellent control in the same year that Bt potato became available to growers; (3) consumer requests to segregate GM potatoes; and (4) trade issues driven by international consumers (Thornton 2003; Grafius and Douches 2008). International trade barriers were more substantial for GM potatoes than for other technology adoptions (Guenthner 2002). Thus, more than 60% of the U.S. market was closed to GM potatoes. This led to the processor and commercial grower discontinuing use, hence the loss of a market for NatureMark potatoes. One additional factor that led to the rapid demise was that only 3% of the U.S. potato area was Bt potatoes (Guenthner 2002). The major impact came when the leading fast food chaing, McDonald’s, decided to ban GM potatoes from its menu. NatureMark dissolved after the 2001 season. This initial failure of GM potatoes shows that consumer acceptance should be regarded as key for adopting transgenic crop technology. Guenthner (2002) indicated that although growers accepted GM potatoes, processors had little to gain by accepting GM raw materials but exposed themselves to significant risk in marketing because they did not have a ‘‘consumer-acceptance accelerator.’’ Nonetheless, the transgenic potato cultivar ‘Elizabeth’, bred at the Center of Bioengineering of the Russian Academy of Sciences and displaying resistance to Colorado potato beetle, was released recently for sale and is being used by the potato industry in Russia (http://www. potatopro.com/Lists/News/DispForm.aspx?ID¼3336). Cooper et al. (2007) evaluated natural host plant resistance mechanisms such as glandular trichomes and Solanum chacoense-derived resistance and genetically engineered resistance (cry3A and cry1Ia1) against Colorado potato beetle in a no-choice cage study. Their research


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suggest that Bt transgenes can provide high levels of host plant resistance and that available S. chacoense–derived resistance mechanisms seems to be variable to control Colorado potato beetle larvae in no-choice situations. The protein avidin—found in the whites of chicken eggs—has a broadspectrum insecticidal activity against arthropod pests, including Lepidoptera, Coleoptera, Diptera, and Acari. Avidin sequesters biotin, which is needed for cell growth and development of insects. Cooper et al. (2009a) incorporated a transgene for this protein in two potato clones: a susceptible genotype and a resistant one with S. chacoense as ancestor. They used leaf bioassays with first-stage Colorado potato beetle larvae to test. Their research showed significantly less survival as well as significantly lower survivors’ mass for larvae feeding on transgenic avidin lines vis- a-vis nontransgenic controls. Significantly fewer larvae fed on transgenic avidin plants survived to adulthood than those feeding on nontransgenic susceptible or S. chacoense–derived plants. The potato tuber moth (PTM, Phthorimaea operculella) or potato tuberworm is the most common and destructive insect pest of potatoes in tropical and subtropical areas worldwide (Visser 2005; Douches and Grafius 2005). The larvae mine the foliage, stems, and tubers in the field and in storage. Significant losses occur after tuber infection because the damaged tubers are attacked by various secondary pathogens and pests. Currently, the only available means to control P. opereculella and avoid major crop losses is the use of chemical insecticides. Combining natural resistance mechanisms with Bt cry genes could be a potential solution to improve potato resistance to PTM. Indeed, potatoes with cry1Ia1, cry1Ac, cry1Ac9, cry5 or cry9Aa2 transgenes—mostly under the transcriptional control of the CaMV 35S promoter but also with the Gelvin super promoter pBIML1-vector or the light-inducible Lhca3 promoter— showed enhanced host plant resistance to PTM larvae in detached-leaf or tuber bioassays (Jansen et al., 1995; Douches et al. 1998, 2002; Wedstedt et al. 1998; Can˜edo et al. 1999; Li et al. 1999; Lagnaoui et al. 2000; Mohammed et al. 2000; Davidson et al. 2002a,b, 2004; Meiyalaghan et al. 2006; Estrada et al. 2007). Field and storage trials in Egypt—under natural infestation—revealed that Bt-cry5 transgenic potatoes derived from the cv. ‘Spunta’ (‘Spunta-G2’, ‘Spunta-G3’, and ‘Spunta-6a3’) had the highest host plant resistance to PTM, with almost 100% of their tubers showing no insect damage in the field and about 90% free of insect damage after 3-month storage (Douches et al., 2004). Likewise, ‘SpuntaG2’ and ‘Spunta-6a3’ were higher yielding than’Spunta’ in field trials in Michigan (USA), but both transgenic clones had lower specific gravity— an important tuber quality trait—than the original cultivar from which they derive. South Africa was selected as the target country for releasing

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and marketing transgenic PTM-resistance potato because of its previous in-country experience with GM potato research (Douches et al. 2008). Multilocation yield trials and PTM efficacy trials (field and storage) have been conducted at six locations in South Africa since 2001. Complete control of the PTM was found at all locations in all years, and there was no infestation in the field when examining the foliage and tubers of ‘Spunta-G2’ and no infestation found in tubers stored up to 6 months. The cry1Ia1 transgene was used to transform two popular South African potato cultivars to ensure a greater impact. Further research by Cooper et al. (2009b) shows that expression of avidin combined with natural host plant resistance (from S. chacoense) in transgenic potato could be useful for controlling PTM. The cry1Ac transgene has been also used to engineer resistance in Andean potato cultivars ‘Diacol Capiro’, ‘Pardo Pastusa’, and ‘Pandeazućar’ to Tecia solanivora, whose larvae attack potato tubers (Valderrama et al. 2007). Bioassays of T. solanivora larvae on transgenic potato tubers showed 83.7% to 100% mortality, whereas the mortality levels on nontransgenic counterparts were 0% to 2.7%. Data indicate the ability of cryAC transgenes to control T. solanivora while reducing the use of insecticides. However, a specific issue needs to be addressed regarding the potential use of these transgenic potatoes in the Andean region—the center of origin of this crop— because of potential gene flow to wild relatives growing near potato crops (Celis et al. 2004). In this regard, Scurrah et al. (2008) suggest the use of sterile cultivars with scarce flower production and lacking seed production to minimize the risk of gene flow from transgenic potato. 2. Late Blight Resistance. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato disease in the world. Transgenic potato plants expressing soybean b-1,3-Endoglucanase gene exhibited enhanced host plant resistance to late blight (Borkowska et al. 1998). The wild diploid potato species S. bulbocastanum is highly resistant to all known races of P. infestans, and derived potato germplasm has shown durable and effective resistance in the field. Song et al. (2003) cloned the major resistance gene RB in S. bulbocastanum using a map-based approach in combination with a long-range (LR) PCR strategy. A four-gene resistance cluster of the coiled coil-nucleotide binding site-Leu-rich repeat class was found within the genetically mapped RB region. Transgenic plants using Agrobacteriummediated transformation containing the full-length gene coding sequence (including the open reading frame and promoter) were integrated into a cultivar (Halterman et al. 2008). All transgenic plants containing


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RB exhibited high foliar resistance whereas RB-containing tubers did not exhibit increased resistance. Furthermore, Kuhl et al. (2007) indicated that RB-transgenic plants showed increased resistance in field trials and variable results in laboratory tests using different P. infestans isolates. They also pointed out that the use of the RB gene for transformation creates a partially cisgenic event in potato because the gene’s native promoter and terminator are used. Such type of genetic transformation provides an opportunity to generate greater public approval of genetically engineered approaches to trait introgression in potato. 3. Host Plant Resistance. There are other transgenic potatoes with host plant resistance to both Potato virus X and Potato virus Y using markerfree and gene-silencing technology (Bai et al. 2009). Virus resistance in GM potatoes will be especially valuable because there are no pesticides for control of viruses, in contrast to insect pests or fungal diseases. Since potatoes are repeatedly propagated by tuber cuttings, viruses are easily spread during the seed multiplication process. Transgenic potatoes resistant to eelworm (Heterodera rostochiensis), to cyst (Globodera spp.) and root knot nematodes (Urwin et al. 2001, 2003; Simon 2003; Lilley et al. 2004) that do not alter susceptibility to nontarget insect herbivores or affect nontarget organisms in the potato rhizosphere (Cowgill et al. 2002a,b, 2003, 2004) and do not possess a risk to human diet (Atkison et al. 2004) are also available. In western Europe, late blight and eelworm resistance could be the primary drivers for market penetration of GM potatoes (Simon 2003). When consumers have confidence in the technology, it will spread quickly due to the environmental and production benefits. These two GM opportunities would greatly reduce pesticide use and would be extremely attractive to potato growers. 4. Other Traits. There are also other research advances on GM potatoes. For example, salt-tolerant potatoes are being bred using the DREB1A transgene driven by the stress-inducible rd29A promoter (Behnam et al. 2006). Likewise, increased nutritive value may be achieved in potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus (Chakraborty et al. 2000), by protein-rich potato expressing the seed protein gene AmA1 (Amaranth Albumin 1) (Chakraborty et al. 2010), or by enhancing levels of carotenoid and lutein due to a crtBtransgene derived from Erwinia uredovora that encodes phytoene synthase (Ducreux et al. 2005). Moreover, tuber quality can be improved by reducing polyphenol oxidase activity associated with reduced woundinducible browning (Arican and Gozukirmizi 2003), or decreasing the

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amount of reducing sugars due to a bacterial-derived transgene coding for phosphofructokinase under a tuber-specific promoter (Navratil et al. 2007). Furthermore, potato researchers are genetically engineering potatoes producing dextran using a dextransucrase DsrS transgene initially isolated from Leuconostoc mesenteroides (Kok-Jacon et al. 2005), inhibiting the expression of the gene for granule-bound starch synthase (GBSS) using antisense constructs (Visser et al. 1991), or expressing full-length spike protein of infectious bronchitis virus (IBV) that may allow a new delivery system of Coronaviridae IBV vaccine (Zhou et al. 2004). Catchpole et al. (2005) used hierarchical metabolomics to demonstrate the substantial compositional similarity between conventional and transgenic potato bioengineered to contain high levels of inulin-type fructans (Hellwege et al. 2000), a prebiotic food supplement. Such comparisons are needed for determining whether any transgenic potato diplays alterations in metabolite composition outside the range of the cultigen pool. Recently the European Union has approved the planting of the amylose-free starch transgenic potato ‘Amflora’, which produces large amounts of pure amylopectin that can be used in technical applications by the paper industry (http://www.foodnavigator.com/news/ng. asp?n¼78450&m¼1FNE724&c¼huokmwqnnbjptez). D. Cucurbits Old World cucurbits, including watermelon (Citrullus lanatus), melon (Cucumis melo), cucumbers and gherkins (Cucumis sativus), and various gourds (Lagenaria and Momordica spp.), and New World cucurbits, such as pumpkin and squash (Cucurbita maxima, C. pepo and C. moschata), are grown throughout the temperate, subtropical, and tropical regions of the world. Cucurbits collectively rank among the top five vegetable crops produced worldwide with nearly 10 million ha devoted to production (FAO 2009). Watermelon ranks first in area (3.3 million ha) with a total production (93.7 million t), followed by cucumbers and gherkins (2.6 million ha and 44.4 million t), pumpkins plus squashes and gourds (1.6 milliom ha and 21.1 million t), other melons (1.2 million ha and 27.8 million ha), and melonseed (1.1 million ha and 0.7 million t). China is the top producer of watermelon (62.2 million t), cucumbers and gherkins (28 million t), and pumpkins plus squashes and gourds (6.3 million t), whereas Indonesia and Nigeria are the top producers of other melons (16.9 million t) and melonseed (490,000 t). Transgenics have become important in four cucurbits: summer squash, watermelon, cucumber, and melon.


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1. Summer Squash. In the United States, declines in yields of summer squash (Cucurbita pepo) due to viruses often range from 20% to 80% with a reported U.S. $ 2.6 million loss alone in the state of Georgia in 1997 (Gianessi et al., 2002). Three of the most important viruses affecting squash production are Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), and CMV (Zitter et al. 1996). Summer squash cultivars with satisfactory resistance to CMV, ZYMV, and WMV are yet to be developed by conventional breeding (Gaba et al. 2004). Control of squash viruses has focused on cultural practices, including delayed transplanting relative to aphid flights, use of reflective film mulch to repel aphids, and application of stylet oil to reduce virus transmission, in combination with insecticides to reduce aphid vector populations (Perring et al. 1999). In the state of Georgia, it is estimated that 10 stylet oil and insecticide sprays are made routinely to control aphids, thereby limiting virus incidence and transmission (Gianessi et al. 2002). Two lines of squash expressing the coat protein (CP) gene of ZYMV, WMV, and CMV were deregulated and commercialized in 1996 (Medley 1994). Subsequently, many squash types and cultivars have been developed, using crosses and backcrosses with the two initially deregulated lines. This material is highly resistant to infection by one, two, or all three of the target viruses (Clough and Hamm 1995; Fuchs and Gonsalves 1995; Ochoa et al. 1995; Tricoli et al. 1995; Fuchs et al. 1998; Schultheis and Walters 1998). Virus-resistant transgenic squash limits virus infection rates by restricting challenge viruses, reducing their titers, or inhibiting their replication or cell-to-cell or systemic movement. Therefore, lower virus levels reduce the frequency of acquisition by vectors and subsequent transmission within and between fields. Consequently, virus epidemics are substantially limited. The adoption of virus-resistant squash cultivars has steadily increased in the United States since 1996. In 2005, the adoption rate was estimated at 12% (approximately 3,100 ha) across the country with the highest rates in New Jersey (25%), Florida (22%), Georgia (20%), South Carolina (20%) and Tennessee (20%) (Shankula 2006). Virus-resistant transgenic squash has allowed growers to achieve yields comparable to those obtained in the absence of viruses with a net benefit of U.S. $ 22 million in 2005 (Shankula 2006). Engineered resistance was the only practical approach to development of cultivars with multiple sources of resistance to CMV, ZYMV, and WMV in those markets where GM squash is allowed. 2. Watermelon. Field trials have been conducted on transgenic watermelons containing genes conferring virus resistance and parthenocarpy (ISB 2007). Virus-resistant lines containing the coat protein genes from

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ZYMV and WMV2 were mechanically inoculated with both viruses and displayed resistance to both ZYMV and WMV2 (Tricoli et al., 2002). Control vines were highly symptomatic, producing very few fruits, and the fruits that were produced were very small in size. 3. Cucumber. Field trials were conducted with transgenic cucumbers engineered for viral resistance, herbicide tolerance, and increased salt tolerance. Slightom et al. (1990) and Gonsalves et al. (1992) tested transgenic cucumber lines derived from ‘Poinsett 76’ expressing the CMV coat protein gene for host plant resistance to CMV. These plants were compared to nontransgenic ‘Poinsett 76’ and the traditionally bred CMV-resistant line ‘Marketmore’. The level of CMV infection was determined at the end of the trial by enzyme-linked immune sorbent assay. The transgenic lines had significantly lower rates of infection than either ‘Marketmore’ or nontransgenic ‘Poinsett 76’. Virus-resistant lines of pickling, slicing, and beit alpha types were tested for resistance to virus in the field using paired plot designs in which each transgenic line was paired with its nontransgenic counterpart. Transgenic lines containing the coat protein genes from CMV, ZYMV, WMV2, and the nuclear inclusion protein A or B from PRSV exhibited high field resistance against all four viruses, including PRSV (Tricoli et al. 2002). 4. Melon. There have been field tests of male-sterile, long-shelf-life and virus-resistant transgenic melon lines (ISB 2007). Field testing of virusresistant transgenic melon lines showed no detrimental effects associated with the transgene. Nutritional analysis conducted on fruits harvested from transgenic and control lines showed no alteration in any of the nutritional components measured. However, in contrast to squash, high levels of virus resistance were achieved only in plants that were homozygous for the transgenes (Clough and Hamm 1995; Fuchs et al. 1997). Plants homozygous for the CMV, ZYMV, and WMV2 coat protein genes (designated CZW-30) never exhibited systemic symptoms of virus, whereas symptoms that did develop late in the season were the result of a single infection of one of the three viruses present in the field as opposed to mixed infections seen in the control lines. Hemizygous lines derived from CZW-30 developed systemic symptoms late in the season but still exhibited a 7.4-fold increase in fruit yields compared to control lines (Fuchs et al. 1997). Very recently, Wu et al. (2009) bred a transgenic melon with resistance to ZYMV by an improved cotyledon-cutting method. This transgenic melon has a great potential for controlling ZYMV in East Asia. Li et al. (2006) provided an update on transgenic approaches for improving quality traits of melon. They indicated that


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transgenic technology seems to be promising for sensory attributes and shelf life of melon fruit. E. Brassicas Vegetable brassicas include cabbage, cauliflower, broccoli, Brussels sprouts, tronchudas, collards, kales, kohlrabi, Chinese kale (B. oleracea), turnip, broccolettos, Chinese cabbage, pak-choi, choy-sum, komatsuna, yellow sarson (B. rapa), rutabaga or swedes, vegetable rape and mustards. They are economically important and grown worldwide for consumption as both fresh and frozen produce. In 2007, their production was 87 million t and the total harvest area of cabbages, cauliflower, and broccoli was 4.5 million ha worldwide (FAO 2009). Of this total area, 80% was grown in the developing world. Cabbages and cauliflower are important vegetable cash crops for low-income farmers throughout Asia, Africa, Latin America, and the Caribbean. They serve as important staple dietary items and are high in folate, vitamins B and C, and other micronutrients. In addition, vegetable brassicas are gaining popularity as they contain glucosinolates with anticancer properties. Protoplast fusion has been investigated as a means to introgress disease and pest resistance from other Brassicaceae species (Christey 2004), but transformation technology offers an alternative approach. 1. Insect Resistance. Lepidopteran larvae are the most problematic insect pests of vegetable brassicas worldwide. The diamondback moth Plutella xylostella is considered the most destructive insect pest of vegetable brassica and has severely limited its production, especially in resource-poor regions (Talekar and Shelton 1993). P. xylostella occurs in excess of 80 countries where brassicas are grown and causes losses to the world economy of over U.S. $1 billion yearly (Talekar and Shelton 1993), a figure that continues to rise. In India, the losses of cabbage and cauliflower due to P. xylostella frequently reach 90% without the use of insecticides (CIMBAA 2010). Significant losses occur and threaten food security even after the frequent use of insecticides. In tropical areas where pest pressure is high, it is not uncommon to apply insecticides every other day or at least 2.5 times per week (Rauf et al. 2004; Sandur 2005). In India, about 6,000 t of active ingredient of insecticides are used annually for diamondback moth control alone (Mohan and Gujar 2003). The cost of cabbage and cauliflower protection is greater than U.S. $168 million annually in India alone. A study undertaken for the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA) found that in a typical production area in the state of Karnataka (India),

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the average number of insecticide applications was 13 per crop (Sandur 2005) or more than 1 weekly. In areas of diamondback moth outbreak, application frequency is often much higher (e.g., up to 30 applications per crop is common). The cost of buying and applying insecticies was 38% of the total variable costs of production. Every year, spraying diamondback moth with insecticide consumes more than 33,000 person-years of labor in India alone. In Sandur’s study, one-third of farmers reported symptoms of pesticide poisoning in the previous year (conjunctivitis, headache, dermatitis, and stomach pains). Farmers widely disregard the ‘‘no-spray periods’’ before marketing brassicas, and twothirds of vegetables tested between 1988 and 1998 were contaminated by pesticide residues, with 11% of the samples exhibiting levels over the maximum residue level (Agnihotri 1999). The situation is undoubtedly worse now, as diamondback moth continues to develop serious resistance to all classes of compounds sprayed to control it, thus increasing the pressure to spray more intensively. Such intense use of insecticides poses hazards to farmers, consumers, and the environment and has caused populations of this insect to become resistant to most of the major insecticides. Diamondback moth has developed resistance to almost all insecticides in many parts of the world. Conventional crop breeding programs have failed to develop cultivars with really useful resistance to diamondback moth. Alternative pest management solutions based on agronomic regimes, biological control, or chemical pesticides have proven effective in some areas, especially in the highlands, but they are insufficient in lowland areas and cumbersome to implement successfully, especially by poorer farmers in developing countries. Various cry genes (cry1A, cry1Ab, cry1Ac, cry1A(b), cry1Ab3, cry1Ba1, cry1C, cry1Ba1, cry1Ia3, and cry9Aa) from Bt have been introduced into cabbage (Metz et al. 1995; Jin et al. 2000; Bhattacharya et al. 2002; Paul et al. 2005; Christey et al. 2006), cauliflower (Khuvshinov et al. 2001; Chakrabarty et al. 2002, Christey et al. 2006), broccoli (Cao et al. 2001, Christey et al. 2006), collards (Cao et al. 2005), Chinese cabbage (Cho et al. 2001), choy-sum (Xiang et al. 2000), and rutabaga or swede (Li et al. 1995). Bt-vegetable brassicas successfully control important insect pests such as diamondback moth (P. xylostella) and other lepidoptera and cabbage white butterfly (Pieris rapae) (Earle et al. 2004). Several field tests with Bt-vegetable brassicas using cry1C and cry1B transgenes, which are effective against P. xylostella, are in progress in Asia, Africa, and Oceania by CIMBAA. Cross-resistance between both cry-derived toxins was not detected (Zhao et al. 2001), Nunhems Inc. Seed Company (one of the partners of CIMBAA) in India has produced cabbage and cauliflower lines that express both proteins at levels sufficient to control


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not only populations of susceptible P. xylostella but also populations of P. xylostella that are resistant to cry1C transgene. None of the populations of P. xylostella has developed resistance to cry1B transgene. Laboratory and greenhouse studies with CIMBAA breeding lines have shown excellent control of P. xylostella and P. rapae. In addition to cry Bt genes, the cowpea trypsin inhibitor has been shown to confer insect resistance in cauliflower (Iingling et al. 2005) and in Chinese cabbage (Zhao et al. 2006). A trypsin inhibitor is a type of protease inhibitor that is capable of controlling a wide spectrum of insect pests. Ding et al. (1998) produced insect-resistant transgenic cauliflower expressing a sweet potato trypsin inhibitor gene. Transgenic cauliflower plants were obtained with a high degree of protection against the lepidoptera pests Spodoptera litura (cut worm) and P. xylostella. 2. Disease Resistance. Downy mildew (Peronospora parasitica), clubroot (Plasmodiophora brassicae), alternaria blight (Alternaria brassicicola), black rot (Xanthomonas campestris pv. campestris), stem rot and watery soft rot (Sclerotinia sclerotiorum), Cauliflower mosaic virus (CaMV), and Turnip mosaic virus (TuMV) are other pathogens affecting vegetable brassicas. Chitin is an important component of fungal cell walls. Chitinase genes cloned from plants and fungi have been transferred into a number of plant species and resistance to a broad range of fungal pathogens obtained. Mora and Earle (2001) produced transgenic broccoli plants expressing an endochitinase gene from the biocontrol fungus Trichoderma harzianum. Transgenic plants were obtained with 14 to 37 times higher endochitinase levels than controls. Transgenic plants inoculated with A. brassicicola showed significantly less severe disease symptoms than controls. Interestingly, polyploid plants were highly susceptible regardless of their endochitinase activity. In contrast, lesion size of plants inoculated with S. sclerotiorum was not statistically different from controls. Braun et al. (2000) introduced antibacterial genes from nonplant sources into cauliflower in an attempt to produce black rot–resistant cauliflower. They introduced the Shiva protein, which is a synthetic analog of cecropin B from the giant silkworm moth, and the magainin II peptide derived from the African clawed frog. In vitro bacterial assays using crude leaf extracts confirmed increased resistance to black rot, but greenhouse screening failed to show any increased resistance compared to controls. Likewise, Zhao et al. (2006) introduced Shiva and cecropin B into Chinese cabbage. Gene expression in the transgenic plants was

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confirmed by Northern blot analysis, but bacterial assays were not undertaken. Glucose oxidase catalyzes the oxidation of glucose generating H2O2 as a by-product. H2O2 effectively inhibits bacterial and fungal growth, and plants transgenic for glucose oxidase show enhanced host plant resistance to pathogens. However, some deleterious phytotoxic effects of glucose oxidase have been noted in transgenic plants. Cabbage expressing a glucose oxidase gene from Aspergillus niger showed enhanced resistance to black rot but also showed phytotoxic effects (Lee et al. 2002). Plants showed significant growth retardation, and seed set was dramatically reduced, with only a few seed produced. Passel egue and Kerlan (1996) transformed cauliflower with two CaMV-derived genes in an attempt to produce CaMV-resistant cauliflower. They used the capsid gene and the antisense gene VI of CaMV. While reverse transcriptase polymerase chain reaction demonstrated the presence of CaMV gene transcripts in all plants, the amount of RNA transcribed was very low, in contrast to the hygromycin resistance (hpt) gene transcript. In plants transformed with the capsid gene, the capsid protein could not be detected. The response to CaMV infection was not tested. In contrast, Zhandong et al. (2007) successfully obtained high levels of resistance to TuMV in Chinese cabbage plants transformed with the antisense TuMV replicase (NIb) gene. Plants were transformed using marker-free A. tumefaciens–mediated floral dipping. 3. Weed Control. The incorporation of herbicide resistance into vegetable brassicas would enable growers to control weeds more efficiently. Basta -resistant broccoli has been produced and field-tested by Christey et al. (1997) and Waterer et al. (2000). Waterer et al. (2000) field-tested six transgenic lines and noted that herbicide application had little effect on head quality and marketable yield of most lines. Christey et al. (1997) field-tested four transgenic lines and also noted that the phenotype was normal although plants were not sprayed in the field. Spraying of Basta on seedlings demonstrated they were resistant in greenhouse trials. 4. Postharvest Quality. Postharvest traits such as transport quality and shelf life are of increasing importance mainly in inflorescence brassicas like broccoli. Postharvest senescence of broccoli is rapid with loss of chlorophyll resulting in yellowing of the head. Ethylene plays an important role in the yellowing of broccoli as chlorophyll loss is associated with an increase in floret ethylene synthesis. Chlorophyll loss can be delayed through the use of inhibitors of ethylene action and biosynthesis. Several groups are conducting transgenic research aimed at


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increasing the shelf life of ethylene-sensitive broccoli. Antisense versions of two key regulatory genes in the ethylene biosynthesis pathway ACC oxidase and ACC synthase have been used to produce transgenic broccoli plants with reduced ethylene synthesis. However, the reduction in ethylene production resulted in increases in shelf life of only 1 or 2 days. In order to delay postharvest senescence in broccoli, Henzi et al. (1999a,b) used A. rhizogenes–mediated transformation to express an antisense ACC oxidase gene from tomato in broccoli. Plants showed reduced ethylene production but little effect on postharvest senescence (Henzi et al. 2000b). Gapper et al. (2002) used A. tumefaciens–mediated transformation to introduce an antisense ACC oxidase gene (driven by the asparagine synthetase promoter from asparagus) from broccoli into broccoli. Several lines were obtained with reduced ethylene production and delayed postharvest senescence (Gapper et al. 2002). Higgins et al. (2006) have also shown that broccoli transgenic for antisense versions of ACC synthase and ACC oxidase have reduced ethylene production, which correlates with delayed chlorophyll loss. Cytokinin has also been shown to be involved with broccoli senescence, as application of cytokinin to broccoli heads can delay postharvest yellowing. Gapper et al. (2002) introduced an isopentenyl phosphotransferase (IPT) gene into broccoli, but their results on the effect of postharvest senescence were not indicated. Some plants had an abnormal phenotype typical of constitutive IPT expression. Chen et al. (2001) also introduced an IPT gene into broccoli under the control of senescence-associated promoters and demonstrated retardation in postharvest yellowing in broccoli heads and leaves. About 31% of transformants exhibited delayed yellowing in detached leaves, 16% in floret branchlets, and 7% in both leaves and floret branchlets. Eason et al. (2005) and Chen et al. (2004) delayed senescence in broccoli through genetic transformation with either an antisense cysteine protease or mutant ethylene response sensor gene, respectively. In both cases, senescence was delayed only by 1 or 2 days. As there are several other genes whose expression is increased at broccoli harvest, it is likely that down-regulation of these genes through antisense or RNA interference will also lead to the production of broccoli with delayed senescence. It is likely that future research will involve the introduction of these genes into green leafy vegetables such as Chinese cabbage and pak-choi, which also show postharvest deterioration due to ethylene. Nutritional quality and health benefits are becoming very important for consumers. Current advances in genetic engineering have enabled the production of plants with alterations in a range of vitamins or amino acids for improved human or animal nutrition. Lu et al. (2006) developed transgenic cauliflower

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with b-carotene accumulation, and Wahlroos et al. (2004) produced oilseed B. rapa with increased histidine content. It is likely in the future that more transgenic vegetable brassicas with altered vitamin or amino acid content also will be developed. 5. Healthy Food. Flavonoids such as anthocyanins are known as antioxidants in vitro and can reduce the risk of many diseases related to aging. However, some vegetable brassicas, such as cauliflower, are low in anthocyanins. In an attempt to manipulate pigment biosynthesis to increase the health benefits of vegetables, the effect of a regulatory locus of flavonoid content was assessed. Agrobacterium tumefaciens– mediated transformation of a Brassica oleracea line, selected for high transformation ability by Sparrow et al. (2004), was used to produce plants transgenic for the maize lc (leaf color) locus. Lc is a regulatory gene in the anthocyanin pathway, and it is expected that its presence will increase the flavonoid content. Seedling explants were cocultivated with Agrobacterium tumefaciens strain LBA4404 containing a binary vector with a neomycin phosphotransferase II (NPTII) gene. Under tissue culture conditions, lc-containing plants were green with no visible increase in anthocyanin production. However, after transfer to the greenhouse, the exposure to high light intensity led to visible signs of pigmentation within one week. Increased pigmentation was apparent in stems, petioles, main leaf veins, and sepals. Lc-containing lines had 10 to 20 times higher levels of total anthocyanins than controls. In addition, antioxidant activity of lc-containing lines was 1.5 times higher than that of controls (Braun et al. 2006). 6. Male Sterility and Self-Incompatability. Hybrid seed production is an important breeding goal in vegetable brassicas as hybrid seed offers many benefits, including increased vigor and greater uniformity. The introduction of male sterility and self-incompatibility (which prevents self-fertilization and promotes outcrossing) into vegetable brassicas would aid production of hybrid seed. Transformation approaches have been used in cabbage, cauliflower, and Chinese cabbage to induce male sterility. In cauliflower, Bhalla and Smith (1998) introduced an antisense pollen-specific gene linked to a pollen-specific promoter and obtained the expected sterility in 50% of the pollen. In Chinese cabbage, introduction of an antisense version of the CYP86MF gene linked to a tapetum-specific promoter induced male sterility (Yu et al. 2004). Lee et al. (2003) also used a tapetum-specific promoter to induce male sterility in cabbage through the introduction of the cytotoxic diphtheria toxin A chain. Self-incompatibility has a number of drawbacks, includ-


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ing breakdown of incompatibility, labor intensiveness, and genetic complexity of the system. In brassicas, self-incompatibility is sporophytically controlled by multiallelic genes at the S-locus. Two genes have been identified at the S-locus: S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK). SLG and SRK encode a secreted glycoprotein in the wall of the stigma papillary cells and a transmembrane receptor kinase, respectively. A self-incompatible response occurs when the same S-allele is expressed in pollen and stigma. Toriyama et al. (1991) introduced an SLG gene from B. rapa S8 homozygote and were able to alter the self-incompatibility phenotype of pollen and stigma. Self-incompatible Chinese kale and partial compatible broccoli plants were fully compatible upon self-fertilization. 7. Plant Stress. Salinity and drought can limit brassica vegetable production. Bhattacharya et al. (2004) produced cabbage plants with enhanced salt tolerance through the introduction of the bacterial glycinebetaine biosynthesis (BetA) gene for biosynthesis of glycinebetaine. Detailed analysis of three independent transgenic lines showed improved growth and development under salt stress compared with the control. Park et al. (2005a) developed transgenic Chinese cabbage with the B. napus late embryogenesis abundant (LEA) gene that showed enhanced tolerance to both drought and salt. F. Lettuce Lettuce (Lactuca sativa) is the most important leafy vegetable worldwide, being grown extensively as a salad crop and consumed primarily in the fresh state. The worldwide estimated production of lettuce is about 23 million t (FAO 2009). In the last decade, the market and area of production for lettuce has expanded quite a bit with more cultivars being cultivated in line with consumer requirements for novel produce. Currently, young leaves of lettuce are becoming popular components (individually or in mixtures) of leafy ‘‘baby salad’’ packs. 1. Disease Resistance. Experiments were undertaken in the 1990s for introducing virus resistance into lettuce using a coat protein genemediated approach. Pang et al. (1996) transferred the nucleocapsid (N) protein gene of the Lettuce Tospovirus (TSWV) into two lettuce breeding lines. Transgenic plants expressing the nucleocapsid (N) protein gene were protected against TSWV. Dinant et al. (1993) introduced a Lettuce mosaic virus (LMV) coat protein (LMV-CP) gene into LMVsusceptible lettuce, but the transgene conferred only poor resistance to

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this virus. Loss of virus resistance was more pronounced in lettuce during subsequent seed generations, an observation also made by Gilbertson (1996). Likewise, Dinant et al. (1997) introduced the coat protein gene from LMV strain O into the virus susceptible cvs. ‘Girelle’, ‘Jessy’, and ‘Cocarde’. Several transgenic plants accumulated LMV-CP. As in other examples of potyvirus sequence-mediated protection, some plants were completely virus resistant, but in others this resistance was not sustained, with the development of viral infection symptoms. The efficiency of this strategy to induce LMV resistance was considered to be related to the developmental stage of the transgenic plants at the time of their inoculation with the virus. Sesquiterpene lactones play a role in host plant resistance to pathogens through the hytoalexin response. Such compounds include lactucin and lettucenin A. Bennett et al. (2002) cloned genes for germacrene A synthases in order to change the profile of these latex-expressed compounds and to enhance host plant resistance to pathogens using sense and antisense approaches. Dias et al. (2006) also introduced the decarboxylase (oxdc) gene from Flammulina spp. into lettuce and showed that leaves from two transgenic plants inoculated with agar plugs of a 2-day old culture of Scierotinia sclerotiorum failed to develop disease symptoms. Transformation of plants with proteinase inhibitors that act on proteolytic enzymes may confer resistance to pests and pathogens. In studies of the role of the proteinase inhibitor II, SaPIN2a, from Solanum americanum, Xu et al. (2004) transformed lettuce with the SaPIN2a gene driven by the CaMV 35S promoter and suggested that this approach may be exploited to counteract pest and pathogen attack. Subsequently, Fan and Wu (2005) also introduced a PIN2 gene into lettuce, while Chye et al. (2006) extended these investigations to show that expression of SaPIN2a in lettuce conferred resistance to cabbage looper (Trichoplusia ni) caterpillars. 2. Weed Control. Lettuce shows a very low competitive ability against weeds. Hence, tolerance to herbicides is a prime target for genetic manipulation in lettuce since weeds cause severe crop losses. Herbicide tolerance by genetic manipulation has been introduced into lettuce from both academic and commercial perspectives. Mohapatra et al. (1999) introduced the bar gene from the bacterium Streptomyces hygroscopicus into seedling cotyledons of the cv. ‘Evola’ by Agrobacterium-mediated transformation with strains 0310 and 1310. The strain 1310 carried the hypervirulent pTOK47 in addition to the binary vector with the nptII and bar genes. Plasmid TOK47 in strain 1310 gave multiple insertions of


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T-DNA into some plants, whereas strain 0310 gave single-gene inserts in all plants analyzed by DNA-DNA hybridization. Axenic seedlings grew on medium with glufosinate ammonium at 5 mg l1, while glasshousegrown plants were resistant to the herbicide when the latter was sprayed on the plants at 300 mg l1. This research confirmed that herbicide resistance can be introduced into lettuce with stable expression in seed generations. 3. Male Sterility. The introduction of male sterility for hybrid seed production is another objective in lettuce breeding. In experiments to induce male sterility in lettuce, a pathogenesis-related b-1,3-glucanase gene linked to a tapetum-specific promoter, A9, was cloned into the binary vector pBIN19 and the latter introduced into A. tumefaciens carrying pGV2260, prior to transformation of the cv. ‘Lake Nyah’ (Curtis et al. 1996b). Transgene expression resulted in dissolution of the callose wall of developing microspores, inhibiting pollen grain development, resulting in male sterility. This or a similar approach may eliminate the need to remove pollen from the stigmatic surface of recipient plants to avoid self-pollination prior to application of donor pollen. 4. Postharvest Quality. Breeding lettuce for postharvest traits, mainly transport quality, shelf life, and cosmetic problems, is of increasing importance. Lettuce used for packaged salads deteriorates rapidly following harvest, requiring a considerable investment of effort to maintain quality and shelf life of cut material. Harvesting increases respiration, thereby stimulating deterioration with an increase in the synthesis of phenylalanine ammonia lyase and phenolic compounds, such as chlorogenic acid, which cause tissue browning (Kang and Saltveit 2003). Delaying leaf senescence in lettuce is therefore an important target for genetic manipulation because lettuce with appropriate transport quality, better shelf life, and good appearance will be preferred by traders and consumers. Curtis et al. (1999b) introduced the T-cyt gene (synonym ipt, tmr, or gene 4) coding for isopentenyl phosphotransferase, which is involved in cytokinin biosynthesis, from the T-DNA of A. tumefaciens on the binary vector pMOG23 into the lettuce cv. ‘Saladin’. Transgenic plants were phenotypically normal following transfer to the glasshouse, set viable seed, and had increased cytokinin and chlorophyll contents in their leaves compared to nontransformed plants. Such results indicated the possibility of delaying senescence in lettuce, possibly reducing the requirement for postharvest controlled environmental conditions to prolong the shelf life of harvested plants. In subsequent research, the ipt gene driven by the senescence-specific promoter SAG12 from

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A. thaliana (pSAG12-IPT) was introduced into the lettuce cv. ‘Evola’. Plants homozygous for the transgene exhibited significantly delayed postharvest leaf senescence (McCabe et al. 2001). Importantly, the transgene was activated only during senescence, particularly when the latter process commenced in the outer (lower) leaves, initiating cytokinin biosynthesis, which inhibited leaf senescence, simultaneously attenuating activity of the pSAG12-IPT gene, thereby preventing cytokinin overproduction. Heads of transgenic plants retained chlorophyll in their outer leaves after harvest 7 days longer than leaves of the nontransgenic plants. Mature plants were morphologically normal at harvest and did not show significant differences in head diameter or fresh weight of leaves or roots compared to their nontransgenic counterparts. Although during storage, heads of transgenic plants showed a threefold increase in the concentrations of acetaldehyde, ethanol, and dimethyl sulfide, increase in the latter compound was paralleled by an accumulation of reactive oxygen species. This research emphasizes the importance of detailed metabolite profiling of plants following transgene insertion, as the integration of a gene to modify one or more traits may affect other biosynthetic pathways. 5. Healthy Food. Nutritional quality as understood by the consumers and available at a moderate price may encourage enhanced consumption, thereby conferring an important marketing incentive to plant breeding. For example, vitamin E—which includes tocopherols—is a lipid-soluble antioxidant. There are a, b, g, and d isoforms of tocopherol with relative vitamin E potencies of 100%, 50%, 10%, and 3%, respectively. Conversion of g-tocopherol to a-tocopherol in food crops could increase their value and importance in human health because vitamin E reduces the risk of several serious disorders (e.g., cardiovascular diseases and cancer), slows aging, and enhances the function of the immune system. Cho et al. (2005) developed transgenic lettuce plants of the cv. ‘Chungchima’ expressing a cDNA encoding g-tocopherol methyltransferase from A. thaliana to improve tocopherol composition. Transgene inheritance and expression in transformed plants increased enzyme activity and conversion of g-tocopherol to the more potent a form. Similalry, resveratrol—a stilbenes—shows cancer chemopreventive activity and may prevent coronary heart disease and arteriosclerosis. Stilbene synthase is the key enzyme in resveratrol biosynthesis, with several stilbene synthase genes being isolated recently. Although lettuce contains the substrates for stilbene synthase, resveratrol is not synthesized. Thus, in order to engineer lettuce for synthesis of this compound, Liu et al. (2006) fused a cDNA encoding


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stilbene synthase from Parthenocissus henryana to the CaMV 35S promoter with the bar gene as a selectable marker. The expression construct was flanked by matrix attachment regions to maximize expression of the gene of interest in transgenic plants. Quantitative analysis showed that resveratrol in transgenic plants was 56.0 5.52 mg1 leaf fresh weight, which is comparable to that in the skin of grapefruit (Citrus paridisi Macfad.). Zinc is also an essential element in human nutrition, as its deficiency severely impairs organ function. In experiments to fortify lettuce with this element, Zuo et al. (2002) used Agrobacterium-mediated gene delivery of a mouse metallothionein mutant b-cDNA in the cv. ‘Salinas 88’. The concentration of zinc in the transgenic plants increased to 400 mg g1 dry weight, which is considerably higher than in wild-type plants. In attempts to reduce bitterness, Sun et al. (2006) cloned the gene for the sweet and taste modifying protein miraculin from the pulp of berries of Richadella dulcifica, a West African shrub. This gene, with the CaMV 35S promoter, was introduced into the cv. ‘Kaiser’ using A. tumefaciens GV2260. Expression of this gene in transgenic plants led to the accumulation of significant concentrations of the sweetenhancing protein. Miraculin, which is active at extremely low concentrations, may be used by people suffering diabetes as a food sweetener. Nitrate content is also important for product quality and consumer health. Accumulation of nitrate in lettuce and other vegetables is undesirable, since in humans, nitrate produces nitrite, which hinders the binding of oxygen to hemoglobin. Lettuce often accumulates nitrate to concentrations that exceed the maximum permitted concentration. Indeed, nitrate concentration is one of several parameters that govern the marketability of this crop, especially in winter, when plants accumulate nitrate because of low light conditions. In experiments to reduce nitrate accumulation in lettuce, Curtis et al. (1999a) introduced the nia2 cDNA for nitrate reductase from tobacco driven by the 35S promoter into the cv. ‘Evola’. Unfortunately, none of the transgenic plants exhibited a reduction in nitrate content compared to the wild-type plants at harvest, although plants with nitrate concentrations slightly less than those of wild-type plants were observed during cultivation. Subsequently, Dubois et al. (2005) investigated nitrate accumulation in the cv. ‘Jessy’ using a similar 35S::nia2 construct. They also concluded that none of the plants carrying the transgene exhibited a reduction in nitrate accumulation, although the transgene was expressed. Transgene-specific silencing extended to the homologous endogenous nitrate reductase mRNA (messenger RNA) resulting in chlorosis and eventual death of the

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transgenic plants, which reveals the difficulty of this approach for reducing nitrate accumulation in lettuce. 6. Plant Stress. Salinity limits crop production in many vegetable areas, particularly in those that have been intensively used. Lettuce is highly sensitive to salinity. In addition, drought and cold restrict its growth in the field. Park et al. (2005b) introduced a late-embryogenesis abundant protein gene from Brassica napus into lettuce. Six transgenic plants generated exhibited enhanced growth compared to nontransformed plants under salt stress imposed by exposure to 100 mM sodium chloride. Furthermore, Curtis et al. (1996a) transformed the lettuce cv. ‘Lake Nyah’ with the rolAB genes from Agrobacterium rhizogenes to stimulate root formation, with the aim of increasing drought tolerance, whereas Pileggi et al. (2001) focused attention on drought, salinity, and cold by transforming the lettuce cv. ‘Grand Rapids’ with a mutated P5CS gene for d-1-pyrroline-5-carboxylate synthase, which catalyzes two steps of proline biosynthesis in plants. This mutated gene is insensitive to feedback inhibition by proline. Increased concentration of proline acts like an osmoprotectant that could confer resistance to drought, salinity, and cold on transgenic plants. Transgenic lettuce plants were tolerant to freezing. Vanjildorj et al. (2005) also targeted drought and cold tolerance in lettuce by overexpressing the Arabidopsis ABF3 gene—encoding a transcription factor for the expression of ABA-responsive genes—in the lettuce cv. ‘Chongchima’. Transgenic plants were phenotypically normal, produced seed, and were more tolerant than wild-type plants to drought and cold stresses. G. Alliums The total production of alliums was 85 million t annually in 2007 including 66 million t for onion and 16 million t for garlic (FAO 2009). Production of these two major allium crops occurs in 175 countries with China the largest world producer, producing 32% of onions and 76% of garlic (FAO 2009). 1. Weed Control. Alliums are monocots and they show a poor competitive ability with weeds. Herbicide-resistant onion germplasm has been bred by using the CP-4-derived gene construct that confers resistance to the systemic herbicide glyphosate (Eady et al. 2003). The enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme, which is involved in the production of aromatic amino acids in plants, is inhibited by glyphosate. Tolerance to glyphosate is achieved either


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through the overexpression of the CP-4 EPSPS enzyme (Hetherington et al. 1999) or by detoxification of the glyphosate by either the glyphosate oxidoreductase (GOX) gene or the glyphosate acetyl transferase (GAT) gene. Onion plants containing a CaMV 35S-bar gene construct and the constitutively expressed CP-4-derived glyphosate resistance gene have been produced. All plants that have been confirmed by Southern analysis as containing the bar or CP-4-derived transgenes (one or two copies) have shown a strong tolerance to the herbicides Buster or Roundup , respectively. Initially, a 0.5% solution of the contact herbicide Buster was sprayed onto the leaves of the transformed onion plants. Plants that tolerated this treatment were then sprayed with commercially recommended concentrations of Buster for generalpurpose weed control to confirm their resistance to this herbicide (Eady et al. 2003). The level of resistance achieved indicated that the commercial production of transgenic onions containing a bar resistance gene is a feasible option for weed control in this crop. Glyphosatetolerant plants produced to date, which were tested in a similar manner (Eady et al. 2003), have proven to be tolerant to twice the recommended field application rates required for general weed control. F1 seed has recently been produced from these plants, and they are currently being field-tested for further assessment. The deployment of glyphosatetolerant lines could substitute for the application of toxic and persistent herbicides. Savings in herbicide usage of up to 75% for this crop have been projected, which equates to an economic saving of about U.S. $250 per hectare (Eady 2001). In addition, glyphosate is a short-lived, lowtoxicity herbicide compared with many of the persistent toxic herbicides that are currently used in many developed countries to control onion weeds. 2. Quality. The unique flavor and odor of alliums is derived from the hydrolysis of ACSOs [S-alk(en)yl-L-cysteine sulphoxides], which produces pyruvate, ammonia, and volatile sulfur compounds (Randle and Lancaster 2002). This reaction is catalyzed by the enzyme alliinase (alliin alkyl-sulphenate-lyase, E.C. 4.4.1.4), which is contained in vacuoles within cells and released upon disruption of the tissue (Lancaster and Collin 1981). Four different ACSOs have been identified in alliums (Bernhard 1970): ( þ )-S-methyl-L-cysteine sulfoxide, ( þ )-Spropyl-L-cysteine sulfoxide, trans-( þ )-S-(1-propenyl)-L-cysteine sulfoxide, and ( þ )-S-(2-propenyl)-L-cysteine sulfoxide (2-PECSO or alliin). Variations in the ratios of these volatile sulfur compounds are responsible for the difference in flavors and odors between Allium species (Randle and Lancaster 2002). Along with health and nutritional

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benefits associated with these compounds, these polyphenols are also major contributors to the bitter taste of some onions (Randle and Lancaster 2002; Almeida 2006). Three sets of transgenic onion plants containing antisense alliinase gene constructs (a CaMV 35S-driven antisense root alliinase gene, a CaMV 35S-driven antisense bulb alliinase, and a bulb alliinase promoter-driven antisense bulb alliinase) have been recently produced (Eady 2002). Results from the antisense bulb alliinase lines have been much more encouraging, and three lines were produced with barely detectable bulb alliinase levels and activity. Progress has been confounded by the poor survival of transgenic plants. Transgenic hybrid onion seed from these transgenic lines has been developed by crossing a nontransgenic open-pollinated parental line with a transgenic parental plant carrying a single transgene in the hemizygous state. Some resulting seed produced by the nontransgenic parents will be hemizygous for the transgene and can be selected to give F1 heterozygous individuals containing the transgene. Self-fertilization of these individuals produces homozygous, hemizygous, and null F2 progeny for the transgene locus. These homozygous individuals can then be used to generate the bulk seed required for the production of commercial transgenic lines. 3. Disease Resistance. Improving resistance to Sclerotium cepivorum, which causes allium white rot, can be achieved by overexpressing a germin protein with oxalate oxygen oxidoreductase (OXO) activity (Bidney et al. 1999) and the antimicrobial magainin (MGD) peptide (Zasloff et al. 1988). OXO degrades oxalic acid, the fungal toxin produced by S. cepivorum and many other plant fungal pathogens, to form carbon dioxide, hydrogen peroxide, and oxygen. The concomitant production of hydrogen peroxide further enhances host plant resistance (Peng and Kuc 1992; Levine et al. 1994). MGD peptides act by preferentially integrating into acidic phospholipid bilayers of microbial membranes, thus destabilizing the cell membranes and causing cell lysis. The tospovirus Iris Yellow Spot Virus is an emerging disease of allium crops that is spreading rapidly (Du Toit et al. 2004; Schwartz et al. 2007). DNA sequence information from this virus has been isolated (Pappu et al. 2006), which, in combination with gene-silencing technology, could be used to control this virus in the future. Multiple tospoviruses have already been targeted and successfully controlled in tomato using gene silencing (Bucher et al. 2006). Garlic mosaic virus, caused principally by the potyviruses Leek yellow stripe virus and Onion yellow dwarf virus, is another major viral disease of alliums (Takaki et al. 2005). The development of gene-silencing technology to combat this virus in garlic would


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revolutionize the production of garlic, which currently relies on costly continual regeneration of virus-free meristematic cultures in order to avoid the proliferation of garlic mosaic virus. 4. Insect Resistance. Transgenic garlic containing the Bacillus thuringiensis cry1Ca and H04 hybrid gene constructs produced at Plant Research International (PRI; Wageningen, the Netherlands) showed complete resistance to beet armyworm (Zheng et al. 2004). H. Sweet Corn Diverse maize types are ingredients of a wide range of traditional diets and provide a means for new market opportunities elsewhere in the world (Ortiz et al. 2007). A wide range of vegetable maize products are also harvested before maturity—most important of these are baby corn, sweet corn, and green-pick maize, of which the first two are traded internationally. Initial estimates of the global value of sweet corn, baby corn, and green maize suggest that maize is one of the five most profitable vegetables in the world (Ortiz et al. 2007). The ‘‘big five’’ producers of vegetable maize are China, the United States, Mexico, Peru, and Thailand. Sweet corn appears as the most popular specialty maize due to its high sugar content, conferred by the homozygous recessive sugary1 (su1) genes, in the kernels at the milky stage, which allows its harvest as vegetable (Ortiz et al. 2007). Another recessive mutant, shrunken-2 (sh2), may double the sugar content of the kernels at the roasting-ear stage. Slowing conversion of sugar into starch at ambient temperature reduces the need for refrigeration of the produce after harvest. Sweet corn genotypes combining the recessive allele sugary-enhancer (se) together with su1 can show twice the sugar content and phytoglycogen levels, thereby conferring a creamy texture. The global retail value of vegetable maize is estimated to range from U.S. $13 billion to U.S. $ 2 billion, which ranks second after tomato (U.S. $56 billion) and compares favorably to watermelon, onions, and brassicas (each worth about U.S. $18 billion). In 2004, about 148 countries were involved in the international trade of sweet corn products with a total value in excess of U.S. $573 million (for a total volume of 603,327 t), whereas frozen sweet corn exports of 65 countries included 244,618 t for a total value exceeding U.S. $212 million. Sweet corn, expressing cry1Ab endotoxin, was introduced commercially in the United States in 1998 into an industry that is highly sensitive to damage to corn ears from lepidopteran pests (Lynch et al. 1999). Research showed that this endotoxin was very effective against the European corn borer (Ostrinia nubilalis) in the state of

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New York, providing 100% clean ears when no other lepidopteran species were present and in excess of 97% when the two noctuids, corn earworm (Helicoverpa zea) and fall armyworm (Spodoptera frugiperda), were also present (Musser and Shelton 2003). Studies in other states in the United States have shown that Bt-sweet corn provided consistently excellent control of the lepidopteran pest complex and the potential for 70% to 90% reductions in insecticide requirement (Lynch et al. 1999; Burkness et al. 2001; Hassell and Shepard 2002; Musser and Shelton 2003; Speese et al. 2005; Rose and Dively 2007). Although it was estimated that of the 262,196 ha of sweet corn (fresh and processing) grown in the United States, less than 5% was Bt-sweet corn in 2006 (NASS, 2007); processors have avoided growing Bt-sweet corn due to concerns about export markets. Since then it has been grown only as a fresh-market vegetable crop. By using appropriately timed insecticide applications with Bt-sweet corn cultivars, fresh-market sweet corn growers in South and North Carolina have been able to extend their production later into the season when populations of H. zea and S. frugiperda are generally too high to control satisfactorily with insecticide applications alone (Hassell and Shepard 2002). Even when two insecticide sprays are required on Bt-sweet corn (e.g., for late season control of H. zea), an economic assessment in Virginia found a gain of US$ 1,777 ha1 for fresh-market sweet corn versus non-Bt-sweet corn sprayed up to six times with pyrethroid insecticides (Speese et al. 2005). Bt-sweet corn has also proven to be soft on the major predators of O. nubilalis, including the lady beetles Coleomegilla maculata and Harmonia axyridis, the hemipteran Orius insidiosus (Musser and Shelton 2003; Hoheisel and Fleischer 2007), and a complex of epigeal coleopterans (Leslie et al. 2007). Overall, Bt-sweet corn was much better at preserving these predators while controlling O. nubilalis than were the commonly used insecticides lambda-cyhalothrin, indoxacarb, and spinosad. Bt-sweet corn can replace the traditional method of controlling Lepidoptera with broad-spectrum insecticides. It may, however, allow secondary pests to arise. Results from these studies led to the development of a decision guide for sweet corn growers that uses information on biological control and advises them on the economic return of using various options, including Bt-sweet corn (Musser et al. 2006). These results demonstrate also that some of the new Bt-sweet corn hybrids allow a truly integrated biological and chemical pest control program in sweet corn, making future advances in conservation, augmentation, and classical biological control more feasible. The use of Bt-sweet corn has proven to be very effective against the targeted lepidopteran key pests, and plantings of Bt-sweet corn


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continue to rise in the United States, with new Bt-fresh-market hybrids being released each year. I. Cowpea Cowpea (Vigna unguiculata) remains as the most important legume of sub-Saharan Africa drylands, accounting for about two-thirds of the world’s harvest of this crop and providing a source of protein in the diet of 200 million Africans. Cowpea leaves as well green pods and peas are eaten as fresh vegetables too. Yield losses due to insect pests, particularly the legume pod borer (Maruca vitrata), can exceed 90% in cowpea (Murdock et al. 2001). A Bt transgene conferring resistance to this pod borer, bred into popular African cowpea cultivars, could eliminate the need for spraying insecticides, with significant advantages to smallholder growers (Simiyu-Wafukho et al. 2008). Popelka et al. (2006) adapted features of several legume and other genetic transformation systems to obtain transgenic cowpeas. These follow Mendelian inheritance for transmitting the transgene to their progeny. The rate of transgenic cowpeas that transmit the transgenes to their progeny was 1 fertile plant per 1,000 explants. Their ultimate aim is to incorporate two or more Bt genes into cowpeas to provide long-term protection against legume pod borer. The International Institute of Tropical Agriculture (IITA; Nigeria) and research partners started preliminary biosafety assessments that consider development of resistance by the target insect pest to the insecticidal protein expressed in the plant, negative effects of the insecticidal protein on nontarget organisms in the same agroecosystem (e.g., natural enemies or pollinators), accidental introduction of the gene expressing the toxic protein into wild relatives of cowpea (i.e., gene flow), and negative effects on human and animal health (Tamo 2009). In this regard, Pasquet et al. (2008) found that bees visited wild and domesticated cowpea populations, thereby mediating gene flow and, in some instances, allowing transgene escapes over several kilometers. Nonetheless, as stated by the authors of this gene flow research, most between-flower flights occur within plant patches, while very few occur between plant patches. Furthermore, when the plant patches are at least 50 m apart, the probability of gene flow by pollen appears to be low. Tamo (2009) indicates that there are several alternative host plants in the wild where M. vitrata is exposed to the attacks of natural enemies throughout the year, thereby providing natural refugia and thus avoiding potential negative impacts of the Bt-toxin from transgenic cowpea.

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J. Root Crops 1. Cassava. The root crop cassava (Manihot esculenta) is among the most important vegetatively propagated food crops for tropical agriculture, especially sub-Saharan Africa, but it is also important in Asia and Latin America (Ortiz 2005). The total production for 2007 was 224 million t, with Nigeria and Brazil accounting together for about onethird of the global production. More than 800 million people depend on this starchy root staple, also known as tapioca, manioc, or yuca (not to be confused with the succulent plant yucca) (Nassar and Ortiz 2010). In Africa and Latin America, cassava is used mostly for human consumption, while in Asia and parts of Latin America, it is also used commercially for the production of animal feed and starch-based products. Roots are processed into granules, pastes, and flours or eaten freshly boiled, fermented, or raw. The leaves are also eaten in Africa and some Asian locations as a green vegetable, which provides protein and vitamins A and B. Fregene and Puonti-Kaerlas (2002) highlight the potential of cassava biotechnology, including the potential for a more rapid and efficient improvement through genetic transformation. The first transgenic cassava plants became available in the mid-1990s (Li et al. 1996; Raemakers et al. 1996; Schopke et al. 1996) as plants with reduced cyanogenic content (Siritunga and Sayre 2003, 2004; Siritunga et al. 2004), resistance to infection by geminiviruses (Chellappan et al. 2003), modified starch content (Raemakers et al. 2003), enhanced starch production (Ihemere et al. 2006), elevated protein content within the storage roots (Zhang et al. 2003), and vitamin A (P. Chavarriaga, CIAT, pers. commun.). Transgenic cassava technology represents proof of concept for traits in this species, but its efficacy has been demonstrated only at the laboratory or greenhouse level (Taylor et al. 2004). The delivery of genetically modified planting materials to cassava farmers remains as a main challenge. The impact of transgenic cassava will depend on successfully transferring this capability to local cultivars. In this regard, there are advances for genetically engineering cassava cultivars from Africa (Hankoua et al. 2006; Ingelbrecht 2009) and Brazil (Ibrahim et al. 2008). 2. Sweet Potato. Sweet potato (Ipomoea batatas), the seventh most important food crop, ranks third among root and tuber staples worldwide, after sweet potato and cassava, with China accounting for 65% of total crop area. The total production for 2007 was about 100 million t. The crop is grown at high density in the central Africa highlands. Elsewhere in the tropics it is grown at a lower density. Protocols for


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genetic engineering of sweet potato to express the coat protein gene of Sweet potato feathery mottle virus (SPFMV)—one of the most serious constraints to the production and the quality of this crop when accompanied by other viruses, such as Sweet potato chlorotic stunt virus (SPCSV)—are available (Okada et al., 2001). Researchers at the Kenya Agricultural Research Institute in collaboration with Monsanto incorporated in the 1990s genes for resistance to SPFMV in eight sweet potato cultivars. Field-testing started in 2001 with materials already screened under containment in the greenhouse (Wambugu 2001). Ex ante analysis suggests a benefit ranging from U.S. $42.31 to U.S. $101.12 per acre (Qaim 1998; Marra 2001). Field trials showed, however, that transgenic sweet potatoes were no less vulnerable than ordinary cultivars to this virus, and sometimes their yields were also lower. Recently Cuellar et al. (2009) showed that transformation of an SPFMV-resistant sweet potato cultivar with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe symptoms. This is similar to the synergism in plants coinfected with SPCSV and SPFMV. They also indicated that RNase3-transgenic sweet potato plants also accumulated higher concentrations of two other unrelated viruses and developed more severe symptoms than nontransgenic plants. Their research provides some insights on how SPCSV causes the significant loss of sweet potato resistance to SPFMV. Transgenic herbicide-resistant sweet potato plants were also produced using the Agrobacterium-mediated transformation system (Jin Choi 2007). This technology may allow a more convenient and efficient weed control in the field than was previously available. Berberich et al. (2005) were able to breed transgenic sweet potato plants producing mouse adiponectin, an anti-diabetic protein whose large-scale production is sought for pharmaceutical applications. The production of adiponectin did not cause obvious differences in growth rate or morphology in the transgenic sweet potato plants. 3. Carrot. Vegetables offer consumers a diverse mixture of nutrients that promote human health more beneficially than dietary supplements. However, the ingestion of plant-based diets rather than diets that rely primarily on animal products could limit the intake of essential nutrients such as calcium (Ca). Consequently, genetically engineering vegetables containing increased Ca levels may boost Ca uptake, thereby reducing the incidence of Ca deficiencies such as osteoporosis. In this regard, Park et al. (2004) modified carrots to express increased levels of the plant Ca transporter sCAX1. These carrot lines were fertile and displayed no

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adverse phenotypes. Further mice and human feeding trials demonstrated increased Ca absorption from sCAX1-expressing transgenic carrots vis- a-vis controls (Morris et al. 2008). This research supports alternative means of biofortifying vegetables with bioavailable Ca.

IV. GM VEGETABLES AND INTEGRATED PEST MANAGEMENT Integrated pest management (IPM) can be defined broadly as ‘‘the careful consideration of all available pest control techniques and subsequent integration of appropriate measures that discourage the development of pest populations and keep insecticides and other interventions to levels that are economically justified and reduce or minimize risks to human health and the environment. IPM emphasizes the growth of a healthy crop with the least possible disruption to agro-ecosystems and encourages natural pest control mechanisms’’ (FAO 2002). Hence, GM vegetable crops that target one or a few key production pests and pack GM pest management technology into the seed provide a useful tool to be included with other IPM-compatible approaches. The excessive use of insecticides in response to a resistance problem also decreases the effectiveness of biological control agents and increases the risk of environmental and human health impacts, increasing the utility of GM vegetable cultivars with resistance to pests. For example, Musser et al. (2006) in New York State show how Bt-sweet corn combined with the action of auxiliary predators can provide a truly IPM system with only one foliar insecticide required. Both conventionally bred and GM insect-resistant plants have been developed with the objective of reducing pest densities below damage thresholds. If successful, reduced pest densities will inevitably lead to a reduction in the abundance of some natural enemies, particularly the parasitoids and predators that are host/prey specific to the target pest(s). This is an obvious and unavoidable consequence of virtually any pest management system, irrespective of the mechanism, and should not be of particular concern related to the use of GM plants (EFSA 2006; Kennedy and Gould 2007; Romeis et al. 2006, 2008a,b) The advantages of GM technology to improve vegetables, reduce pesticide use, increase yields, add health benefits, and lower production costs should provide incentive for integration of this technology into vegetable breeding and commercial crop production, if consumer resistance can be overcome or mollified. It would be expected that as consumers become more accustomed to other GM crops, concerns about GM vegetables are likely to lessen, and markets will accept the new products.


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However, the opposition to GM crops is well organized by advocates of the environmental and the organic movements. Vegetables, as minor crops by themselves, are unable to lead the campaign to change consumer acceptance of GM vegetables The production of vegetables worldwide tends to be on smaller areas and in more diversified holdings than field crops such as cotton, maize, soybean, rice, and wheat. Vegetables are often in more complex agricultural systems where insects may move from one crop to the next within the same farm. How this will impact the use and effects of GM vegetable plants in the agricultural landscape can be complex. Growing multiple insect-resistant GM vegetable plants in the same area and exposed of a polyphagous insect to the same Bt protein expressed in the different vegetable species will challenge conventional strategies developed for GM cotton or maize cultivars. Thoughtful consideration therefore will be needed before choosing which toxins vegetable plants should express. The selection should be based not only on what will be an effective toxin against the target insect but what toxins are already in use in other vegetable crops that may be hosts for the target insect. Additionally, the difficulty of sampling insect populations for resistant alleles will take on a higher level of complexity in a diversified vegetable system. Further consideration should also be given to the effects on nontarget organisms within diversified GM vegetable plantings. In a study conducted in the northeastern United States, Hoheisel and Fleischer (2007) investigated the seasonal dynamics of coccinellids and their food (aphids and pollen) in a vegetable farm system containing plantings of Bt-sweet corn, Bt-potato, and GM insect-resistant squash. Their results indicated that the transgenic vegetable crops provided conservation of cocinellids and resulted in a 25% reduction in insecticide use. In a similar study with these same crops, Leslie et al. (2007) compared the soil surface–dwelling communities of Coleopetera and Formicidae in the transgenic crops and their isolines and found no differences in species richness and species composition but found that the transgenic vegetables required fewer insecticide applications. Such results make clear that GM technology can be introduced within vegetable IPM systems and that GM vegetables can offer novel and effective ways of controlling insects and the pathogens they transmit. Virus-resistant transgenic plants are particularly valuable if no genetic source of resistance has been identified or if host resistance is difficult to transfer into elite cultivars by traditional breeding approaches due to genetic incompatibility or links to undesired traits. In such cases, engineered resistance may be the only viable option to develop virusresistant cultivars. Growers can also use virus-resistant transgenic

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vegetables as a trap crop by growing it as a border around the non-GM vegetable crop and allowing it to cleanse viruliferous aphids, as it is done in Hawaii with Papaya ringspot virus (PRSV)-resistant transgenic papaya (Gonsalves 1998; Fuchs and Gonsalves 2007). In Hawaii, the papaya industry—after two decades of field and biosafety testing—can now produce and market both transgenic and conventional papaya in the same field and even organic papaya in adjacent fields if other organic practices are performed. This is a case in which organic agriculture directly benefits from GM crops, which are not allowed as part of the organic production philosophy. In small, diversified vegetable plantings typical of those found throughout the developing world, the challenges for regulatory oversight of GM plants are immense. In these countries, farmers will likely save GM seed and move GM seed between locations, and some GM products may move into markets that do not permit these products. These concerns will be lessened if GM vegetable plants are consumed locally and in accordance with national biosafety regulatory policies. However, it is likely that violations will occur, and this will challenge legal systems. While each vegetable has its own set of one or more key pests, other pests can also be problematic. Traditional broad-spectrum insecticides often control a suite of pest insects. Thus, when Bt- or other GM vegetables are introduced into production systems, other methods of control will have to be applied or developed for secondary pests. Because the current GM technologies have proven to be less harmful to natural enemies, biological control of secondary pests may be more achievable, but other tactics, such as the use of selective insecticides, applied either as seed treatments or foliar sprays, may be necessary (Romeis et al. 2006, 2008a,b). In conclusion, GM vegetables can have a major role in the management of insects and the diseases they transmit. When the markets have allowed the production of GM plants, farmers have readily adopted the technology as part of their pest management practices. This is likely to continue with GM vegetables. IPM could benefit from some herbicide-resistant crops, if alternative nonchemical methods can be applied first to control weeds and the specific herbicide could be used later, only when and where the economic threshold of weeds is surpassed (Krimsky and Wrubel 1996). Generally, though, the use of herbicide-resistant crops will lead to increased use of herbicides and environmental and economic problems (Altieri 1998; McCullum et al. 1998; Pimentel and Ali 1998). Repeated use of herbicides in the same area may also create problems of weed herbicide resistance (Wrubel and Gressel 1994). In addition, farmers will suffer because of the high costs of employing herbicide-resistant


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crops—in some instances, weed control with herbicide-resistant crops may increase weed control costs for the farmer twofold (Pimentel and Ali 1998).

V. OUTLOOK Both pesticides and biotechnology have definite advantages in reducing crop losses to pests. At present, pesticides are used more widely than biotechnology, and thus they are playing a greater role in protecting world food supplies. In terms of environmental and public health impacts, pesticides probably have a greater negative impact at present because of their more widespread (and sometimes careless) use. GM vegetable crops for resistance to insect pests and plant pathogens could, in most cases, be environmentally beneficial, because these more resistant crops could allow a reduction in the use of hazardous insecticides and fungicides in crop production. In time, there may also be economic benefits to farmers who use genetically engineered crops; this will depend, though, on the prices charged by the biotechnology firms for these modified, transgenic crops. There are, however, some environmental problems associated with the use of genetically engineered crops in agriculture, as discussed. A major environmental and economic concern associated with genetically engineered crops may be the development of herbicide-resistant crops. Although in rare instances herbicide-resistant crops may result in a beneficial reduction of toxic herbicide use, it is more likely that the use of herbicide-resistant crops can increase herbicide use and environmental pollution. Of great interest and importance for the future of GM vegetables will be the course set by developing countries, since nearly 60% of the world’s vegetables are grown in China and India, which account for nearly 40% of the world’s population and where pest and viruses are severe. Both countries have readily adopted Bt-cotton, and it is likely that Bt-rice will be commercialized in China in the very near future, since China is the first country in the world to give biosafety approval (BSA) for the development of Bt-rice cultivars. Acceptance of GM field crops in these two large, highly populated countries will make it more likely they will adopt GM vegetables. This in turn likely will hasten their adoption in other parts of the world and allow farmers to use this technology. With the eventual acceptance of GM technology, it is expected that the costs associated with deregulation will become more affordable and that the biotechnology industry, in the hands of huge corporations, will become

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more interested in developing GM vegetables, especially for the developing world, where more than 80% of the world’s 6.8 billion people live. These populations will increase rapidly in the next several decades. Reasonable profit margins are necessary to pay back the research-fordevelopment costs, to fund future research on developing even better GM vegetable cultivars, and to stay competitive. The proportion of the economic benefits that accrue to the farmer, the consumer, and the technology company or corporation also varies among countries, depending on the degree of protection provided for intellectual property rights and the degree of government control over commodity prices. In the United States, plant breeding companies can take advantage of the U.S. utility patent law to protect not only the cultivar itself but all of the plant’s parts. Such product protection options have presented a business incentive to corporations to invest in the seed industry, leading to an enormous increase in private research and development, insuring strong competition in the marketplace among the major seed companies. Such patenting must be moderated to eliminate coverage so broad that it stifles innovation. Direct health benefits accrue from the reductions in insecticide use on Btcrops and other virus-resistant transgenic vegetables as a result of lower pesticide residues in food and water, and reduced exposure of farm workers and vegetable growers during pesticide applications. These benefits are especially great in developing countries in which pesticide regulation is weak, the education of farmers is generally low, and pesticides are usually applied manually. The full potential of GM technology to reduce exposure to pesticide residues in foods as not yet been realized because pesticide residues on food are of greatest concern in vegetables, and few insect-resistant GM vegetable crops are commercially available. Developing country public research is focused on crops for the poor and traditionally has been considered a government investment, with returns coming back to the public in the form of food security, better health, and greater subsistence farmer income, compared to product research by the private sector. Much of this research, however, has been supported by foundations and governments in the developed world. Unfortunately, support resources have diminished in the last few decades. The challenge, therefore, especially for developing country regulatory agencies, is to examine where data collection requirements can be reduced or streamlined without compromising the level of safety achieved by current developed world regulatory requirements, in order that the investments made by governments are fully realized. The GM eggplant in India is an excellent working example of a model pro-poor


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philanthropic-public-private sector partnership. Bt-egg plant technology has been generously donated by its private sector developer, Mahyco, to public sector institutes in India, Bangladesh, and the Philippines for incorporation in open-pollinated cultivars of eggplant for the use of farmers and especially small, resource-poor farmers. The regulation of Bt-eggplant in India is the key barrier that denies growers in this country timely access to the significant benefits that this biotech vegetable crop offers. Sharing of knowledge and of experience with the regulation process could greatly simplify and lighten the regulatory burden by eliminating duplication of this significant effort, thereby contributing to the important goal of harmonizing simplified, responsible, and appropriate regulations among countries. Molecular tools will be useful for selecting resistance genes and increasing quality, nutritional value, and yields. These value-added traits plus food safety will be important aspects of future GM vegetable breeding efforts. Biotechnology provides the ability to produce a broad array of insect-resistant and pathogen-resistant cultivars that also express a variety of other value-added traits, such as nutritional and postharvest traits. As the number of value-added, GM traits increases, the number of potential combinations of traits that could be stacked within individual cultivars increases geometrically, as to the cost associated with maintaining inventories of geographically adapted cultivars expressing different combinations of traits. Consequently, we can expect that commercially available, GM vegetable cultivars of the future will express multiple, unrelated, transgenic traits, and farmers in many cases likely will not have the option of planting cultivars expressing only single traits. To the extent that this occurs, insect-resistant GM vegetable crops are likely to be widely used in situations where they are neither needed nor appropriate. Then the decision to use an insect-resistant GM vegetable crop must be made prior to planting. It involves weighing the cost of implementing the technology against the risk of experiencing a yield-suppressing infestation of the targeted pest species during the season. The costs of using a GM vegetable crop for crop protection include both the fee premium charged for the GM trait and the costs, if any, associated with any undesirable agronomic characteristics of the GM cultivar compared to non-GM cultivars. The availability of transgenic vegetable crops does not, however, ensure that they will be adopted by growers (Ortiz and Smale 2007). To be widely adopted, the benefits of their adoption must exceed their costs for a large proportion of vegetable growers from one season to the next. As suggested by this chapter, the most promising traits seem to be host plant resistances to insects and pathogens,

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especially for vegetables such as eggplant, potato, summer squash, sweet corn, and tomato. Many current vegetable breeding efforts remain underfunded and disorganized. There is a great need for a more focused, coordinated approach to efficiently utilize funding, share expertise, and continue progress in horticultural GM technologies and programs. GM vegetable production can help the poor escape poverty and malnutrition in this 21st century, but only if enough investments are made to improve and sustain breeding and productivity of vegetable crops. Policy makers and investors (including international aid and philanthropy) have to turn their attention to enhanced funding for the vegetable and horticultural sector, allowing growers to compete with their products on a world market increasingly determined by market quality standards and phytosanitary concerns and regulations. Only then will the silent vegetable and horticultural revolution currently under way benefit a significant portion of the world’s poor nations, growers, and landless laborers and enable us to overcome poverty and malnutrition. We should not be fundamentalists. We must ensure that society will continue to benefit from the vital contribution that plant breeding offers, using both conventional and biotechnological tools. These efforts must be coordinated with continuing striving to bring vegetable growers and consumers into the modern world to share enjoyment of the fruits of modern biotechnology. LITERATURE CITED Abdeen, A., A. Virgo´s, E. Olivella, J. Villanueva, X. Avil es, R. Gabarra, and S. Prat. 2005. Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors. Plant Mol. Biol. 57:189–202. ABSP II (Agricultural Biotechnology Support Project II). 2009. www.absp2.cornell.edu/. Acciarri, N., G. Vitelli, S. Arpaia, G. Mennella, F. Sunseri, and G.L. Rotino. 2000. Transgenic resistance to the Colorado potato beetle in Bt-expressing eggplant fields. HortScience 35:722–725. Acord, B.D. 1996. Availability of determination of non-regulated status for a squash line genetically engineered for virus resistance. Federal Register 61:33484–33485. Agnihotri, N.P. 1999. Pesticide safety, evaluation and monitoring. All India Co-ordinated Research Project on Pesticide Residues, IARI. New Delhi, India. Alan, A.R., A. Blowers, and E.D. Earle. 2004. Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease. Plant Cell Rep. 22:388–396. Almeida, D. 2006. Manual de culturas hortıcolas. Vol. I. Editorial Presen¸ca, Lisbon. Altieri, M.A. 1998. The environmental risks of transgenic crops: an agroecological assessment. 5th Ann. ESSD Conference Proceedings. World Bank, Washington, DC. Alvarez, M.L., H.L. Pinyerd, J.D. Crisantes, M.M. Rigano, J. Pinkhasov, M. Amanda, A.M. Walmsley, H.S. Masona, and G.A. Cardineau. 2006. Plant-made subunit vaccine


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5 Millets: Genetic and Genomic Resources Sangam Dwivedi, Hari Upadhyaya, Senapathy Senthilvel, and Charles Hash International Crops Research Institute for the Semi-Arid Tropics Patancheru PO Hyderabad 502324, AP, India Kenji Fukunaga Faculty of Life and Environmental Sciences Prefectural University of Hiroshima 562 Nanatsuka, Shobara Hiroshima 727-0023, Japan Xiamin Diao Lab of Minor Cereal Crops Institute of Crop Sciences Chinese Academy of Agricultural Sciences 12 Zhongguancun South Street, Haidian Beijing 100081, People’s Republic of China Dipak Santra University of Nebraska–Lincoln Panhandle Research and Extension Center 4502 Avenue I Scottsbluff, Nebraska 69361, USA David Baltensperger Soil and Crop Sciences Texas A&M University 2472 TAMU College Station, Texas 77843-2474, USA Plant Breeding Reviews, Volume 35, First Edition. Edited by Jules Janick. 2012 Wiley-Blackwell. Published 2012 by John Wiley & Sons, Inc. 247

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Manoj Prasad National Institute of Plant Genome Research Aruna Asaf Ali Marg JNU Campus, PO Box 10531 New Delhi 110067, India

ABSTRACT Small-grained millets, comprising ten annual grasses from the family Poaceae and grown for grain, contribute 13% of annual global cereal production. Some are widely grown, while cultivation of others is restricted. They differ in ploidy, genome size, and breeding system, but their grains are all highly nutritious. Their most common nonfood uses are in brewing and as livestock feeds. Millets are C4 plants adapted to marginal lands in hot, drought-prone arid and semiarid regions. Selection for plant phenology and architecture, panicle shape, spikelet structure and reduced shattering, seed dormancy, and seed coat hardness contributed to their domestication. Approximately 161,708 millet accessions are preserved in gene banks globally. These show exceptional diversity associated for phenology, photoperiod sensitivity, tolerance to abiotic stresses, resistance to biotic stresses, seed storability and shelf life, and specific grain characteristics associated with end user preferences. Contributions from wild relatives’ toward enhancing cultivated gene pools have been limited to pearl millet and foxtail millet. Core or minicore/reference collections have been used to identify new sources of biotic stress resistances and abiotic stress tolerances. Waxy mutants have been selected in barnyard millet, foxtail millet, and proso millet for specific food uses. Pearl millet hybrids and open pollinated varieties (OPVs) with high iron and zinc grain densities will soon be available in India. While no transgenic work has reached field level, DNA markers are routinely used to assess millets’ population structure and genetic diversity. Genetic maps of varying density are reported in finger millet, foxtail millet, pearl millet, proso millet, and tef. Major quantitative trait loci associated with resistance to downy mildew, rust, and blast and tolerance to terminal drought stress have been backcrossed into elite inbred pearl millet hybrid parents. Markerassisted backcrossing has been used to improve downy mildew resistance in pearl millet. Cytoplasmic-genetic male sterility (CMS)–based hybrids of pearl millet are extensively cultivated, and CMS systems for foxtail millet are under development. An aligned genome sequence of foxtail millet will be released in the near future as this millet is closely related to several polyploid bioenergy grasses. This foxtail millet genome sequence is highly syntenic with those of rice, sorghum, and maize, which should allow comprehensive surveys of genetic diversity for identifying and conserving diversity in grass germplasm with bioenergy crop potential.

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KEYWORDS: diversity; domestication; genetic markers; genome synteny; phylogeny; population structure; quantitative trait loci; stress tolerance LIST OF ABBREVIATIONS I. INTRODUCTION II. NUTRITIONAL QUALITY AND FOOD, FEED, MEDICINAL, AND OTHER USES III. DOMESTICATION, PHYLOGENETIC, AND GENOMIC RELATIONSHIPS IV. ASSESSING PATTERNS OF DIVERSITY IN GERMPLASM COLLECTIONS V. IDENTIFYING GERMPLASM WITH BENEFICIAL TRAITS A. Resistance to Biotic Stresses 1. Phenotypic Screening 2. Natural Genetic Variation 3. Pathogen Variability, Mechanism, and Genetics of Resistance B. Tolerance to Abiotic Stresses 1. Drought 2. Salinity 3. Low Temperature 4. Lodging 5. Waterlogging C. Seed Quality VI. GENOMIC RESOURCES A. Markers and Genetic Linkage Maps B. Characterization and Functional Validation of Genes Associated with Important Traits C. Genomic and Genetic Tools to Sequence the Foxtail Millet Genome VII. ENHANCING USE OF GERMPLASM IN CULTIVAR DEVELOPMENT A. Core, Mini-Core and Reference Sets for Mining Allelic Diversity and Identifying New Sources of Variation B. Assessing Population Structure and Diversity in Germplasm Collections C. Promoting Use of Male Sterility as an Aid in Crossing VIII. FROM TRAIT GENETICS TO ASSOCIATION MAPPING TO CULTIVAR DEVELOPMENT USING GENOMICS A. Markers/QTL Associated with Agronomic Traits, Abiotic Stress Tolerance, Biotic Stress Resistance, and Product Quality B. Marker-Aided Introgressions of Disease Resistance C. Marker-Aided Introgressions to Enhance Drought Tolerance D. Use of Rice, Maize, Sorghum, and Foxtail Millet Genome Sequences to Strengthen Molecular Breeding Tools E. Exploiting Variation at Waxy Locus to Diversify Food Uses F. Foxtail Millet, Sorghum and Maize Genome Sequences as Resources for Identifying Variation Associated with High Biomass Production in Bioenergy Grasses IX. CONCLUSIONS AND FUTURE PROSPECTS ACKNOWLEDGMENTS LITERATURE CITED

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LIST OF ABBREVIATIONS AFLP AICPMIP BEP BP Bp cDNA cDNA–AFLP CISP CMS CO2 DArT DNA DM EST FAO WHO Fe GBSS I GCP HDL ISSR ITS LDL LG MABC Mbp MRL mRNA Na NC7 NSSL OA PACCAD PCR PGQO P5C QTL QTL-NIL

Amplified fragment length polymorphism All India Coordinated Pearl Millet Improvement Project Bambusoideae, Ehrhartoideae, Pooideae Before present Base pair Complementary deoxyribonucleic acid Complementary deoxyribonucleic acid–amplified fragment length polymorphism Conserved intron scanning primers Cytoplasmic-genetic male sterility Carbon dioxide Diversity arrays technology Deoxyribonucleic acid Downy mildew Expressed sequence tag Food and Agriculture Organization World Health Organization Iron Granule-bound starch synthase I Generation Challenge Program High-density lipoprotein Inter-simple sequence repeats Internal transcribed spacer Low-density lipoprotein Linkage group Marker-assisted backcrossing Million base pair Maximum root length Messenger ribonucleic acid Sodium North Central Regional PI Station National Center for Genetic Resource Preservation Osmotic adjustment Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae, Aristidoideae, Danthonioideae Polymerase chain reaction Plant Germplasm Quarentine Program Pyrroline-5-carboxylate Quantitative trait loci QTL near-isogenic line


RAPD rDNA RFLP RILs S9 SNP SSCP–SNP SSR TILLING Tr UPGMA VPD W6 WUE Zn

251

Rapid amplified polymorphic DNA Ribosomal deoxyribonucleic acid Restriction fragment length polymorphism Recombinant Inbred Lines Southern Regional PI Station Single-nucleotide polymorphism Single-strand conformation polymorphism–single nucleotide polymorphism Simple sequence repeat Targeting Induced Local Lesions in Genomics Transpiration rate Unweighted pair group method arithmetic mean Vapor pressure deficit Western Regional PI Station Water use efficiency Zinc

I. INTRODUCTION Cereals (rice, wheat, maize, barley, sorghum, millets, oats, rye, and triticale) contributed on average 255.1 million tonnes annually to world food production during the period from 2004 to 2008, of which the millet share was 12.7% (32.3 mt). Millets are comprised of a number of smallgrained, annual cereal grasses that include several distinct species: pearl millet (Pennisetum glaucum), finger millet (Eleusine coracana), foxtail millet (Setaria italica), proso millet (Panicum miliaceum), little millet (Panicum sumatrense), barnyard millet [Echinocloa crus-galli (Japanese) and E. colona (Indian)], kodo millet (Paspalum scrobiculatum), tef (Eragrotis tef), fonio [Digitaria exilis (white fonio) and D. iburua (black fonio)], guinea millet (Brachiaria deflexa), and Job’s tears (Coix lacrymajobi). Taxonomically, these millets belong to the Poaceae but differ either at species, genus, tribe, or subfamily hierarchy; ploidy levels (pearl millet and foxtail millet are diploids; finger millet, proso millet, tef, fonio, and Job’s tears are tetraploids; barnyard millet is hexaploid); genome size [foxtail millet has the smallest genome, 490 million base pair (Mbp) (Bennett et al. 2000) while finger millet, 2509 Mbp (Bennett and Leitch 1995) and pearl millet, 2352 Mbp (Bennett et al. 2000) have the largest genomes among other millets studied for genome size variation); and breeding systems (pearl millet being highly outbreeding, Job’s tears with mixed mating—inbreeding and outbreeding, and the

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remaining millets with high levels of inbreeding with some outcrossing (0.3%– 4%) in foxtail millet, Setaria italica, and its wild ancestor, S. virdis (Li et al. 1945; Till-Bottraud et al. 1992) (Table 5.1). Natural outcrossing in the range of 0.2% to 1% has also been reported for tef (Ketema 1993). Wild relatives of these millets possess even greater taxonomic diversity. For example, barnyard millet relatives vary from tetraploid to octaploid; those of finger millet are all diploid; relatives of foxtail millet and Job’s tears vary from diploid to octaploid; those of pearl millet from diploid to hexaploid; while kodo millet, little millet, proso millet, and tef are tetraploid (Table 5.2). Furthermore, both sexual and asexual (apomictic) forms of reproduction have been reported among pearl millet’s wild relatives. Most of these wild species are annuals; however, some of the foxtail millet and pearl millet wild relatives have both annual and perennial life-forms (Table 5.2). Other minor millets include Brachiara ramosa, Setaria glauca, Echinochloa turneriana, Echinochloa oryzicola, and Panicum hirticaule var. hirticaule (Hirosue and Yabuno 2002; Kimata et al. 2000). Brachiara ramosa is cultivated in pure stands while Setaria glauca in mixed stands along with little millet, and the grains are used as traditional foods in southern India (Kimata et al. 2000). The cultivated form of E. oryzicola is characterized by large spikelets with nonshattering habit and no innate dormancy (Hirosue and Yabuno 2002). The millets have abundant within-species racial diversity. In finger millet, there are five races (coracana, which resembles the subsp. africana, vulgaris, compacta, plana, and elongata) (Dida and Devos 2006) and 10 subraces (laxa, reclusa, and sparsa in elongata; seriata, confundera, and grandigluma in plana; liliacea, stellata, incuriata, and digitata in vulgaris). The race compacta in finger millet has no subraces. Foxtail millet has three races (moharia, maxima, and indica) and ten subraces (aristata, fusiformis, and glabra in moharia; compacta, spongiosa, and assamense in maxima; and erecta, glabra, nana, and profusa in indica). Proso millet has five races: miliaceum, patentissimum, contractum, compactum, and ovatum, while little millet (subsp. sumatrense) has two races, nana and robusta, each with two subraces: laxa and erecta in the former and laxa and compacta in the latter. Barnyard millet has two cultivated species, the Indian barnyard millet (Echinocloa colona) and Japanese barnyard millet (E. crus-galli), each with two ssp.: colona and frumentacea in the former and crus-galli and utilis in the latter. Subspecies colona has no races, while ssp. frumentacea has four races: stolonifera, intermedia, robusta, and laxa. Both ssp. crus-galli and utilis each have two races: crus-galli and macrocarpa in the former and utilis and intermedia in the latter. The three races in kodo millet are

253

Subfamily

Panicoideae

Chloridoideae

Panicoideae

Panicoideae

Maydeae

Panicoideae Panicoideae

Panicoideae

Panicoideae

Chloridoideae

Barnyard millet

Finger millet

Fonio

Foxtail millet

Job’s tears

Kodo millet Little millet

Pearl millet

Proso millet

Tef

Eragrosteae

Paniceae

Paniceae

Paniceae Paniceae

Andropogoneae

Paniceae

Paniceae

Eragrosteae

Paniceae

Tribe

Eragrostis

Panicum

Pennisetum

Paspalum Panicum

Coix

Setaria

Digitaria

Eleusine

Echinochloa

Genus

E. tef

P. miliaceum

P. glaucum

P. scrobiculatum P. sumatrense

C. lacryma-jobi

S. italica

D. exilis D. iburua

E. colona E. crus-galli E. coracana

Species

Taxonomic relationships of ten cereals belonging to millets group of crops.

Common name

Table 5.1.

Tetraploid

Tetraploid

Diploid

Tetraploid

Tetraploid

Diploid

Tetraploid

Tetraploid

Hexaploid

Ploidy

40

36

14

36

20

18

36

36

36

Chrom. no.

Wanous 1990; de Wet et al. 1983 Wanous 1990; Bisht and Mukai 2001 Adoukonou-Sagbadja et al. 2007; Wanous 1990 Wanous 1990; Bennett et al. 2000 Clayton 1981; Wanous 1990 Wanous 1990 Wanous 1990; Hiremath et al. 1990 Wanous 1990; Bennett et al. 2000 Baltensperger 1996; Hiremath et al. 1990; Zeller 2000 Wanous 1990; Ingram and Doyle 2003

Reference

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Finger millet E. indica (A genome) E. floccifolia E. tristachya E. intermedia E. verticillata E. multiflora E. jaegeri E. coracana subsp. africana E. spontanea E. kigeziensis

E. oryzoides

E. crusgalli

Barnyard millet E. colona

Species

18 18 18 18 18 16 20 36 Not reported 36

Diploid Diploid Diploid Diploid Diploid Diploid Tetraploid

Not reported Tetraploid

36

36, 54

36, 54, 72

Chromosome number

Diploid

Tetraploid, hexaploid, octaploid Tetraploid, hexaploid Tetraploid

Ploidy

Sexual Not reported

Not reported Not reported Not reported Not reported Not reported Not reported Sexual

Sexual

Sexual

Sexual

Reproductive behavior

Inbreeder Not reported

Not reported Not reported Not reported Not reported Not reported Not reported Inbreeder

Not reported

Inbreeder

Inbreeder

Mating system

Annual Perennial

Perennial Annual Perennial Not reported Annual Perennial Annual

Annual

Annual

Annual

Life form

Table 5.2. Differences in ploidy level, chromosome number, reproductive behavior, mating system and life form among selected wild relatives of millets species.

NRC 1996; Bisht and Mukai 2001; Neves et al. 2005; Anderson and de Vicent 2010

Wanus 1990; de Wet et al. 1983

Reference

255

Foxtail millet S. virdis (A genome) S. faberii (AB genome) S. verticillata (AB genome) S. glauca (S. pumela) S. adhaerans (B genome) S. holstii S. woodii S. chevalieri S. incrassata S. leiantha S. neglecta

Fonio D. longiflora D. ternata D. lecardii D. ciliaris reported reported reported reported

Sexual Sexual

36, 54 36–72 18 18 18 36 36 36 36

Tetraploid and hexaploid Complex ploidy

Diploid

Diploid Diploid Tetraploid Tetraploid Tetraploid Tetraploid

Sexual Sexual Sexual Not reported Not reported Not reported

Sexual

Sexual

36

Sexual

Not Not Not Not

Tetraploid

reported reported reported reported

18

Not Not Not Not

Diploid

Not reported Not reported Not reported Not reported

Inbreeder Not reported Not reported Not reported Not reported Not reported

Inbreeder

Inbreeder

Inbreeder

Inbreeder

Inbreeder


Perennial Not reported Perennial Not reported Not reported Not reported

Annual

Not reported

Annual

Annual

Annual


(continued )

Hacker 1967; Till-Bottraud et al. 1992; Le Thierry d’Ennequin et al. 1998; Benabdelmouna et al. 2001a; Wang et al. 2007b; Jia et al. 2009a; Wang et al. 2009; http://database. prota.org

Adoukonou -Sagbadja et al. 2007

256

palmifolia parviflora sphacelata macrostachya pumila

Job’s tears C. aquatica C. aquatica C. aquatica C. aquatica

S. finita S. sphacelata S. grisebachii (C genome) S. queenslandica (AA genome) S. verticillata (B genome) S. leucopila (A genome)

S. S. S. S. S.

Species

Table 5.2 (Continued)

18 18

Diploid

Diploid

10 20 30 40

36

Tetraploid

Diploid Tetraploid Hexaploid Octaploid

Not reported Not reported 18

36 36 18 to 90 54 36, 54

Chromosome number

Tetraploid Tetraploid Complex ploidy Hexaploid Tetraploid and hexaploid Not reported Not reported Diploid

Ploidy

Not Not Not Not

reported reported reported reported

Sexual

Not reported

Sexual

Not reported Not reported Sexual

Not reported Not reported Sexual Not reported Sexual



Inbreeder

Not reported

Inbreeder

Not reported Not reported Inbreeder

Not reported Not reported Outbreeder Not reported Inbreeder

Mating system


Annual

Not reported

Annual

Not reported Not reported Annual

Perennial Perennial Perennial Perennial Annual

Life form

Reviewed in Han et al. 2004

Reference

257

Tetraploid Triploid Hexaploid Tetraploid Tetraploid Tetraploid Diploid Hexaploid

P. P. P. P. P. P. P. P.

28 27 54 36 36 36 16 54

14 14 10

Diploid Diploid Diploid

purpureum setaceum setaceum villosum pedicellatum orientale mezianum squamulatum

14

36 36

49

Diploid

Tetraploid Tetraploid

Tetraploid

P. glaucum ssp. monodii P. violaceum P. mollissimum P. ramosum

Pearl millet

Little millet P. sumatrense P. psilopodium

Kodo millet Paspalum scrobiculatum

Sexual Sexual sexual and apomictic Sexual Apomictic Apomictic Apomictic Sexual Sexual Sexual Apomictic

Sexual

Sexual Sexual

Sexual

Inbreeder Inbreeder Inbreeder Inbreeder Inbreeder Inbreeder Inbreeder Inbreeder

Inbreeder Inbreeder Inbreeder

Inbreeder

Inbreeder Inbreeder

Inbreeder

Annual Annual Annual, Biennial Perennial Perennial Perennial Perennial Annual Perennial Perennial Perennial

Annual

Annual Annual

Annual

(continued )

Martel et al. 1997; http:// cropgenebank. sgrp.cgiar.org

Wanous 1990; Hiremath et al. 1990; Wanous 1990

Wanous 1990

258

Tetraploid

Tetraploid Not reported Not reported Not reported Not reported Not reported Not reported Not reported

Tef E. pilosa E. cilianensis E. ciliaris E. curvula E. cylendriflora E. gengetica E. tremula E. turgida

Ploidy

Proso millet P. miliaceum

Species


40 Not Not Not Not Not Not Not

36

reported reported reported reported reported reported reported

Chromosome number

Not Not Not Not Not Not Not Not

reported reported reported reported reported reported reported reported

Sexual


Not reported Not reported Not reported Not reported Not reported Not reported Not reported Not reported

Inbreeder

Mating system

Not reported Not reported Not reported Not reported Not reported Not reported Not reported Not reported

Annual

Life form

http://database. prota.org

Baltensperger 1996

Reference


259

regularis, irregularis and variabilis. All these races and subraces can be recognized by variation in panicle morphology (Prasad Rao et al. 1993). The two most recognized and widely cultivated species in fonio are white and black fonio, differentiated by seed color (Murdock 1959). The millets growing area worldwide has declined by 18% over a period of 45 years, from the average of 43.7 million ha in 1964 to 1968 to 35.82 million ha in 2004 to 2008; however, production during the same period has increased by 20.5%, from 26.9 million t in 1964 to 1968 to 32.3 million t in 2004 to 2008, largely due to increased productivity, which raised from 0.61 t ha1 in 1964 to 1968 to 0.9 t ha1 in 2004 to 2008 (Table 5.3). Globally, the millets are grown in 90 countries (http:// faostat.fao.org/). The major countries for production of millets are India, China, Nepal, Pakistan, and Myanmar in Asia; Burkina Faso, Cameroon, Chad, Ghana, Kenya, Mali, Namibia, Niger, Nigeria, Senegal, Sudan, Tanzania, Togo, Uganda, and Zimbabwe in sub-Saharan Africa; and Argentina and the United States on the American continent (Table 5.4). The production trends of 45 years (1964–2008) from these countries reveal interesting patterns. For example, China recorded the highest average annual production of 8.4 million t during the 1969–1973 period, which gradually declined to 1.7 million annual t in the period between 2004 and 2008. In contrast, India has shown a consistently upward trend in millets production, with marginal variation, increasing from 7.8 million t annually in the 1964 to 1968 period to 11.1 million t annually between 2004 and 2008 (i.e., an increase of 43%). The increased production of millets in India, particularly pearl millet with substantial production, is due to large-scale adoption of hybrid cultivars with inherent resistance/tolerance to biotic and/or abiotic stresses, which Table 5.3. Five-yearly averages of world area, production, and productivity of millets for the period from 1964 to 2008. Year 1964–1968 1969–1973 1974–1978 1979–1983 1984–1988 1989–1993 1994–1998 1999–2003 2004–2008

Area (million ha)

Production (million tons)

Yield (t ha1)

43.71 44.25 40.61 37.26 36.75 37.10 36.59 35.77 35.82

26.84 29.94 27.52 26.67 27.26 28.12 27.81 28.57 32.34

0.61 0.68 0.68 0.72 0.74 0.76 0.76 0.80 0.90

Source: http://faostat.fao.org.

260

1964–1968


South and Southeast Asia Afghanistan 0.024 Bangladesh 0.050 China 7.946 India 7.791 Myanmar 0.044 Nepal 0.108 Pakistan 0.386 Sub-Saharan Africa Burkina Faso 0.325 Cameroon 0.082 Chad 0.290 Ghana 0.074 Kenya 0.133 Mali 0.433 Namibia 0.020 Niger 0.875 Nigeria 2.435 Senegal 0.427 Sudan 0.299 Tanzania 0.119 Togo 0.133 Uganda 0.545 Zimbabwe 0.215 American continent Argentina 0.188 USA 0.137 CIS countries Ukraine 0.000 Russia 0.000

Country 0.035 0.043 6.454 9.491 0.050 0.137 0.304 0.360 0.090 0.241 0.128 0.129 0.485 0.030 0.947 3.175 0.521 0.458 0.231 0.096 0.598 0.186 0.278 0.088 0.000 0.000

0.319 0.083 0.236 0.113 0.130 0.418 0.026 0.894 3.041 0.400 0.382 0.131 0.132 0.701 0.186

0.167 0.137

0.000 0.000

1974–1978

0.029 0.056 8.356 9.901 0.044 0.132 0.335

1969–1973

0.000 0.000

0.214 0.112

0.401 0.090 0.163 0.117 0.062 0.511 0.036 1.307 2.570 0.486 0.339 0.358 0.047 0.473 0.131

0.032 0.055 6.299 9.677 0.122 0.120 0.248

1979–1983

0.000 0.000

0.112 0.164

0.617 0.047 0.238 0.135 0.050 0.775 0.047 1.274 3.780 0.565 0.304 0.304 0.072 0.467 0.169

0.026 0.088 5.300 8.754 0.172 0.147 0.222

1984–1988

0.260 1.331

0.081 0.178

0.726 0.061 0.216 0.140 0.059 0.752 0.043 1.675 4.624 0.567 0.245 0.235 0.071 0.598 0.106

0.023 0.064 3.814 10.009 0.129 0.239 0.176

1989–1993

0.220 0.617

0.051 0.190

0.791 0.064 0.282 0.175 0.044 0.760 0.063 1.848 5.572 0.534 0.622 0.274 0.055 0.565 0.076

0.022 0.057 3.143 10.102 0.146 0.274 0.192

1994–1998

0.268 0.773

0.035 0.291

0.972 0.052 0.378 0.160 0.057 0.885 0.060 2.328 5.948 0.575 0.588 0.189 0.045 0.591 0.040

0.021 0.028 2.106 10.227 0.168 0.282 0.207

1999–2003

0.200 0.660

0.014 0.319

1.106 0.060 0.497 0.163 0.068 1.136 0.060 2.874 7.745 0.469 0.644 0.226 0.043 0.707 0.048

0.017 0.016 1.746 11.142 0.181 0.288 0.251

2004–2008

Table 5.4. Five-yearly averages of the millets production from the major millets producing countries in South and Southeast Asia, sub-Saharan Africa, the American continent, and CIS countries for the period from 1964 to 2008.


261

have shown 25% to 30% yield advantage over open-pollinated varieties (Gowda and Rai 2006), while maize largely replaced millets in large acreage in China mainly due to its high yield potential, ease of cultivation, and better agronomic management practices including use of herbicides, thus reducing production cost (Diao 2007). Production of millets in Nepal almost tripled from the 1964–1968 period to the 2004–2008 period. In sub-Saharan Africa, Burkina Faso, Chad, Niger, Nigeria, Mali, Senegal, and Uganda are the largest producing countries, recording consistently increasing production. For example, millets production increased by 218% in Nigeria and by 240% in Burkina Faso, largely because of increased productivity (Table 5.4). Although the millet production in Niger and Mali increased by 228% and 162%, respectively, this increase probably was largely due to increased acreage. In many other sub-Saharan African countries, however, production either remained stagnant or has declined since the 1960s. The millets in these countries are still grown on marginal lands, low in soil fertility, poor crop management practices adopted, and unavailability of seeds of improved cultivars. The millets production in Argentina and America also showed variable trends. Production in Argentina reached its highest peak in the 1970s and then declined rapidly, with an average annual production of only 14,000 t for the 2004–2008 period. Annual millets production in the United States, except for periods in the 1970s and early 1980s, largely remained between 137,000 t to 319,000 t, and the highest average annual production was recorded for the period between 2004 and 2008. Millets production in Ukraine remained at below 300,000 t annually for the last 20 years while production declined by 50.4% in Russia. The economic development around the world brought dietary changes—those of hunter-gatherers containing large amounts of fiber and low amounts of sugar and fat to energy diets composed predominantly of highly processed foodstuffs, driven by a variety of culturally specific factors, including the increased production, availability, and marketing of processed foods and the complex effects of urbanization (Drewnowski and Popkin 1997; Popkin 2004, 2006; Finnis 2007). Global food consumption patterns have been shifting from food grains to highvalue crops/animal products in developing countries while it is from animal/fish-based to crop-based foods in the developed countries. Worldwide, per-capita cereal consumption declined by 5.6% between 1990 to 2003 while fruit consumption increased by 55% and vegetable consumption by 26% during the same period, with more pronounced effect noted in developing than developed countries. While meat, dairy, and seafood/fish consumption increased remarkably—55%, 29%, and

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44% in developing countries—it declined by 1.2%, 0.6%, and 11.5% in developed countries (https://www.ifama.org/events/conferences/2010/ cmsdocs/a72_pdf). Women’s opportunity cost of time—that is, the extent of women working outside the home generating income for the family— has also emerged as a key determinant in the shift from coarse-grain cereals to nontraditional grains (wheat and rice) and convenience foods (Senauer et al. 1986; Kennedy and Reardon 1994). For example, sustained economic growth, increasing population, and changing lifestyles has caused significant changes in the Indian food basket, away from staple foodgrains toward high-value horticultural products (Kumar et al. 2007; Mittal 2007). More important, the production of minor millets, for example, in the Kolli Hills region of Tamil Nadu, India, has declined substantially due to changing consumption preferences in favor of other crops, such as cassava, rice, and pineapple (Gruere et al. 2009). The erratic rainfall and drudgery associated with processing of minor millets also contributed to decline in production of these millets species (S.B. Ravi, MSS Research Foundation, Chennai, India). The changes in the dietary pattern also led to an increased demand of food grains as feed (Dikshit and Birthal 2010), with a steeper decline in per-capita consumption of coarse-grain cereals than that of rice and wheat, in both rural and urban India (Kumar et al. 2009). Millets productivity in the last five decades showed consistent increases in China, India, Burkina Faso, Nigeria, Uganda, Argentina, and the United States (Table 5.5). However, the percentage increase varied— 76% in China; 132% in India; 183% in Nigeria; 80% in Uganda; 40% in Argentina; and 20% in the United States. In Kenya, productivity remained on average at 1.7 t ha1 until the 1970s, but then substantially declined to 40% and 71% for the early 1980s and the last decade. In contrast, millets productivity remained constant at around 1 t ha1 in Nepal. Millet yield in Namibia among the African countries remained the lowest (0.20–0.30 t ha1) (Table 5.5). Isolated cases of very high grain yield under reasonably good management conditions have also been reported: finger millet grain yield as high as 4.2 t ha1 in Uganda (Odelle 1993), 6 t ha1 in Zimbabwe (Mushonga et al. 1993), 3.7 t ha1 in Ethiopia (Mulatu and Kebebe 1993), and 4 to 6 t ha1 in India (Seetharam and Prasada Rao 1989; Bondale 1993); foxtail millet grain yield as high as 9 t ha1 in China (Diao and Cheng 2008), and up to 11 t ha1 in breeding trial with the newly released hybrid cultivar ‘‘Zhangzagu 8’’ (Diao 2007). Pearl millet, finger millet, foxtail millet, and proso millet are grown widely (pearl millet in south Asia and sub-Saharan Africa; finger millet in South and Southeast Asia and East Africa; foxtail millet in South and

263

1964–1968


South and Southeast Asia Afghanistan 0.847 Bangladesh 0.870 China 1.150 India 0.404 Myanmar 0.289 Nepal 1.108 Pakistan 0.455 Sub-Saharan Africa Burkina Faso 0.443 Cameroon 0.750 Chad 0.586 Ghana 0.571 Kenya 1.788 Mali 0.745 Namibia 0.225 Niger 0.482 Nigeria 0.563 Senegal 0.459 Sudan 0.503 Tanzania 0.636 Togo 0.482 Uganda 0.911 Zimbabwe 0.557 American continent Argentina 1.106 USA 1.284 CIS countries Ukraine 0.000 Russia 0.000

Country 0.848 0.680 1.323 0.517 0.315 1.111 0.488 0.426 0.790 0.508 0.619 1.618 0.706 0.232 0.394 0.864 0.587 0.390 0.836 0.686 1.184 0.502 1.217 1.217 0.000 0.000

0.400 0.688 0.559 0.552 1.710 0.736 0.226 0.399 0.633 0.452 0.458 0.631 0.714 1.121 0.491

1.041 1.308

0.000 0.000

1974–1978

0.836 0.764 1.249 0.502 0.273 1.125 0.481

1969–1973

0.000 0.000

1.178 1.327

0.473 0.701 0.531 0.659 0.883 0.718 0.251 0.429 1.293 0.543 0.303 1.151 0.624 1.519 0.417

0.860 0.718 1.567 0.546 0.648 0.966 0.495

1979–1983

0.000 0.000

1.240 1.439

0.575 0.949 0.502 0.652 0.649 0.859 0.309 0.394 1.255 0.590 0.178 0.996 0.831 1.401 0.572

0.866 0.750 1.724 0.544 0.933 0.933 0.448

1984–1988

1.335 0.814

1.453 1.501

0.599 1.044 0.403 0.697 0.610 0.658 0.285 0.378 1.026 0.635 0.200 0.881 0.522 1.542 0.406

0.839 0.724 1.836 0.683 0.693 1.147 0.422

1989–1993

1.173 0.791

1.233 1.501

0.682 1.005 0.418 0.935 0.482 0.720 0.236 0.367 1.048 0.606 0.240 1.033 0.499 1.410 0.282

0.815 0.701 2.073 0.774 0.641 1.060 0.449

1994–1998

1.102 0.960

1.689 1.431

0.739 1.004 0.495 0.805 0.552 0.691 0.252 0.428 1.221 0.663 0.243 0.798 0.585 1.519 0.274

0.821 0.693 1.792 0.823 0.703 1.081 0.515

1999–2003

1.151 1.169

1.547 1.546

0.853 1.134 0.541 0.869 0.650 0.743 0.245 0.463 1.596 0.599 0.301 0.799 0.700 1.645 0.226

0.905 0.693 2.023 0.937 0.772 1.100 0.548

2004–2008

Table 5.5. Five- yearly averages of the millets productivity (t ha1) from the major millets-producing countries in South and Southeast Asia, sub-Saharan Africa, the American continent, and CIS countries for the period from 1964 to 2008.

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Southeast Asia; proso millet in Asia, Europe, and North America), while other millets are mostly confined to specific geographic regions: for example, fonio in West Africa; tef predominantly in Ethiopia; Job’s tears and barnyard millet in South and Southeast Asia; and little millet and kodo millet in South Asia (Table 5.6). India is the largest producer of pearl millet and finger millet, while China is the largest producer of foxtail and proso millet. Millets species are known by different vernacular names across regions and countries within regions (Table 5.7). Like other cereals, millets are also adversely affected by diseases, including downy mildew, rust, smut, ergot, and leaf blight in pearl millet; blast (leaf, neck, and finger) and leaf blight in finger millet; blast, downy mildew, rust, and leaf spot in foxtail millet; and rust, head smudge, and damping-off diseases in tef (Table 5.8). Major insect pest damage has been limited in millets but does impact regions of production. Proso millet is limited to less humid environments of the United States by chinch bugs, and this impact has been reported to impact pearl millet as well (Ni et al. 2009). Stem-boring insects have also been reported in proso, foxtail, and pearl millet (Adugna and Hofsvang 2000). Aphids have been limiting to grain and forage production and interact with the spread of plant viruses (www.ars.usda.gov/Research/ docs.htm?docid¼8927). Foraging insects, such as grasshoppers, also occasionally have been severe for proso millet in the U.S. Great Plains (Lyon et al. 2008) and pearl millet in Mali (Coop and Croft 1993). Some pest damage has been reported in tef and fonio from Africa, or during storage conditions. Additionally, the millets grain retains viability for long periods even under poor storage conditions. Most of the millets species are considered to be hardy crops adapted to marginal lands in the hot, drought-prone arid and semiarid regions of Africa, Asia, and the American continent (http://www.underutilized-species.org/documents/ millet_mssrf.pdf); however, drought and heat stresses adversely affect millets productivity. For example, postflowering drought stress in pearl millet causes substantial grain and stover yield losses (Mahalakshmi et al. 1987), and tef is highly sensitive to water stress during grain filling (Mengistu 2009). Lodging adversely affects finger millet, foxtail millet, proso millet, tef, and fonio production. Parasitic weeds, Striga spp., are serious constraints to finger millet, pearl millet, and fonio cultivation in Africa. Millets are C4 plants (Roder 2006; Osborne and Freckleton 2009), which have competitive advantage (better adaptation) over C3 plants under conditions of drought, high temperature, and nitrogen or carbon dioxide (CO2) limitation. C4 plants utilize their specific leaf anatomy, known as Kranz anatomy, to fix CO2 around rubisco, thus reducing photorespiration (Osborne and Beerling 2005). Millets are considered to provide more grain


Table 5.6.

265

Major regions/countries with substantial millets production.

Major geographical regions and countries with substantial production Barnyard millet South and Southeast Asia: China, Korea, Japan, India Finger millet South and Southeast Asia: India, China, Nepal, Myanmar, and Sri Lanka Eastern Africa: Uganda, Kenya, Sudan, and Eritrea Southern Africa: Zimbabwe, Zambia, Malawi, and Madagascar Central Africa: Rwanda and Burundi

Reference Prasad Rao et al. 1993

Prasad Rao et al. 1993; http:// afriprod.org.uk/ paper02obilana.pdf

Fonio West Africa: Benin, Burkina Faso, Chad, Guinea, Gambia, Mali, Nigeria, Senegal, and Togo

http://underutilized-species.org

Foxtail millet China, South and Southeast Asia: India, Nepal, Afghanistan, Korea, and Japan East Asia: China Other regions/countries: Russian Federation, USA, and France

http://hort.purdue.edu/ newcrop/proceedings1997/ v3-182html; Prasad Rao et al. 1993

Job’s tears South and Southeast Asia: Burma, China, India, Malaysia, the Philippines, Thailand, and Taiwan South America: Brazil

Venkateswarlu and Chaganti 1973; Wanous 1990 iat.sut.ac.th/food/FIA2007/ FIA2007/paper/P1-07-CP.pdf

Kodo millet South Asia: widely grown in India

Prasad Rao et al. 1993

Little millet South Asia: India (Eastern Ghats), Nepal, Myanmar, and Sri Lanka Pearl millet South Asia: India (Rajsthan, Gujarat, Maharashtra, Haryana and Uttar Pradesh), Afghanistan, Bangladesh, Myanmar, and Pakistan Sub-Saharan Africa: Grown in 28 countries with Nigeria, Niger, Burkina Faso, and Mali being the largest producers Proso millet India, China, Japan, Russia, Afghanistan, Iran, Iraq, Syria, Turkey, Mongolia, Romania, and USA (Nebraska, South Dakota, and Colorado)

Prasad Rao et al. 1993

Yadav 1996a; afriprod.org.uk/ paper02obilana.pdf

http://hort.purdue.edu/ newcrop/proceedings1997/ v3-182html; Wanous 1990 (continued )

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S. DWIVEDI ET AL.

Table 5.6 (Continued) Major geographical regions and countries with substantial production Tef Eastern and southern Africa: Ethiopia the major grain producer and the highlands of Eritrea; South Africa (both forage and grain), northern Kenya Europe and North America (small-scale grain production): USA, Canada, and the Netherlands Oceania: Australia (both grain and forage) Other countries: tef as forage in Morocco, India, and Pakistan

Reference database.prota.org

per unit of water than other cereals (Briggs and Shantz 1914; Felter et al. 2006). Millets grains are nutritious (see Section II) and commonly used for food in Asia and Africa, while in Europe and on the American continent, they are predominantly used as poultry feed. However, proso millet is a common ingredient in high-priced artisan breads sold in the United States, where there is a new ‘‘ancient grains’’ marketing niche. Millets straws are important sources of fodder in developing countries. Millets are also grown on the American continent as forage crops on light-textured or acidic soils throughout the tropical and subtropical lowlands and increasingly as a mulch component in no-till soybean production on the acidic soil savannahs of Latin America (http://www.cgiar.org/impact/research/millet.html). Millets are an underresearched crop commodity, especially compared with maize, which continues to push into previous millet cropping systems. Pearl millet, and to a lesser extent proso millet, finger millet, foxtail millet, and tef, have received greater attention from the research community to developing genetic and genomic resources for use in breeding, while in others only limited progress has been realized to date. This chapter is focused primarily on domestication and evolution of millets vis- a-vis other cereals; nutritional quality to diversify food uses; germplasm resources; sources of resistance to biotic and abiotic stresses and of agronomic and seed quality traits; diversity pattern in germplasm collections and formation of reduced subsets representing diversity present in entire germplasm collection of a given species to identifying new sources of variation; promoting use of male sterility to exploit heterosis; and genomic resources as an aid to marker-aided

267

Japanese barnyard millet (Echinocloa crus-galli), Indian barnyard millet (E. Colona), cockspur grass, Korean native millet, prickly millet, sawa millet, and watergrass Ragi in Hindi; tailaban in Arabic; petit mil and coracan in French; fingerhirse in German; wimbi and ulleji in Swahili; dagussa in Ethiopia; telebun in Sudan; bulo in Uganda; African millet, birdsfoot, hansa ragi, koracan, maduwa Hungry rice in English, fonio in French; acha in Nigeria, eboniaye in Senegal, findo in Gambia, podgi in Benin; crabgrass, fundi, and raishan. Italian millet; German millet; Russian millet; Hungarian millet; awa in Japanese; Siberian millet, dawa in Indonesia, shao-mi, su and kou wei tsao in China; mohar in Russia; millet des oiseaux and millet d’Italie in French; panico, milho panico, and milho panico de Italica in Portaguese; kimanga in Swahili Hortus, magharu, shoriew, mim (arora), trigo tropical (Joyal), attabi (Bodner), walln€ ofer adlay in the Philippines; hatomugi, mayuen, or Chinese pearl barley in China kodo in Hindi, khoddi in Urdu, arugu in Telugu, and varagu in Tamil, all Indian languages; African bastard millet grass, arika, haraka, ditch millet in New Zealand, and mandal in Pakistan Samai in Tamil (India); sama (little or slender) (India)

Bajra, bajri, bulrush millet, cattail millet, babala, bulrush, seno, spiked millet, cumbu, gero, munga; dukhun in Arabic; mil a chandelles in French; mijo perla in Spanish Broomcorn millet, common millet, hog millet, Hershey millet, white millet, creeping paspalum, ditch millet, Indian paspalum, water couch, brown corn, Russian millet; huang mi, mi tzu, and shu in Chinese Tef, t’ef, teff grass, and/or Williams lovegrass in English, French and Portuguese; tahf in Arabic

Barnyard millet

Pearl millet

Tef

Proso millet

Little millet

Kodo millet

Job’s tears

Foxtail millet

Fonio

Finger millet

Other vernacular names

Common name

Arunachalam et al. 2005; Wanous 1990 Yadav 1996a; Wanous 1990; http://www.sik.se/traditional grains/review/ Prasad Rao et al. 1993; Wanous 1990; http://www.sik. se/traditional grains/review/ http://database.prota.org; NRC 1996; Wanous 1990

http://plantsforuse.com; http:// iat.sut.ac.th/food/FIA2007/ FIA2007/paper/P1-07-CP.pdf Prasad Rao et al. 1993; de Wet et al. 1983; Wanous 1990

http://database.prota.org; Wanous 1990; Austin 2006

NRC 1996; Wanous 1990

Wanyera 2007; NRC 1996; Wanous 1990

Prasad Rao et al. 1993; Wanous 1990

Reference

Table 5.7. Vernacular names of barnyard millet, finger millet, fonio, foxtail millet, Job’s tears, kodo millet, little millet, proso millet, and tef as known in different regions.

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Table 5.8. Major biotic constraints reported in barnyard millet, finger millet, fonio, foxtail millet, Job’s tears, kodo millet, little millet, pearl millet, proso millet, and tef. Biotic stress Barnyard millet Grain smut (Ustilago panici-frumentacei Brefeld) Finger millet Leaf, neck and finger blast (Pyricularia grisea); leaf blight (Heliminthosporium nodulosum); shoot fly (Atherigona milliaceae) and pink stem borer (Sesamia inferens) Fonio Insect causing severe leaf and stem damage Foxtail millet Blast (Pyricularia setariae); downy mildew (Sclerospora graminicola); rust (Uromyces setariae-italiae); smut (Ustilago crameri); leaf spot (Helminthosporium spp.); shoot fly (Atherigona spp.); seed smut (Sorosporium bullatum), kernel smut (Ustilago paradoxa); and wheat curl mite (Eriophyes tullipae Keifer) and wheat streak mosaic virus reported from USA Job’s tears Leaf blight (Pseudocochlibolus nisikadoi)

Kodo millet Head smut (Sorosporium paspali); rust (Puccinia substriata Ellis and Barht); smut (Ustilago crus-galli, U. paradoxa and U. panici-frumentacei) Little millet Rust (Uromyces linearis) Pearl millet Downy mildew (Sclerospora graminicola); smut (Moeszimyoces penicillariae); ergot (Clavisceps fusiformis); leaf blight (Pyricularia grisea and Bipolaris setariae); rust (Puccinia substriata); head caterpillar (Heliothis albipunctella); scarab beetle (Pachnoda interrupta (Olivier)), stem borer (Acigona ignefusalis (Hamps.), and striga (Striga hermonthica) Proso millet Head smut (Sphacelotheca destruens); bacterial spot (Pseudomonas syringae), smut (Sphacelotheca panici milliacei), wheat curl mite (Eriophyes tullipae) and wheat streak mosaic virus reported from USA

Reference Gupta et al. 2009a Sreenivasaprasad et al. 2007; cropgene bank.sgrp.cgiar.org

Adoukonou-Sagbadja et al. 2006 Brink 2006; Siles et al. 2004; http://www.hort.purdue.edu; http://database.prota.org

http://www.nilgs.affrc.go.jp/db/ diseases/contents/de40. htm#cm%20leaf%20blight Viswanath and Seetharam 1989

Viswanath and Seetharam 1989 crop.sgrp.cgiar.org; de

ianpubs.unl.edu/live/ec137/ build/ec137.pdf; Ilyin et al. 1993; Baltensperger 1996

5. MILLETS: GENETIC AND GENOMIC RESOURCES Table 5.8

269

(Continued)

Biotic stress Tef Diseases: Rust (Uromyces eragrostidis); head smudge (Heliminthosporium miyakei); damping off (Drechslera spp., and (Epicoccum nigrum) Pest: Wollo bush-cricket (Decticoides brevipennis); red tef worm (Mentaxya ignicollis); black tef beetle (Erlangerius niger); grasshoppers, ants, and termites

Reference database.prota.org

gene introgression of food, feed, and bioenergy traits for product development.

II. NUTRITIONAL QUALITY AND FOOD, FEED, MEDICINAL, AND OTHER USES Millets grains are nutritionally equivalent or superior to other cereals (Mengesha 1965; FAO 1972). The grains contain high amounts of carbohydrates, proteins, minerals, and vitamins. For example, high levels of protein, calcium, iron, and zinc are found in finger millet, foxtail millet, and fonio; methionine, iron and zinc in pearl millet; methionine and/or cysteine in finger millet and fonio; iron in tef; tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucine, and isoleucine in foxtail millet (Ode et al. 1993; de Lumen et al. 1993; NRC 1996; Malleshi and Klopfenstein 1998; Fernandez et al. 2003; Khairwal et al. 2004; Alaunyte et al. 2010; database.prota.org; http://www. underutilized-species.org/documents/millet_mssrf.pdf). Millets gains are therefore recommended for lactating women and for diabetic (non-insulin-dependent) and sick people (Kumari and Sumathi 2002). Diets containing proso millet protein concentrate raise plasma levels of high-density lipoprotein (HDL) cholesterol without causing an increase in low-density lipoprotein (LDL) cholesterol levels in rats and mice (Nishizawa et al. 1990; Nishizawa and Fudamoto 1995; Shimanuki et al. 2006; Park et al. 2008). Furthermore, Nishizawa et al. (2009) reported the beneficial effects of dietary Japanese barnyard millet protein on plasma levels of adiponectin, high-density lipoprotein (HDL) cholesterol, glucose, and triglycerides in obese diabetic mice. Foxtail millet grain has high protein and iron contents compared to rice, wheat, and maize. Not only is the biological value of digestible

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protein higher than in rice and wheat, seven of the eight essential amino acids, which cannot be synthesized by the human body, are higher in foxtail millet (Zhang et al. 2007a). Edible fiber is important for intestine and stomach health. Foxtail millet grain contains 2.5 times the edible fiber found in rice and thus is a promising source for edible fiber (Liang et al. 2010). Foxtail millet bran contains 9.4% crude oil and is rich in linoleic (66.5%) and oleic (13.0%) acids (Liang et al. 2010). Millets fodders are highly nutritious and palatable and are fed to animals in Asia, Africa, and the American continent. From ancient times (>7000 years BP), foxtail millet has been in use for grain (for use by human) and hay production (for cattle and horse feeding) in China (Diao 2007). Some of the foxtail millet cultivars specifically bred for hay production in China contain as high as 15% protein (Zhi et al. 2011). Some brown-midrib (bmr) mutants in pearl millet have shown increased in vitro dry matter digestibility compared to normal cultivars (Cherney et al. 1988; Akin and Rigsby 1991), and have potential as sources of improved forage quality. Millets being C4 plants have great potential for biomass production; for example, biomass of pearl millet can yield 6 to 12 t ha1 on a dry-weight basis in less than 100 days (Khairwal et al. 2004). Hall et al. (2004) reported substantial genetic variation for stover quality and quantity without detrimental effect on grain yield in pearl millet. Millets are also considered sacred crops in some communities/ regions, where they play a central role in social events and celebrations. Because of its long cultivation history and great contribution to Chinese ancient civilization, foxtail millet was named ‘‘first’’ among the ‘‘Five Grains of China’’ (Austin 2006), which also include proso millet, rice, soybean, and wheat. Foxtail millet is used even today in ancestor worship ceremonies. In developing countries, in both Africa and Asia, the dry stalks of millets are used for fuel, thatching houses, constructing fences, and making mats. Job’s tears seeds are used as decorative beads to make necklaces and rosaries (Table 5.9). Substantial variations in seed composition of proso millet, finger millet, and foxtail millet cultivars have been reported. Ravindran (1991) reported higher seed protein (14% to 16%) and crude fat (5% to 8%) in proso millet and foxtail millet than in finger millet (protein 10% and crude fat 1.6%). Finger millet, however, had higher carbohydrate (81%) levels than those reported for proso millet and foxtail millet (70% to 74%), while all three millets had similar (4%) fiber contents. Ravindran (1991) also reported high calcium and potassium contents in finger millet grains, while other minerals, such sodium, magnesium, and phosphorous, were similar across these three millets. Regarding the

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Finger millet Flour to make bread (chapatti); porridge; popped grains as snacks; whole grains cooked as khichadi; sprouted grains; dosa, a thin fermented pancake containing blackgram

Barnyard millet Flour to make bread (chapatti); porridge; popped grains as snacks

Food

Both grain and/or stover used for animal feed including caged birds and poultry

Straw superior to rice and oat straw because of high protein and Ca content (Yabuno 1987)

Feed

Highly recommended diet for lactating women, diabetic people, and sick people

Unknown

Medicinal uses

Grains brewed for beer

Unknown

Beverage

Unknown

Unknown

Other uses

(continued )

Taylor and Emmambux 2008

Reference

Table 5.9. Food, feed, medicinal and industrial uses of barnyard millet, finger millet, fonio, foxtail millet, Job’s tears, kodo millet, little millet, pearl millet, proso millet and tef grains, and stover.

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Foxtail millet Dehusked grain for steamed food and porridge or gruel; flour to make bread (chapatti); porridge; popped grains as snacks

Fonio Porridge; tuwo; fonio-beans prepared on special occasions; couscous; both black fonio and white fonio used to make couscous ‘‘wusu-wusu’’; bread; popped

Food


Both grain and/or stover used for animal feed including caged birds and poultry; hay production

Straw and chaff used a fodder; hay

Feed

Pregnant and lactating women; prevention of diabetics; bran oil for skin diseases; dietary fiber for prevention of stomach and intestinal diseases

Grain is regarded as medicinal (i.e., antithyroid, chronic diarrhea, dysentery, chickenpox, stomachache, asthma) and healing properties; highly recommended diet for lactating women, diabetic people, and sick people

Medicinal uses

Huangjiu or yellow wine—-alcoholic drink; xiaomiyin— nonalcoholic drink

Grains brewed for beer, named locally as tchapalo, tchoukoutou, pito and burukuto

Beverage

Decoration; thatching houses

Straw and chaff mixed with clay to build houses; sacred crop that plays central role in social events/ celebrations; grains used as an important part of dowry in Sahelian communities

Other uses

Sema and Sarita 2002; Li 2005; Austin 2006; Diao 2007; Zhang et al. 2007a

http://www. underutilizedspecies.org; NRC 1996; AdoukonousSagbadia et al. 2006

Reference

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Little millet Flour to make bread (chapatti); porridge; popped grains as snacks

Kodo millet Flour used to make chapatti or flat cake/bread

Job’s tears Porridge


Straw as fodder

Foliage as green fodder to animals

Not known

Not known

Anodyne; anthelmintic, antiinflammatory; antipyretic; antirheumatic; antispasmodic; cancer; hypoglycemic; diuretic; pectoral; sedative; tonic; warts; appendicitis; rheumatoid arthritis; menstrual disorders

Not known

Not known

Tea from boiled seed as drink to cure warts; soup; grains for brewing beer ‘‘dzu’’; vines; coffee made from roasted grains

Not known

Not known

Seeds as decorative beads to make necklaces and rosaries; stems to make matting

(continued )

http://www.pfaf. org/database/ plants.php? Coix þ lacrymajobi; http://www. waynesword. palomar.edu/ plapr99.htm

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Tef A flat, spongy, and slightly sour bread, injera; porridge; gruel (muk)

Proso millet Flour to make bread (chapatti); porridge; popped grains as snacks

Pearl millet Flour to make bread (chapatti); porridge; boiled and/or roasted grains; baked food; weaning mixture; diabetic product; couscous or arraw (steamed product)

Food


Tef straw as animal feed



Feed

Gluten-free grains for health food

Birdseed

Gluten-free grains to use in health food

Medicinal uses

Grains brewed to make alcohol

A popular alcoholic beer, bosa, in Balkans, Egypt, and Turkey

Nonalcoholic— oshikundu in Namibia and kunun zaki in Nigeria Alcoholic— ndlovo beer in Bulawayo and Zimbabwe

Beverage

Hay

Hay

Dry stalks used for firewood, thatching houses, constructing fences, and making mats

Other uses

http://database. prota.org; Stallknecht et al. 1993

Baltensperger 1996; Lyon et al. 2008

Andrews and Kumar 1992; Khairwal et al. 2004; Taylor and Emmambux 2008

Reference


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trace elements, both proso millet and foxtail millet had high manganese, zinc, and iron contents, while all the three millets had similar copper contents. Most millets grains contain some antinutrients in their seeds. The major antinutrients include polyphenols, phytic acid, and oxalic acid. Phytates decrease the bioavailability of minerals such as calcium, iron, and zinc, while oxalic acid reduces calcium availability (Reddy et al. 1982). Ravindran (1991) found that finger millet grains have less phytic acid than that present in proso millet and foxtail millet, while foxtail millet grains contain high amounts of oxalate. To date, no antinutrients from barnyard millet and kodo millet have been reported. Among all millets, Kodo millet has the highest free radical quenching potential, thus possessing good antioxidant property (Taylor and Emmambux 2008). Some people are allergic to gluten present in cereals; for example, gluten in wheat causes severe allergies. Unlike foxtail millet (Sakamoto 1987), pearl millet, tef, some proso millet, fonio, and barnyard millet grains are gluten-free and therefore offer good opportunities for their use as health foods (NRC 1996; Gulia et al. 2007b; Hoshino et al. 2010). The association of a mycotoxin with ‘‘kodua poisoining’’ was reported when kodo millet (Paspalum scorbiculatum) grains infected with Aspergillus flavus or A. tamarii were used as food or feed. Both fungi produce cyclopiazonic acid, which results in kodua poisoning in man (Rao and Husain 1985), which result sleepiness, tremors and guiddiness (Bhide 1962). Grain from millets has also shown high potential for milling, popping, and malting. Malleshi and Desikachar (1985) demonstrated that millets could be milled to remove the outer bran (husk) and such milled grains could be easily cooked for consumption. The popped products have potential for use in development of breakfast and specialty foods (Srivastava and Batra 1998; Srivastava et al. 2001; Singh and Sehgal 2008). The millets grains, especially pearl millet, finger millet, foxtail millet, proso millet, and Job’s tears, are locally brewed, both in Africa and Asia, to produce alcoholic and nonalcoholic beverages (Table 5.9). Malting and fermentation processes result in malted and brewed alcoholic or nonalcoholic products. Huangjiu, an alcoholic drink made from brewing foxtail millet or proso millet grain, was very popular in ancient China and is still popular in some parts of northern China. Malted pearl millet and finger millet are used in brewing of the traditional opaque African beer in southern and eastern Africa. Finger millet provides the best-quality malt, which is used in the brewing industry in southern and eastern Africa as well as in south and southeast Asia and for making highly digestible nutritious foods.

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Foods prepared from millets are of several types that differ between countries and regions (Table 5.9). Because of their long cultivation and use as food, a number of different methods of consumption have been developed using foxtail millet and proso millet in China. The most popular dish from these millets are dehusked grain (referred to as miaomi) steamed or used to make gruel and porridge. Flour from foxtail millet and proso millet is used to make bread, pancakes, chapattis, and snacks. Steamed bread made from composite flour containing foxtail millet, wheat, and soybean has gained prominence in northern China; it not only tastes good but is also nutritious (Diao 2007). Food dishes from pearl millet in western Africa vary by countries: thick porridge (tuwo) is most popular in Sahelian countries while thin porridge and steamed products (couscous) are also consumed in Francophone countries. Tef and fonio are mostly used for porridges and flat breads. For example, injera, the soft, spongy, thin pancakelike bread with a sour taste made from tef flour, is the major staple food in Ethiopia. This traditional milletbased food has recently gained ground in Europe, North America, and Israel. Traditional foods made from pearl millet in India include chapatti or roti, porridges, and roasted/boiled grains eaten as snacks (Khairwal et al. 2004). European and American multigrain breads frequently use dehulled proso millet. Grain color is an important seed quality trait that influences the overall grain quality that determines the end use pattern of millets. Grain color in pearl millet ranges from ivory, to cream, to gray and brown. The major grain colors in other millets include white and black in fonio; white, red, and brown in tef; white and brown in finger millet; yellow, red, gray, black, and white in foxtail millet; white, cream, straw, olive, red, black, and brown in proso millet; and straw, olive, brown, and gray in little millet. Moreover, variation in grain color is associated with variation in quality traits and trade value. For example, tef grains with dark color are rich in flavor (NRC 1996); white-colored finger millet grains contain higher protein and iron contents but are lower in fiber and tannins (Seetharam et al. 1984; Rao 1994); black-colored finger millet grains contain only half as much iron and one-tenth as much molybdenum as reported for white-colored finger millet grains (Fernandez et al. 2003; Glew et al. 2008); dark-colored proso millet grains have higher tannin contents than those with light color (Lorenz 1983). White-grained finger millet and foxtail millet grains get high premiums in trade (C. R. Ravishanker, pers. commun.). Red- and brown-seeded tef are harvested from plants that are hardier, faster maturing, and easier to grow (NRC 1996).


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Millets have medicinal values for treating complex diseases (Table 5.9). Foxtail millet is widely used not only as an energy source for pregnant and lactating woman but also for sick people and children and especially for diabetics. It is reported to reduce blood sugar concentration in female diabetics (Sema and Sarita 2002). Job’s tears grains are most popularly used in Chinese traditional medicine because of their anti tumor and anti-allergenic, probiotic, and hypolipidomic properties while fonio reportedly has healing properties. It is suggested that the low incidence of anemia in the Ethiopian population can be attributed to the high consumption levels of tef, which has high iron content (NRC 1996). Utilization of whole-meal cereals including the seed coat in food formulations is increasing worldwide, since these are rich sources of phytochemicals and dietary fiber, which offer several health benefits. Regular consumption of finger millet is known to reduce the risk of diabetes (Gopalan 1981) and gastrointestinal tract disorders (Tovey 1994), which could be attributed to polyphenols and dietary fiber present in its grains. In China, foxtail millet is used to cure rheumatism. Proso millet protein concentrate, when fed for 21 days to rats, was shown to increase plasma levels of HDL cholesterol without an increase in lowdensity lipoprotein (LDL) cholesterol compared with a casein diet, which (HDL) may have a beneficial effect against the risk of coronary heart disease (Shimanuki et al. 2006). Furthermore, finger millet and proso millet may prevent cardiovascular disease by reducing plasma triglycerides in hyperlipidemic rats; in contrast, sorghum increases total cholesterol and HDL and LDL cholesterol concentrations (Lee et al. 2010). Inhabitants of southeast Asia and eastern Asia prefer sticky food. Amylose is an important starch in cereals including millets. Foods made from waxy grains are much stickier than those obtained from nonwaxy grains due to differences in amylose content. Large variations in the waxy phenotype has been reported in several cereals including foxtail millet, proso millet, and Job’s tears. This presents opportunities to diversify food uses of millets using allelic variation at the waxy locus (see Section VIII.E).

III. DOMESTICATION, PHYLOGENETIC, AND GENOMIC RELATIONSHIPS The comprehensive overview of grass phylogenetic relationships stems from the Grass Phylogeny Working Group (GPWG 2001). A simplified representation of one of the combined analyses, using morphological

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BEP clade

Bambusoideae

bamboos

Ehrhartoideae

Rice (cultivated and wild)

(Brachypodieae)

brachypodium

(Aveneae) Pooideae (Poeae)

oat

(Triticeae)

wheat, barley, and rye

ryegrass and fescue

© 60-80 mya Aristoideae

Danthonieae Arundinoideae

PACCAD clade

Chloridoideae

finger millet and tef

Centothecoideae (Paniceae) Panicoideae

foxtail millet, pearl millet, and common millet (Proso millet)

(Andropogoneae)

maize, sorghum, sugarcane, and Job’s tears

Fig. 5.1. Phylogenetic relationships of the crown group of grasses. Taxon terminal names are subfamilies, with tribes in parentheses. (Source: Adapted from Doust 2007).

and molecular data sets, revealed that the earliest diverging lineages of basal grasses were from a few species and that cereal and forage crops were domesticated from many different grass groups (Fig. 5.1). The members of ‘‘crown’’ (C) group of grasses, which have two large clades, the BEP and PACCAD (acronyms composed of the initial letters of the included subfamilies), diverged from one another 60 to 80 million years ago (Crepet and Feldman 1991; Prasad et al. 2005). The BEP clade is comprised of the basal subfamily Bambusoideae (bamboos) sister to Ehrhartoideae (wild and cultivated rice) and Pooideae (wheat, oats, barley, etc.). This large group of 4200 species is sister to another clade (PACCAD clade) comprised of the Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae, Aristidoideae, and Danthonioideae subfamilies. The Panicoideae has two tribes, the Paniceae, containing


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the foxtail millet, pearl millet, and proso millet, and the Andropogoneae, containing sorghum, maize, sugarcane, and Job’s tears. The Chloridoideae subfamily includes finger millet and tef (Doust 2007). In the first 15 to 20 million years of the 60 to 80 million years of evolution, when the main cereal grass lineage separated from other flowering plants, there was little molecular divergence among grass genomes. However, marked genomic divergence has occurred in the last two-thirds (45–60 million years) of this period (Paterson et al. 2004), resulting in genome size differences that range from rice at 420 Mb to wheat at 16,000 Mb (Goff et al. 2002). Genomic evolution in grasses has been complex, with a number of rounds of genome duplications followed by gene deletions (Kellogg 2003; Malcomber et al. 2006). Cereal genomes have shown a high level of macrocollinearity (Gale and Davos 1998), while microcollinearity was disrupted or incomplete at sequence level (Xu and Zhang 2004). Finger millet, foxtail millet, and pearl millet among the millets were the only species studied for collinearity with other cereal genomes. The rice genome has shown a high degree of conserved macrocollinearity against that of foxtail millet and finger millet (Devos et al. 1998; Srinivasachary et al. 2007), while the pearl millet genome has undergone many rearrangements compared to foxtail millet and rice (Devos et al. 2000; Gale et al. 2005). Pearl millet (Pennisetum glaucum) belongs to the genus Pennisetum, which has five sections: Penicillaria, Brevivalvula, Gymnothrix, Heterostachya, and Eu-Pennisetum (Stapf and Hubbard 1934) and 80 to 140 species (Donadıo et al. 2009), with haploid chromosome numbers of 5, 7, 8, or 9 (Jauhar 1981) and ploidy levels ranging from diploid to hexaploid. Phylogenetic analyses revealed that Pennisetum (excluding P. lanatum) is paraphyletic as it is nested with the closely related genus Cenchrus. Sections Pennisetum and Gymnothrix are polyphyletic. The domesticated species P. glaucum, P. purpureum (napiergrass), P. squamulatum, P. nervosum, and P. sieberianum are closely related, suggesting potential use of these species in crop improvement (Martel et al. 2004; Donadıo et al. 2009). The wild progenitor of pearl millet is Pennisetum glaucum ssp. monodii (Harlan 1975; Brunken 1977). Some believe that pearl millet is the product of multiple domestications (Harlan 1975; Port eres 1976) while others propose a single domestication (Marchais and Tostain 1993). Evidence suggests that pearl millet domestication took place in Africa, although different geographical origins have been proposed along the Sahelian zone from Mauritania to Sudan (Harlan 1975; Port eres 1976; Marchais and Tostain 1993). The earliest archaeological evidence for pearl millet domestication is from northern Ghana, some 3,500 years BP (D’Andrea and Casey 2002). Studies on

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isozyme and simple sequence repeat (SSR) markers have further confirmed a monophyletic origin of pearl millet in West Africa (Ibrahima et al. 2005; Mariac et al. 2006a,b; Oumar et al. 2008; Kapila et al. 2009). Using microsatellite data from wild and cultivated accessions from Africa and Asia, Oumar et al. (2008) detected significantly higher diversity in the wild pearl millet group. The phylogenetic relationship among accessions not showing introgressions support a monophyletic origin of cultivated pearl millet in West Africa, with eastern Mali and western Niger as the most likely region of pearl millet domestication. Introgression has played a major role in evolution of pearl millet (Brunken et al. 1977; Ibrahima et al. 2005; Miura and Terauchi 2005; Mariac et al. 2006a,b; Oumar et al. 2008). There seems to be a putative supergene or gene complex involved in the domestication syndrome that differentiates weedy and cultivated types (Miura and Terauchi 2005). Quantitative trait loci (QTL) analyses involving F2 populations derived from crosses of cultivated pearl millet and Pennisetum glaucum ssp. monodii revealed two genomic regions on linkage groups (LGs) 6 and 7, which controlled most of the key morphological differences (Poncet et al. 1998, 2000, 2002). The importance of these two LGs reveals their central role both in the developmental control of spikelet structure and in the domestication process of pearl millet, and these genomic regions may correspond with quantitative trait loci (QTL) involved in domestication of other cereals, such as maize and rice (Poncet et al. 2000, 2002). Foxtail millet (Setaria italica) is a diploid species, and its wild ancestor is S. virdis (Kihara and Kishimoto 1942; Li et al. 1945; Wang et al. 1995; Le Thierry d’Ennequin et al. 2000). Vavilov (1926) suggested east Asia, including China and Japan, to be the principal center of diversity for foxtail millet, while other views suggest independent domestication in China and Europe based on archaeological, isozyme, 5S rDNA, and morphological evidence (Harlan 1975; de Wet et al. 1979; Jusuf and Pernes 1985; Li et al. 1995a,b, 1998; Benabdelmouna et al. 2001a). However, diversity studies using different DNA marker systems do not support the hypothesis of two domestication centers. Using 16 restriction fragment length polymorphism (RFLP) probes, Fukunaga et al. (2002a) classified 62 landraces into five groups, with no clear geographical structure. Le Thierry d’Ennequin et al. (2000) used 160 polymorphic amplified fragment length polymorphism (AFLP) loci data on 39 S. italica (foxtail millet) and 22 S. virdis (green foxtail millet) accessions. Neither cultivated nor wild accessions showed a clear differentiation of population structure, but both domesticated and wild accessions from China were the most genetically diverse, which supports the monophyletic origin of foxtail millet in China. Previous studies


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involving rapid amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers (Schontz and Rether 1998, 1999) or the analysis of either waxy or prolamine genes (Nakayama et al. 1999; Fukunaga et al. 2002b) were also not conclusive in supporting hypotheses of two domestication centers of foxtail millet. QTL mapping of candidate genes revealed that tillering and panicle shape were involved in domestication (Doust et al. 2004, 2005), while human selection contributed to the origin of waxy phenotype in foxtail millet (see Section VIII.D). The genus Setaria, which also includes foxtail millet, has approximately 125 species widely distributed in warm and temperate parts of the world. The genome of foxtail millet and S. viridis is designated as AA genome (Li et al. 1945). Weedy tetraploid species S. faberii and S. verticillata have AABB genome, probably originated from a natural cross between S. viridis and another diploid species, S. adhaerans (Benabdelmouna et al. 2001a,b). S. grisebachii from Mexico has been identified as CC genome diploid species (Wang et al. 2009). S. queenslandica is the only autotetraploid (AAAA genome) species in genus Setaria (Wang et al. 2009) whereas other polyploid species such as S. pumila and S. pallide-fusca do not contain the AA genome (Willweber-Kishimoto 1962; Benabdelmouna et al. 2001a,b; Benabdelmouna and Darmency 2003). Cultivated finger millet, E. coracana subsp. coracana, was domesticated some 5,000 years ago from the wild E. coracana subsp. africana (2n ¼ 4x ¼ 36) in the highland that stretches from Ethiopia to Uganda (Hilu and de Wet 1976; Hilu et al. 1979; Werth et al. 1994). Subsp. africana is the result of a spontaneous hybridization event between the diploid E. indica (AA genome) and an unknown B-genome donor (Hilu and Johnson 1992; Hiremaths and Salimaths 1992; Salimaths et al. 1995; Neves et al. 1998; Bishit and Mukai 2000). Neves et al. (2005) assessed the phylogenetic relationships in finger millet, a tetraploid species, using nuclear (internal transcribed spacer [ITS] region of the 18S-26S ribosomal DNA repeat and the 5.8S RNA gene) and plastid (trnT-trnF) DNA sequences, which strongly support a monophyletic origin, but basal relationships in the genus remain uncertain, with either E. jaegeri or E. multiflora the first diverging lineage. Further, two putative ITS homologues loci (A and B loci) were identified in finger millet. E. coracana and its putative ‘‘A’’ genome donor, the diploid E. indica, are close allies, while the sequence data contradict the hypothesis that E. floccifolia is its second genome (B) donor. Thus, the ‘‘B’’ genome donor remains unidentified and may be extinct. More recently, Dida et al. (2008) analyzed phylogeny of finger millet landraces from Africa

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and India and their wild ancestor with microsatellite markers. They confirmed that finger millet was domesticated in East Africa and dispersed into India, which became the secondary center of diversity for this crop. Proso millet (Panicum miliaceum) and little millet (P. sumatrense) are tetraploid species (Sakamoto 1988) that belong to the genus Panicum, a cosmopolitan genus with approximately 450 species. Panicum is a remarkably uniform genus in terms of its floral characters but exhibits considerable variation in anatomical, physiological, and cytological features. Proso millet probably originated from a weedy variety, Panicum miliaceum var. ruderale, distributed from northeast China to eastern Europe (Sakamoto 1987). Vavilov (1926) suggested that China is the center of diversity for proso millet, while Harlan (1975) opined that proso millet probably was domesticated in China and Europe together with foxtail millet. Further study revealed that proso millet was domesticated somewhere in the region ranging from central Asia to northwestern India together with foxtail millet (Sakamoto 1987). Current evidence suggests that proso millet was the first millet domesticated, some 10,000 years BP in Neolithic China, where it appears to have been the earliest dry-farming crop (Lu et al. 2009). Using molecular data of the chloroplast ndhF gene, Aliscioni et al. (2003) assessed infrageneric classifications and proposed a robust phylogenetic tree of Panicum; however, genome origin of proso millet and little millet has not been analyzed. RAPD analysis differentiated North American wild proso and cultivated species (Colosi and Schaal 1997). Barnyard millets Echinochloa crus-galli (Japanese) and E. colona (Indian), both hexaploid species, are from eastern Asia and India. E. crus-galli originated from the hybridization between tetraploid E. oryzicola and an unknown diploid species. The genetic relationship between E. crus-galli and E. oryzicola using nuclear DNA (nrDNA) ITS and the chloroplast DNA (cpDNA) trnT-L, trnL intron, and trnL-F regions clearly separated the New World E. crus-galli from Eurasian E. crus-galli and showed a close relationship to the American taxa, E. crus-pavonis and E. walteri. The nuclear DNA ITS sequences further indicated no differentiation between the Eurasian E. crus-galli and E. oryzicola, in contrast to their clear divergence in the cpDNA sequence, suggesting that E. oryzicola is the male donor of E. crus-galli (Aoki and Yamaguchi 2008). Further, phylogenetic analysis of the homologous copy sequences of Oryza sh4 gene (controlling shattering nature of the spikelets) in Echinocloa showed genomic relationship between the Asian Echinocloa species, which supports the theory that the allohexaploid E. crus-galli shares two genomes with its parental donor, E. oryzicola. The Asian


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perennial tetraploid species, E. stagnina, shares one genome with E. oryzicola and possesses an unknown genome. E. crus-pavonis, from the New World, shows a close affinity of two genomes with E. crus-galli and E. oryzicola, while E. colona sows distinct affinities in all homologous copies (Aoki and Yamaguchi 2009). Ethiopia is the center of origin and diversity for tef (Eragotis tef ) (Vavilov 1951), and farmers in Ethiopia have greatly contributed to domesticating this unique cereal as a food crop. Tef is an allotetraploid cereal crop whose origin within the large genus Eragrostis was investigated by Ingram and Doyle (2003). Phylogenetic analysis of sequence data from the nuclear gene waxy and the plastid locus rps16 strongly supports the widely held hypothesis of a close relationship between tef and E. pilosa, a wild allotetraploid. Eragrostis heteromera, another previously proposed progenitor, is shown by the waxy data to be a close relative of one of the tef genomes. Other putative progenitors included in the taxon sample were not supported as closely related to tef. The waxy phylogeny also resolves the relationships among other allopolyploids, supporting a close relationship between the morphologically similar disomic tetraploid species E. macilenta, E. minor, and E. mexicana. Eragrostis cilianensis, another morphologically similar disomic polyploid, appears to have shared one diploid progenitor with these species but derived its other genome from an unrelated diploid. Both E. tef and E. pilosa are disomic tetraploid species, cross compatible, and have similarity in karyotype and morphological traits; however, the two differ in spikelet shattering. The multifloreted spikelets of E. pillosa readily break apart at maturity as a natural mechanism of seed dispersal, whereas they remain attached to the rachis at maturity in E. tef (Phillips 1995). Job’s tears (Coix lacryma-jobi), a native to tropical Asia, belongs to the Andropogoneae tribe. The genus Coix consists of four species, Coix aquatica, C. gigantea, C. lacryma-jobi, and C. puellarum. C. lacryma-jobi is further divided into four taxa, var. mayuen, var. lacryma-jobi, var. monilifer, and var. sternocarpa. C. lacryma-jobi is widely distributed in Africa, Oceania, east Asia, and America (Bor 1960; Koyama 1987). Var. mayuen is cultivated as a cereal or medicinal plant in east Asia, southeast Asia, and south Asia, whereas other taxa are wild and some are used as medicine or beads. Murakami and Harada (1958) reported that mayuen is cultivated as a cereal and domesticated from lacryma-jobi, but the two differ in hardness of seed coats; mayuen is softer than lacrymajobi. Job’s tears probably were domesticated as a cereal in the continental parts of southeast Asia (Arora 1977; Sakamoto 1988). Enomoto et al. (1985) used restriction endonuclease of cpDNAs to study the phylogenetic relationship among crops in tribe Gramineae and

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showed that the phylogenetic tree is in complete agreement with that reported by Tateoka (1957) except that the genetic distance between the chloroplast genomes of sorghum (Sorghum bicolor) and maize (Zea mays)/Job’s tears (Coix Lacryma-jabi), is closer than that between maize and Job’s tears despite sorghum belongs to different tribe from maize and Job’s tears. Thus, the two genera, Zea and Coix, should be placed in separate tribes. More recently, Leseberg and Duvall (2009) also demonstrated that the position of Job’s tears in a phylogenetic tree coincides with the broadly delimited Andropogoneae (GPWG 2001) but contradicts earlier studies that classified Job’s tears in a putative sister tribe, Maydeae, with Zea mays (Kellogg and Birchler 1993). The genus Digitaria has 230 species, widely distributed in the tropics and subtropics (Clayton and Renvoze 1986). Of these species, D. exilis (white-seeded fonio) and D. iburua (black-seeded fonio) are domesticated and cultivated in West Africa (Port eres 1976), with the former being most diverse and widely cultivated, while the latter is restricted to northern Nigeria, Benin, and Togo (Murdock 1959; NRC 1996). The putative wild relatives of cultivated fonio are probably D. horizontalis and D. longiflora; the latter has many interesting agronomic traits (erect habit, resistant to lodging, long panicle full of grains and large-size seeds) and appears useful for improving cultivated fonio (Dansi et al. 2010).

IV. ASSESSING PATTERNS OF DIVERSITY IN GERMPLASM COLLECTIONS Ex situ seed storage is the most widely used method to conserve millets genetic resources. To date, 161,708 accessions of millets species are preserved in gene banks across the globe, 98.1% cultivated and 1.9% wild types (Table 5.10). Finger millet, foxtail millet, pearl millet, and proso millet form the largest collection of cultivated millets germplasm, while fonio and Job’s tears form the smallest (Tables 5.11–13). In addition, the U.S.-based GRIN database contains 306 accessions of 18 Echinocloa species from 33 countries housed at the National Center for Genetic Resources Conservation (Fort Collins, Colorado; NSSL); 1,468 accessions of eight Eleusine species from 20 countries housed at NSSL and Southern Regional PI Station (Griffin, Georgia; S9); 1,014 accessions of 36 Setaria species from 52 countries housed at the North Central Regional PI Station (Ames, Iowa; NC 7); 1,616 accessions of 38 Panicum species from 52 countries housed at NC 7, NSSL, the Plant Germplasm Quarantine Program (Beltsville, Maryland; PGQP), S9, and the Western


285

Table 5.10. List of cultivated and wild relatives of barnyard millet, finger millet, fonio, foxtail millet, Job’s tears, kodo millet, little millet, proso millet, and tef germplasm preserved worldwide in national and international gene banks in Africa, America, Asia, Europe, and Oceania. Crop

Africa

Cultivated germplasm Barnyard millet Finger millet 7,766 Fonio 285 Foxtail millet 985 Kodo millet Job’s tears Little millet Pearl millet 11,105 Proso millet Tef 4,747 Total 24,888 Wild relatives Barnyard millet 27 Finger millet 930 Foxtail millet 143 Job’s tears Pearl millet 286 Tef 1 Total 1,387

America

Asia

1,453

749 24,308

1,368 1 13,213 1,134 768 1,7937

19 21 57 5 102

38,429 4,025 154 1,017 13,252 8,547 420 90,901

Europe

48 4,643

Oceania 67 21 336 227

4 4,088 14,918 46 23,747

130 388 8 1,025 1 1,552

252 245 20 1,168

1 1 24 25

1

Total 816 33,596 285 45,761 4,252 159 1,017 41,910 24,844 6,001 158,641 27 1,079 552 9 1,369 31 3,067

Source: http://apps3.fao.org/wiews/germplasm_query.htm.

Regional PI Station (Pullman, Washington; W6); and 1,401 accessions of 69 Paspalum species from 44 countries housed at NSSL, PGQP, and S9 gene banks (http://www.ars-grin.gov/npgs/stats/). The largest collections of finger millet can be found in India in Asia and in Ethiopia, Kenya, and Uganda in Africa; China, France, India, and Japan have the largest collections of foxtail millet; China, Russia, and Ukraine have the largest collections of proso millet; India has the largest collections of kodo millet and little millet; India and Japan have the largest collections of barnyard millet; Benin has the largest collection of fonio; Japan has the largest collection of Job’s tears; Brazil, Canada, China, France, India, Namibia, Niger, Nigeria, and Pakistan have the largest collections of pearl millet; and Ethiopia has the largest collections of tef germplasm. Evidence suggests that some of the fonio germplasm has already been lost. The main reason for fonio genetic erosion is due to difficulties in its harvesting and postharvest processing (Adoukonou-Sagbadja et al. 2004). Likewise, diversity in barnyard millet has fast eroded due

286

Nepal

Japan

India

Asia Bangladesh China

Country

Bangladesh Agr. Res. Inst., Joydebpur, Gazipur Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences (ICGR-CAAS), Beijing All India Coordinated Small Millet Project, UAS, Bangalore CSK HP Krishi Vishvavidyalaya, Palampur, Himachal Pradesh CCS Haryana Agricultural University, Hisar, Haryana Indian Grassland and Fodder Research Institute(IGFRI), Jhansi, Uttar Pradesh International Crop Research Institute for the Semi-Arid Tropics, Patancheru Indian Grass and Fodder Research Institute National Bureau of Plant Genetic Resources (NBPGR), New Delhi Regional Station Akola, NBPGR, Maharashtra Regional Research Center, Jodhpur Department of Genetic Resources I, National Institute of Agrobiological Sciences (NIAS), Tsukuba-shi National Grassland Research Institute (NGRI), Nasu-gun, Tochigi-ken Plant Germplasm Institute, Faculty of Agriculture, Kyoto University (KYOPGI), Mozume-cho - Muko-shi, Kyoto Central Plant Breed. & Biotechnol. Division, Nepal Agric. Res. Council (CPBBD), Khumaltar, Kathmandu

Institute

4,330 349

9,522 455

869

58

74

30

274

2,450

1,488

5,852

565

2,512

515 26,233

Foxtail millet

6,257 30

300

Finger millet

5,772 133

568 3,294

875 734

103

Pearl millet

No. accessions

Table 5.11. Number of cultivated germplasm accessions of finger millet, foxtail millet, pearl millet and proso millet preserved globally in national and international gene banks.

16

62

296

849

577

209 6,517

Proso millet

287

Senegal

Nigeria Namibia

Niger

Malawi Mali

Ethiopia Kenya

Burkina Faso

Benin Botswana

Africa Angola

Thailand

Sri Lanka

Pakistan

Centre National des Resources Phytogenetiques, Ministere de l’agriculture et du Developpement Rural (CNRF), Luanda Centre de Recherches Agricoles Sud (CRAS), Attogon Department of Agricultural Research, Sebele Agricultural Research Station, Gaborone Centre de Recherches Agricoles de Farako-Ba (CRA), Bobo-Dioulasso Inst. Biodiversity Conserv (IBC). Addis Ababa Natl. Gene Bank of Kenya, Crop Plant Genet. Resour. Centre (KARI-NGBK), Muguga Chitedze Agr. Research Station Station de Recherche Agronomique de Cinzana (SRAC), Cinzana, Segou Institut National de la Recherche agronomique du Niger (INRAN), Niamey ICRISAT, Niamey Nat. Centre Genet. Resour. Biotechnol., Moor Plantation—Ibadan National Plant Genetic Resources Center, National Botanical Research (NPGRC) Institute Unite de Recherche en Diversite Genetique et Culture In-vitro (URCI), Dakar

Plant Genetic Resources Institute, Natl. Agric. Res. Centre, Islamabad Fodder Research Institute, Sargodha Seed Conservation Unit, Plant Genetic Resources Centre, Gannoruwa, Peradeniya Dry Zone Agricultural Research Institute, Maha-Illuppallma National Corn and Sorghum Research Center, Kasetsart University, Pak Chong - Nakhon Ratchasima

2,156 2,875

31

295

45 5

772

110

138

44

2,817 46 1,416

2,052

47 243

166 499

112

27 61

135

63

333

1,377

(continued )

21

288

USA

Mexico

Canada

Americas Brazil

Embrapa Milho e Sorgo (CNPMS), Sete Lagoas Embrapa Recursos Geneticos e Biotechnologia (CENARGEN), Brasilia Plant Genet. Resour. of Canada, Saskatoon Research Centre, Agr. & Agri-Food Canada, Saskatoon, Saskatchewan Estacioń de Iguala, Instituto Nacional de Investigaciones Agrıcolas, (INIA), Iguala North Central Reg. Plant Introd. Station, USDA-ARS, NCRPIS, Iowa State Univ. Ames, IA National Center for Genetic Resources Preservation, Fort Collins Colorado Plant Genetic Resources Conservation Unit, Southern Regional Plant Introduction Station, University of Georgia, USDA-ARS, Griffin, GA

Division of Plant and Seed Control, Dept. Agriculture, Pretoria National Plant Genetic Resources Centre (NPGRC), Arusha Serere Agric. & Animal Prod. Res. Inst.,(SAARI) Soroti Mt. Makulu Central Res. Station, Chilanga SADC Plant Genet. Resour. Centre, Lusaka Zambia Agriculture Research Institute (ZARI), Chilanga Genetic Resources and Biotechnology Institute, Ministry of Agriculture, Mechanization and Irrigation Development (GRBI), Causeway—Harare

South Africa Tanzania Uganda Zambia

Zimbabwe

Institute

Country


748

702

3

3 74 1,231 390 1,037

Finger millet

1,000

350

18

122

41

Foxtail millet

2,063

3,764

7,225 161

785 323 73

69 48 2,142

Pearl millet

No. accessions

713

400

21

Proso millet

289

Australian Medicago Genetic Resources Centre, South Australian Research and Development Institute (AMGRC), SARDI, PRC GPO Box 397, Adelaide Australian Tropical Crops & Forages Collection, Australian Plant Genetic Resource Information Service, Biloela

AGES Linz—Austrian Agency for Health and Food Safety/Seed Collection, Wieningerstrasse 8, Linz Inst. Plant Genet. Resour. ‘‘K.Malkov’’ (IPGR), Sadovo, Plovdiv Res. Inst. Crop Production, Praga Biologie Vegetale Appliquee, Institut Louis Pasteur (IUT), 3 rue de l’Argonne-Strasbourg ORSTOM-MONTP, Montpellier Cedex Gene Bank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, Gatersleben Institute for Agrobotany (RCA), Kulsomezo 15, Tapio´szele Bot. Garden of Plant. Breed. & Acclimatization Inst., Bydgoszcz Res. Inst. Cereals and Technical Plants Fundulea, Fundulea, Calarasi N.I. Vavilov All-Russian Scientific Res. Inst. of Plant Industry, St. Petersburg Res. Inst. Plant Production, Piestany Inst. Plant Prod. V.Y. Yurjev of UAAS, Kharkiv Ustymivka Experimental Station of Plant Production, S. Ustymivka Institute of Biological, Environmental & Rural Sciences, Aberystwyth University (IBERS-GRU), Ceredigion, Wales


Total

Oceania Australia

United Kingdom

Russian Federation Slovakia Ukraine

Hungary Poland Romania

Germany

Bulgaria Czech Republic France

Europe Austria

33,596

13

8

11

27

10

46,070

336

12

14

27 82

3,500 124

34 850

41,910

252

4,059 29

24,844

245

53 1,046 3,976

8,778

20 721 65

97 162

290

S. DWIVEDI ET AL.

Table 5.12. Number of cultivated germplasm accessions of barnyard millet, kodo millet, and little millet preserved globally in national and international gene banks. No. accessions Country Asia India

Oceania Australia

Total

Institute All India Coordinated Minor Millet Project, UAS, Bangalore ICRISAT, Patancheru NBPGR, New Delhi NBPGR Regional Station, Akola, Maharashtra Tropical Crops & Forages Collection, Australian Plant Genetic Resource Information Service, Biloela

Barnyard millet

Kodo millet

Little millet

1,111

544

749

665 2,170 79

473

67

227

816

4,252

1,017


to considerable reduction in acreage and changing sociocultural and economic dimensions of the farming community in India (Maikhuri et al. 2001). Foxtail millet, finger millet, and pearl millet have extensive collections of their wild relatives preserved in ex situ seed gene banks. No wild relatives are reported for fonio, kodo millet, and little millet (Table 5.14). In addition, some of the pearl millet wild relatives are maintained by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in an ex situ field gene bank at Patancheru, India, as they do not set seed. Among global gene banks, China has the largest collection of wild relatives of foxtail and proso millet; India has the largest collection of finger millet; and France and India have largest collections of pearl millet. A German gene bank contains the largest number of the few accessions of tef’s wild relatives available. Descriptor lists were developed and used to characterize barnyard millet (IPGRI 1983), finger millet (IBPGR 1985a), foxtail millet (IBPGR 1985b), kodo millet (IBPGR 1983), proso and little millets (IBPGR 1985c), pearl millet (IBPGR/ICRISAT 1993), and tef (Ketema 1997) germplasm for sets of morphological and agronomic traits. This information, along with passport data, was used to assess patterns of diversity in millets germplasm collections and has revealed many interesting facts about the utility of such germplasm in millets breeding and


291

Table 5.13. Number of cultivated germplasm accessions of fonio, Job’s tears, and tef millets preserved globally in national and international gene banks. No. accessions Country Asia China

India

Japan

Africa Ethiopia Benin Ghana Kenya South Africa Americas Brazil USA

Europe Germany

UK

Hungary Oceania Australia

Institute

Fonio

National Key Laboratory of Crop Genetic Improvement, Huazhong Agr. Univ., Wuha National Bureaue Plant Genetic Resources, New Delhi CCS Haryana Agr. Univ., Hissar Department of Genetic Resources I, National Institute of Agrobiological Sciences (NIAS) National Inst. Crop Sci., Tsubuka Institute of Biodiversity Conservation, P.O.Box 30726 Laboratory of Genetics and Biotechnology, Univ, Aboney-Calvi, Cotonou Sabana Agr. Res. Inst., Tamale National Gene Bank of Kenya, Crop Plant Genetic Resources Centre, Muguga Division of Plant and Seed Control, Dept. Agr, Technical Service

Job’s tears 14

253

140

4,741 261 24 3 3

400 368 1

Gene Bank, Leibniz Institute of Plant Genetics and Crop Plant Research Federal Center for Breeding Researcg on cultivated plants (BAZ), Braunschweig Welsh Plant Breeding Station, Genetic Resources Unit, Institute of Grassland and Environmental Research Institute for Agrobotany

12 30 2

2

2

2

Australian Tropical Crops & Forages Genetic Resources Centre


137

30

Centro de Pesquisa Agropecuaria dos Cerrados (CPAC), Planaltina Western Regional Plant Introduction Sta., USDA-ARS, Washington State Univ. North Central Regional Plant Introduction Station, USDA-ARS, NCRPIS

Total

Tef

20 285

159

6,001

292

Malawi South Africa Tanzania Zambia

Kenya

Pakistan Yemen Africa Ethiopia

Japan

India

China

Asia Armenia

Country

Int. Livestock Res. Inst. (ILRI), Addis Ababa Institute of Biodiversity Conservation (IBC), Addis Ababa Agricultural Research Centre (KARI), Kitale National Gene Bank of Kenya, Crop Plant Genetic Resources Centre(KARI-NGBK), Muguga Chitedze Agricultural Research Station, Lilongwe RSA Plant Genetic Resources Centre, Pretoria National Plant Genetic Resources Centre (NPGRC), Arusha SADC Plant Genet. Resour. Centre, Lusaka Zambia Agriculture Research Institute, Chilanga

Laboratory of Plant Gene Pool and Breeding(LPGPB), Yerevan Scientific Center of Agrobiotechnology (SCAPP), Echimiadzin Institute of Crop Germplasm Resources, Chinese Academy of Agricultural Sciences, Beijing National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuha International Crops Research Institute for the Semi Arid Tropics, Patancheru National Bureaue Plant Genetic Resources, New Delhi CCS Haryana Agric. University, Hissar Department of Genetic Resources I, National Institute of Agrobiological Sciences (NIAS), Tsukuba-shi Plant Genet. Resour. Inst., Natl. Agric. Res. Centre, Islamabad Agricultural Research and Extension Authority (AREA), Dhamar

Institute

27

Barnyard millet

383

156 21 286

56

11 17

25

105

Finger millet

119 6 13 5

18

81

62

54

173

Foxtail millet

1

7

Job’s tears

No. accessions

8 10

59

203

78 875

42 30

Pearl millet

Table 5.14. Number of wild relative accessions of barnyard millet, finger millet, foxtail millet, Job’s tears, pearl millet, and tef preserved globally in national and international gene banks.

1

1

Tef

293


American continent Canada Plant Genetic Resources of Canada, Saskatoon Res. Center, Agric., and Agri-Food Colombia CIAT, Cali, Valle del Cauca USA Western Regional Plant Introduction Station, USDA-ARS, Washington State University Plant Genetic Resources Conservation Unit, Southern Regional Plant Introduction Station, University of Georgia, USDA-ARS, Griffin Uruguay INIA La Estanzuela Europe Germany Gene Bank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, Gatersleben Austria AGES Linz—Austrian Agency for Health and Food Safety/Seed Collection, Wieningerstrasse 8, Linz France Biologie Vegetale Appliquee, Institut Louis Pasteur (IUT), Strasbourg ORSTOM-MONTP, Montpellier Cedex Hungary Institute for Agrobotany (RCA), Kulsomezo 15, Tapio´szele Slovakia Botanical Garden, University of Agriculture, Nitra United Kingdom Seed Conservation Department, Royal Botanic Gardens (RBG), Kew, Wakehurst Place Oceania Australia Australian Medicago Genetic Resources Centre, South Australian Research and Development Institute (AMGRC), SARDI, PRC GPO Box 397, Adelaide Australian Tropical Crops & Forages Genetic Resources Centre (ATCFC), Biloela Total 27

2

18

906

57

23

1,133

15

250

30

21

4

9

16

3

9

1 1,781

7

250

131

31

5

27

25

32

1

1

1

22

2

3

294

S. DWIVEDI ET AL.

genetics (Table 5.15). For example, accessions belonging to laxa race of barnyard millet, endemic to Sikkim state of India, are not represented in the ex situ collections preserved at the ICRISAT gene bank in Patancheru, India. Thus, there is an urgent need to collect this race before it becomes extinct. Likewise, the germplasm accessions from tef-growing regions of Hararghe, Arsi, Wellega, and Bale in Ethiopia are not represented in the gene bank of the Institute of Biodiversity Conservation in Ethiopia (Demissie 2001). Fonio landraces collected from Ghana and Togo have immense diversity with respect to agroecological adaptation and preferences of the tribes that maintain and cultivate these landraces: for example, landraces from the northern zone of Togo are better adapted to dry conditions; those from the Kara region in the north had the most landrace diversity, with greatest landrace diversity being maintained by the Lamba tribe. The later-maturing fonio landraces from Ghana have lighter seeds (1,000-seed weight) while early-maturing types have heavier seeds. Furthermore, earliness, ease in processing, and long shelf life (e.g., seeds of ‘‘Saranu’’ landrace could be stored up to eight years without loss of quality or viability) were the basis for farmer selection of landrace variability in fonio. More recently, Dansi et al. (2010) grouped 15 farmernamed landraces collected from the fonio production zones of Benin into five morphotypes, of which four belong to D. exilis (white fonio) and one to D. iburua (black fonio), and identified eight preference criteria of farmer-preferred fonio varieties: earliness, culinary characteristics, ease of harvesting and processing, productivity, grain size, storability, and drought tolerance. This study further revealed that farmers preferred the early-maturing landrace ‘‘Tintinga’’ as it help them to bridge the food shortage period when no other crops are ready for harvest and consumption. Likewise, the preference for the ‘‘S embr e’’ landrace was mainly due to its ease in processing (husking) of the grains, while most farmers disliked landraces ‘‘Tamaou’’ and ‘‘Foˆloˆm’’ because of their long growth period and difficulties in husking their grains. Foxtail millet (Setaria italica) accessions from Afghanistan, Iran, and Lebanon, one of the three possible (putative) centers of domestication and diversity in foxtail millet, resemble green foxtail millet (S. virdis), the wild progenitor species of cultivated S. italica. In pearl millet, landraces from Yemen are a source of variation for early maturity, cold tolerance, short stature, and large seeds. Landraces from western and central Africa show exceptional buffering against environmental variability, and landraces from Cameroon, Togo, and Ghana are good sources for earliness and/or large seeds. Early flowering, profuse tillering, more panicles plant1, and larger seed size are the characteristics of some landraces from northwestern India. Some of the landraces from this Indian region exhibit no trade-off

295

11 landraces from farmers barns in Ghana

Fonio 13 landraces from Ghana and 5 traits

Finger millet 909 germplasm from southern and eastern Africa and 7 traits

Barnyard millet 194 accessions from India and 14 traits

Accessions/traits studied

Phenology—a major determinant of diversity among landraces: those from Nyankpala matured earlier than those from eastern part of northern region; late-maturing types had lighter 1000-seed weight while early-maturing types heavier seeds Earliness, ease of processing and storage quality the basis for farmers’ selection of landrace variability, i.e., Nomba, Fefeka, and Kiyo landraces selected for early maturity; Yadema for ease in processing; Sarannu for long shelf life (8 years without loss of quality and viability); and Nankapando for drought resistance

Early-flowering accessions from Kenya while later-flowering types from Tanzania and Zaire; accessions with narrowest inflorescence width from Kenya and Zimbabwe while those with the widest inflorescence width from Nepal, Ethiopia, and Tanzania; accessions with no panicle exertion can be found in Kenya, Nepal, and Zimbabwe while those with full panicle exertion from Tanzania and Zaire

Assessing pattern of phenotypic diversity among accessions collected from different ecogeographical regions of India revealed no accession represented race laxa, endemic to Sikkim in India

Pattern of diversity discerned

(continued )

Clottey et al. 2006b

Clottey et al. 2006a

Upadhyaya et al. 2007a

Gupta et al. 2009

Reference

Table 5.15. Summary of the pattern of diversity as assessed in barnyard millet, finger millet, fonio, foxtail millet, pearl millet, proso millet, and tef germplasm.

296

20,844 germplasm from 51 countries and 23 traits

169 landraces from India evaluated for grain and stover yield

Pearl millet 145 inbreds derived from 122 WCA landraces

2907 accessions from 16 provinces of China þ 22 countries and 9 traits

Flowering, relative response to photoperiod and panicle length significantly impacted, population structure differentiation but not the environmental factors such as latitude, temperature, or precipation Significant differences among landraces for biomass, grain, and stover yield; several landraces outperformed controls in both grain and stover yields; no trade-off between stover and grain yields under arid zone conditions Diversity in flowering ranges from 33 to 159 days; plant height from 30 cm to 490 cm; tillers from 1 to 35; 100-seed weight from 1.5 to 21.3 g; forage type 141 accessions; 9 panicle shapes, 5 seed shapes, and 10 seed colors

Greater diversity for flowering in Sri Lankan germplasm, while narrowest in Russian germplasm; accessions from China dwarf while those from India tall; accessions with maximum panicle exertion from Russia; accessions with longest and widest inflorescence from India Accessions of Chinese origin highly diverse, while those from Afghanistan, Iran, and Lebanon less diverse and characterized by short plant height with more tillers and smaller panicles, resembling green foxtail millet (wild type)

Landraces from the northern zone better adapted to dry conditions than those cultivated in the south, which are adapted to a relatively wet climate; landraces from Kara region in the north have the most diversity followed by Plateaux in the south and Savanes in the north; at ethnic level, the Lamba tribe maintained maximum landrace diversity followed by the Akposso, LossoNwada, and Tamberma

95 accessions representing 34 landraces collected from 7 ethnic groups in Togo

Foxtail millet 1535 accessions from 26 countries and 6 traits

Pattern of diversity discerned



Upadhyaya et al. 2007b

Yadav and Bidinger 2008

Stich et al. 2010

Li et al. 1995a

Reddy et al. 2006

Adoukonou-Sagbadja et al. 2004

Reference

297

227 landrace populations from Ghana and 18 traits

918 accessions including wild relatives from Cameroon and 8 traits

105 landraces from northwestern India and 8 traits

229 germplasm from Yemen and 12 traits

424 landraces from West and Central Africa (WCA) evaluated for flowering

5197 germplasm from India and 8 traits

Climate variables impacted pattern of diversity: arid zone as the promising source of early flowering, short height, and large seeds; semiarid zone for thick panicles and high panicle exertion; subhumid zone for tall and long panicles Exceptional buffering capacity (both at individual and population level) against environmental variability, due to variation in photoperiod sensitivity and intravarietal heterogeneity for flowering, confer adaptive advantages under variable climatic conditions, thus, a good resource to enhance adaptation of pearl millet under similar scenarios in other agroecological zones as found in WCA Yemen has extreme variation in elevation, temperature, and rainfall, which significantly impacted variability in pearl millet: germplasm from high elevation good source for early maturity, cold tolerance, short plant height, and large seeds; accessions from lower elevation have longer panicle while increasing elevation have accessions with thinner panicle Large variation in flowering, plant height, panicle length and panicles plant1 among landraces; more than 2-fold difference in grain and stover yield; phenotypic diversity spread into 9 clusters, some with specific attributes: i.e., landraces from cluster 9 were highest yielding due to early flowering, more panicle plant1, and larger seed size while cluster 4 landraces provided highest stover yield but flowered late and produced less grain A good source for more reproductive tillers, large compact spikes, and larger ivory- and cream-colored grain besides its potential for forage; early-maturing types (Mouri) adapted to low rainfall, while late-maturing types (Yadiri) in high rainfall regions Mixtures of various morphological types were the common features of landrace populations grown by the farmers and good source of genes for earliness and large grain size (continued )

Rao et al. 1985

Rao et al. 1996

Yadav et al. 2004b

Reddy et al. 2004

Haussman et al. 2007

Upadhyaya et al. 2007c

298

1080 germplasm (36 populations) from 6 central/northern regions of Ethiopia and 14 traits

60 germplasm and 6 traits

3000 panicle derived lines from 60 germplasm of Ethiopia and 17 traits

Tef 144 heterogeneous germplasm from Ethiopia and 18 traits

Proso millet 842 germplasm from 27 countries and 9 traits



Regions and altitudes have had no substantial effect on genetic diversity; higher intraregional genetic diversity (between tef germplasm from the same region and altitude) than interregional diversity Detected regional and clinal (altitude zone) diversity patterns in tef germplasm; all the 6 regions remain separate and unclustered at 75% similarity, while at 50% level of similarity Shewa, Wellega, and Keffa clustered together and the remaining 3 regions remained distinct and ungrouped Germplasm from high altitudes (>2400 m.a.s.l.) differed significantly from those either lowland (300 marker loci. Over 200 DArT markers were mapped in more than one population, and their mapping positions were reasonably consistent across maps. Among these, 32 DArT markers representing all seven pearl millet linkage groups were mapped in all three RIL populations, permitting the development of a well-saturated pearl millet consensus linkage map combining DArT and SSR markers. Recently some DNA markers from rice, wheat, oat, and barley have shown polymorphism in proso millet (Hu et al. 2009). More recently, Reddy et al. (2010) isolated 41 resistant gene homologues from a popular finger millet cultivar, ‘UR762’, which showed strong homology to NBSLRR type R-genes of other crop species. The molecular cloning of these resistant gene homologues may provide new ways to deploy these genes against biotic stresses. Clearly, more directed efforts are needed to develop markers in other millets. One way to overcome the paucity of DNA markers in these millets is to try markers from other cereals, as both macro- and micro-synteny have been reported among cereals (Devos et al. 2000; Srinivasachary et al. 2007; Yadav et al. 2008; also see Section VIII.D). Recent work on switchgrass (Panicum virgatum) has shown


317

Table 5.19. Summary of genetic linkage maps reported in finger millet, foxtail millet, pearl millet, and tef from 1994 to 2007. Summary of linkage maps reported Finger millet 131 markers mapped to 16 LGs on A genome, with a total map distance 721.4 cM, while 196 markers to 9 LGs on B genome covering 786.8 cM map distance 332 loci from 266 primers mapped into 26 LGs. 13 on A-genome and 9 on B-genome LGs assembled into 9 homologous groups, 6 six of these corresponding to a single rice chromosome each, while remaining 3 were orthologous to 2 rice chromosomes; gene orders between rice and finger millet highly conserved Foxtail millet A high-density genetic map with 1000 SNPs evenly mapped to all 9 chromosomes; a number of chromosomal rearrangements, including several previously unknown rearrangements, relative to sorghum and rice genomes

81 SSR and 20 RFLP markers mapped to 9 LGs, with a total map length of 1654 cM, and marker density of 16.4 cM 160 RFLP loci mapped to 9 LGs, with a total map distance of 964 cM Job’s tears 80 AFLP and 10 RFLP markers mapped to 10 LGs, with a total map length of 1339.5 cM, average marker density 14.88 cM Pearl millet A map with 55 RFLP and 32 genomic SSR and 17 EST-SSR loci spanning 675 cM An integrated genetic map, based on 4 crosses, mapped 353 RFLP and 65 SSRs into 7 linkage groups (LGs), 85% of the markers occupying less than a third of the total map length A map with 61 RFLP and 30 SSR loci spanning 476 cM 181 RFLP loci mapped to 7 LGs, with a total map length of 303 cM and 2 cM marker density A map with 38 RFLP markers covering 280 cM Tef 252 SSR loci mapped to 30 LGs, with a total map length of 1277.4 cM (78.7% genome coverage), averaged marker density 5.7 cM 156 loci from 121 markers (RFLP, SSR, SNP/INDEL, IFLP, ISSR) mapped to 21 LGs, with a total map length of 2081.5 cM and 12.3 cM marker density 166 markers (AFLP, ISSR, and SSR) mapped to 20 LGs, covering 2112.3 cM and marker density of 12.7 cM.

Reference Dida et al. 2007

Srinivasachary et al. 2007

http://www. plantbio.uga. edu/media/ 2010_grad_ symposium(1). pdf Jia et al. 2009b Wang et al. 1998

Qin et al. 2005

Senthilvel et al. 2008 Qi et al. 2004

Yadav et al. 2004a Liu et al. 1994 Jones et al. 1995 Zeid et al. 2010

Yu et al. 2006a

Chanyalew et al. 2005 (continued )

318 Table 5.19

S. DWIVEDI ET AL. (Continued)

Summary of linkage maps reported

Reference

149 RFLP loci mapped to 20 LGs, with a total map distance of 1489 cM and marker density of 9.99 cM; alignment of tef RFLP map with the rice RFLP map shows synteny and collinear gene order between the 2 genomes 211 AFLP loci mapped to 25 LGs, with a total map distance of 2149 cM, marker density of 10.4 cM

Zhang et al. 2001

Bai et al. 1999

many common expressed sequence tag (EST) markers with proso millet (Tobias et al. 2008). B. Characterization and Functional Validation of Genes Associated with Important Traits A number of QTLs have been identified and mapped for resistance to downy mildew, drought tolerance, grain yield and yield components, and for stover quality in pearl millet and for agronomic traits in foxtail millet and tef (see Section VIII.A). Linkage analysis in most of these studies allowed identification of genes/QTLs at a distance as large as 10 to 40 cM from the nearest markers, which may not be suitable for either marker-assisted breeding or for identification/cloning of candidate genes. Unlike other cereals such as rice, maize, and barley (Table 5.20), the only studies reported on functional validation of genes associated with agronomic traits in millets are for the tb1 and ba1 genes associated with branching (basal and axillary) in foxtail millet (Doust and Kellogg 2006); PHYC gene associated with flowering time and morphological variation (spike length and stem diameter) (Sa€ıdou et al. 2009); a major drought-tolerance QTL on linkage group 2 (Sehgal et al. 2009) in pearl millet; and the SiOPRI gene associated with osmotic adjustment and improved drought tolerance in foxtail millet (Zhang et al. 2007b). Further, toward identifying candidate genes for salt tolerance in foxtail millet, Jayaraman et al. (2008) used the cDNA–AFLP technique to compare gene expression profiles of salt-tolerant and salt-sensitive cultivars in foxtail millet, and identified 27 nonredundant differentially expressed cDNAs unique to genes involved in metabolism, cellular transport, cell signaling, transcriptional regulation, messenger ribonucleic acid splicing, seed development and storage in the salttolerant cultivar ‘Prasad’. The expression patterns of seven such genes showed a significant increase in ‘Prasad’ after 1 hour of salt stress in comparison to the salt-sensitive cultivar ‘Lepakshi’. More recently,


319

Table 5.20. Summary of quantitative trait loci (QTL) or gene association with important traits and their validation in barley, foxtail millet, maize, pearl millet, and rice from 1995 to 2009. Trait Barley Flowering time Foxtail millet Drought (osmotic adjustment) Vegetative branching (basal and axillary) Maize Plant architecture Yield Pearl millet Flowering time, plant and spike morphology Rice Heading time Grain number Seed shattering Salt tolerance UV resistance Submergence tolerance

QTL/gene

Validation

References

Ppd-H1

Association

Stracke et al. 2009

SiOPR1

Zhang et al. 2007b

tb1 and ba1

Doust and Kellogg 2006

Tb1 lcyE

Complementation Mutagenesis

Doebley et al. 1995, 1997 Harjes et al. 2008

PHYC

Association

Sa€ıdou et al. 2009

Hd1/Se1 Hd3a Gn1/CKX2 qSH-1/RPL sh4 SKC1 qUVR-10 Sub1

Transformation Transformation Transformation Complementation Transformation Transformation Transformation Transformation

Yano et al. 2000 Kojima et al. 2002 Ashikari et al. 2005 Konishi et al. 2006 Li et al. 2006 Ren et al. 2005 Ueda et al. 2005 Xu et al. 2006

Lata et al. (2010) detected above 2.5-fold variation in nine up-regulated transcripts between drought-tolerant and susceptible cultivars upon dehydration stress. The induction of these genes suggests their function in regulation of dehydration tolerance in foxtail millet. These researchers therefore initiated cloning of full-length copies of some of the known and unknown up-regulated genes and will analyze their functions to identify candidate genes for drought tolerance in foxtail millet. In summary, the limited published research on QTL mapping and validation among millets has been restricted only to foxtail millet, pearl millet, and tef and research on gene expression for abiotic stresses tolerance has been limited to pearl millet and foxtail millet, largely because of the nonavailability of DNA markers or sequences in most of the other millets. Clearly, more efforts should be directed toward the development of large numbers of genic and genomic markers to conduct

320

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association genetics for identification and validation of candidate genes associated with important traits. C. Genomic and Genetic Tools to Sequence the Foxtail Millet Genome Foxtail millet has a highly conserved genome structure relative to the ancestral grass lineage (Devos et al. 1998). It is a diploid grass with a relatively small genome (490 Mb) and is closely related to bioenergy grasses, such as switchgrass (Panicum virgatum), napiergrass (Pennisetum purpureum), and pearl millet. It is an ideal model crop to investigate plant architecture, genome evolution, and physiology in the bioenergy grasses (Doust et al. 2009). In 2008, the Joint Genome Institute of the U.S. Department of Energy announced support for developing genomic and genetic tools to complement sequencing of the foxtail millet genome and for the improvement of biomass production for bioenergy crops (http:// GenomicScience.energy.gov/research/DOEUSDA). Four U.S. universities along with the Hudson Alpha Institute for Biotechnology of Huntsville, Albama, and the Joint Genome Institute of Walnut Creek, California, are involved in sequencing of the foxtail millet genome and development of the complementary tool sets. The latest report from this group revealed that draft genome sequencing of foxtail millet has been completed to 8.3 coverage, with the aligned sequence showing a high degree of synteny to rice and sorghum, even though these lineages last shared a common ancestor more than 50 million years ago (Mitros et al. 2010). The ongoing genetic and genomic research on foxtail millet includes annotation and mining of the full genome sequence, development of foxtail millet bacterial artificial chromosome (BAC) and expressed sequence tag (EST) resources, comparative analysis with sorghum and rice, characterization of orthologous copies of genes controlling biomass in other grass groups, establishment of efficient transformation protocols, creation of new mapping populations, and QTL analyses to identify new candidate genes for plant architectural variation. In addition, resequencing of several diverse green foxtail millet accessions will provide a data set that allows measurement of the overall genetic variability present within the wild and cultivated crop and will a source of markers for mapping and biodiversity studies (Doust et al. 2009, 2010; see Section VII.A). Other genomic tools available for foxtail millet research include the availability of >100 SSRs and the genetic map (see Section VI.A), 1,500 SNPs, the genome sequences from other cereals (see Section VIII.D), the QTL associated with agronomic traits (see Section VIII.A), and candidate genes


321

associated with agronomic traits (see Section VI.B). All of these resources are expected to support molecular breeding in foxtail millet. VII. ENHANCING USE OF GERMPLASM IN CULTIVAR DEVELOPMENT A. Core, Mini-Core and Reference Sets for Mining Allelic Diversity and Identifying New Sources of Variation Core (10% accessions of the entire collection) and mini-core (10% accessions of the core collection or 1% of entire collection) collections are cost-effective sources to identify accessions with desirable agronomic traits, including resistance to biotic and abiotic stresses. To date, core and mini-core collections (based on phenotypic characterization and evaluation data) are reported in finger millet, foxtail millet, little millet, pearl millet, and tef (Table 5.21). Limited evaluation of finger millet and foxtail millet core collections has resulted in identification of germplasm accessions that mature early, produce more grain or fodder in comparison to control cultivars, or differ in panicle shape and size and seed color and of a few accessions tolerant to drought or salinity. Many of accessions with grains having high seed protein, calcium (Ca), iron (Fe), and/or zinc (Zn) contents were also identified (ICRISAT 2009). Moreover, the core or mini-core collections are dynamic in nature, and these must be augmented, as recently done in pearl millet. Researchers at ICRISAT have developed a global composite collection in pearl millet, finger millet, and foxtail millet, which were genotyped (using SSRs and high-throughput assay, ABI3700) to determine population structure and diversity prior to formation of reference germplasm sets. This reference set captured between 87% to 95% allelic diversity of the composite collections (www.generationcp.org; ICRISAT 2009). Clearly, more research is needed to develop these subsets in other millets or to augment the existing subsets to make them more relevant to the changing needs of crop breeding. B. Assessing Population Structure and Diversity in Germplasm Collections Vast collections of millets germplasm are maintained worldwide in gene banks (see Section IV), and in many cases core or mini-core collections have been formed (see Section VII.A), representing diversity present in the entire collection of a given species. Such reduced subsets are ideal resources to dissect population structure and diversity (both at

322

S. DWIVEDI ET AL.

Table 5.21. Core collection, mini-core subset, and genotype-based reference set reported in finger millet, foxtail millet, little millet, pearl millet, and tef. Crop Core collection Finger millet Foxtail millet Little millet Pearl millet Tef Mini-core collection Finger millet Foxtail millet Pearl millet Genotype-based reference set Finger millet Foxtail millet Pearl millet

No. accessions 622 551 155 55 1600 2094 (revised core) 320

Reference Upadhyaya et al. 2006 Gowda et al. 2007 Upadhyaya et al. 2008 Gowda 2008 Bhattacharjee et al. 1997 Upadhyaya et al. 2009 http://www.database.prota.org

80 35 238

Upadhyaya et al. 2010 ICRISAT unpublished data Upadhyaya et al. 2011

300 200 300

ICRISAT unpublished data ICRISAT unpublished data ICRISAT unpublished data

phenotypic and molecular level), to identify new sources of variation, and to conduct association mapping, which provides insights to markertrait association. In the last few years, there have been greater efforts to develop PCR-based markers, especially microsatellites and SNPs, and/ or DArT markers (see Section VI.A), which were employed to assess population structure and diversity in barnyard millet, common millet, finger millet, foxtail millet, Job’s tears, pearl millet, and tef germplasm collections (Table 5.22). For example, barnyard millet accessions belonging to var. esculenta were less diverse than those of var. crus-galli or var. formosensis (Nozawa et al. 2006), and the molecular profile of tetraploid E. oryzicola is different from that of hexaploid E. crus-galli var. formosensis (Nozawa et al. 2004). Microsatellites differentiated finger millet subsp. africana accessions from those of subsp. coracana originating either from Africa or Asia (Dida et al. 2008). Wang et al. (2010) detected a low level of genetic diversity in Setaria virdis (green foxtail millet) in comparison to its cultivated form, Setaria italica. In addition, they also found that despite a 55% loss of its wild diversity, S. italica still harbors a considerable level of diversity when compared to rice and sorghum. Likewise, the level of linkage disequilibrium in S. italica extends to 1 kb; it decayed rapidly to a negligible level within 150 bp in S. virdis. The 17 SSRs differentiated most of the Chinese Job’s tears accessions from those of Korean accessions, and the Chinese accessions

323

Foxtail millet 77 S. italica and 40 S. virdis accessions, rDNA IGS

Finger millet 109 accessions including wild types and 45 SSRs

170 accessions and 13 SSRs

Barnyard millet 155 accessions and 3 SSRs

Accessions and markers

PCR-based length polymorphism and sequence polymorphism of rDNA intergenic spacer (IGS) clearly demonstrated genetic differentiation between cultivated and wild forms from northern Pakistan and Afghanistan; cultivated forms to some extent showed genetic differentiation between diffient areas, while wild forms clearly showed differentiation between regions in northern Pakistan.

E. coracana germplasm grouped into 3 distinct clusters: subsp. africana, subsp. coracana originating from Africa, and subsp. coracana originating from Asia, with few accessions showing introgression between the African and Asian cultivated germplasm pools, and lower diversity in Asian subpopulation probably due to small number of founder plants involved in its origin.

The 155 accessions included 49 from var. esculenta, 94 from var. crusagalli, and 12 from var. formosensis. SSR markers clustered the var. esculenta accessions into 2 groups (either from central and northeastern Japan or northern and southern Japan), crusa-galli accessions into 12 groups, and formosensis accessions into 6 groups. E. esculenta were less diverse than either of crusa-galli or formosensis accessions. The var. esculenta accessions grouped into 2 classes, while those from var. crus-galli into 11 classes. Marker EC1 discriminated E. oryzicola (a tetraploid species) from the hexaplopid species E. crus-galli var. formosensis.

Pattern of population structure and diversity

(continued )

Fukunaga et al. 2010

Dida et al. 2008

Nozawa et al. 2004

Nozawa et al. 2006

Reference

Table 5.22. Assessment of population structure and diversity as reported in barnyard millet, common millet, finger millet, foxtail millet, and tef germplasm.

324

Job’s tears 79 accessions (Korea and Japan) and 17 SSRs

39 Setaria species and 19 RAPD markers

81 accessions and AFLP markers

Most Chinese accessions genetically distinct from Korean accessions; genetic relatedness and place of collection not related; greater within population polymorphism in Chinese accessions, potentialy a reservoir of novel alleles for crop improvement.

DNA sequence variation at 9 loci revealed low level of genetic diversity in wild green foxtail (q ¼ 0.0059). Despite of a 55% loss of its wild diversity, the cultivated foxtail millet still harbored a considerable level of diversity (q ¼ 0.0027) compared to rice (q ¼ 0.0024) and sorghum (q ¼ 0.0034). LD in domesticated foxtail millet extends to 1 kb, while it decayed rapidly to a negligible level within 150 bp in wild green foxtail millet. Landraces grouped into 5 major clusters: cluster I and II and to some extent cluster IV contain landraces from East Asia including China; cluster III from subtropical and tropical regions in Asia; cluster V from central and western regions of Eurasia; Chinese landraces highly variable among the germplasm studied. Chinese accessions were highly diverse, consistent with the hypothesis of a center of domestication in China, while accessions from eastern Europe and Africa form 2 distinct clusters. The genetic relatedness within S. virdis or between S. virdis and S. italica is probably due to consequence of geneflow between the two subspecies. RAPD analysis revealed that S. italica more closely related to S. virdis, supporting idea that the former originated from the latter. Setaria italica and S. glauca differ considerably. S. glauca and S. sphacelata distinct from S. italica, implying that it will be difficult to transfer some of the beneficial traits from S. glauca and S. sphacelata to S. italica.

50 S. italica and 43 S.virdis accessions, sequence variation at 9 loci

62 landraces and 16 RFLP markers

Pattern of population structure and diversity

Accessions and markers


Ma et al. 2010

Li et al. 1998

Le Thierry d’Ennequin et al. 2000

Fukunaga et al. 2002b

Wang et al. 2010

Reference

325

72 inbreds (70 B-lines and 2 R-lines) and 34 SSR primer pairs

22 inbreds and 627 markers

2000 lines and 24 SSRs

Pearl millet 145 WCA inbreds and 20 SSRs

STRUCTURE analysis detected 5 subgroups and 1 admixed group. Plotting the STRUCTURE results on the geographic map revealed no obvious association either of country of origin or agroecological zone of origin. Established a diversity panel of 288 genotypes, 4 maturity groups representing the whole breadth of genetic variation in the pearl millet germplasm pool from Africa and Asia. 267 of the 627 markers (100 pearl millet genomic SSRs, 60 pearl millet EST SSRs, 410 intron sequence haplotypes, and 57 exon sequence haplotypes) were polymorphic among the 22 inbred lines, which were grouped into 3 clusters with most of the inbreds derived from landrace Iniadi in cluster I; high correlation (r >0.97, P

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