No title

ADVANCES IN GENETICS VOLUME 19 Edited by E. W. CASPARI Department of Biology University o f Rochester Rochester, New York

1977 ACADEMIC PRESS

NEW YORK SAN FRANCISCO LONDON

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1977, nY ACADEMIC PRESS, INC.

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LIBRARY OF CONGRESS CATALOG CARD NUMBER:47-30313 ISBN 0-12-017619-X PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 19 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

EDWARD G. BARRY(133), Department of Botany, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina ETTAKAFER(33), Department of Biology, McGill University, Montreal, Canada CALVIN F. KONZAK (407), Department of Agronomy and Soils, and Program in Genetics, Washington State University, Pullman, Washington N. C. MISHRA(341), Department of Biology, University of South Carolina, Columbia, South Carolina P. A. PARSONS ( l ) ,Department of Genetics and Human Variation, L a Trobe University, Bundoora, Victoria, Australia DAVIDD. PERKINS (133) , Department of Biological Sciences, Stanford University, Stanford, California RUTHSAGER(287), Department of Microbiology and Molecular Genetics, Harvard Medical School, and Sidney Farber Cancer Institute, Boston, Massachusetts

vii

MEIOTIC AND MITOTIC RECOMBINATION IN Aspergillus AND ITS CHROMOSOMAL ABERRATIONS Etta Kafer Department of Biology, McGill University, Montreal, Canada

I. Introduction . . . . . . . . . . . . . . . . . . 11. Meiotic Crossing-over and Nondisjunction: Effects of Chromosomal Aberrations, Especially Translocations . . . . . . . . . . . A. Meiotic Crossing-over in Standard Crosses, . . . . . . . . B. Reduction of Crossing-over by Chromosomal Aberrations, Especially Reciprocal Translocations . . . . . . . . . . . . . C. Aneuploids from Controls, and from Crosses Heteroeygous for Reciprocal Translocations . . . . . . . . . . . . . . . . . D. Meiotic Nondisjunction Frequencies in Crosses Heteroeygous for Reciprocal Translocations . . . . . . . . . . . . . E. Interchromosomal Effects of Translocations in Meiosis . . . . . 111. Mitotic Recombination . . . . . . . . . . . . . . . A. Mitotic Crossing-over and Nondisjunction in Standard Diploids . . B. Crossing-over in the Centromere Area of Group I and Genetic Differences between Strains . . . . . . . . . . . . . . . . C. Mitotic Recombination in Triploids . . . . . . . . . . I). Induced Mitotic Recombination in Standard Diploids . . . . . E. Mitotic Recombination in Translocation Heterozygotes . . . . . F. Mitotic Crossing-over in Disomics from Translocation Crosses, Especially in “Stable” Disomics . . . . . . . . . . . IV. Genetic Mapping and the Use of Translocations . . . . . . . . A. Genetic Mapping of Mutants in Aspergilhs nidulans and Effects of Translocations . . . . . . . , . . . . . . . . . B. Frequencies, Detection, and Mapping of Translocations . . . . . C. Mapping of Centromeres and the Use of Translocations for Sequencing of Meiotic Fragments. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

33 35 35 40

43 50 56 58 58

67 74 81 85 97 100 101 107 116 124

I. Introduction

When mitotic recombination and the parasexual cycle were first demonstrated in Aspergillus nidulans, it was expected and hoped that these processes would permit mapping and breeding in commercially used asexual fungi, like Penicillium chrysogenum, Aspergillus oryzae, etc. 33

34

ETTA KAFER

(Pontecorvo et al., 1953 ; Pontecorvo, 1956). Indeed the feasibility of such work, and the existence of somatic fusion of cells and nuclei followed by mitotic segregation, have been demonstrated in a large number of species (Roper, 1966). These include not only important parasitic fungi, like Ascochyta imperfecta (Sanderson and Srb, 1965) or Ustilago hordei (Megginson and Person, 1974), but also mammalian cells and recently even those of higher plants (Gamborg et al., 1974). However, most of these investigations have not yet progressed to any practical stage, since various unexpected problems were encountered [for example, scarcity of spontaneous recombinants in Penicillium (Macdonald, 1971 ; Ball. 1973) 1. From the experience with Aspergillus nidulans (Kafer, 1965), it seems likely that chromosomal aberrations, especially translocations, which are induced simultaneously with desired mutations and recombinants, produce a major problem for genetic analysis by mitotic recombination, even though these do not explain all unexpected features of mitotic segregation encountered with asexual species. Such aberrations are frequently induced in fungi by practically all commonly used mutagens, especially the relatively high and multiple doses of irradiation which have been applied in many asexual species (even low doses of UV induce a considerable frequency of translocations in Neurospora, as reported by Perkins, 1974). Since heterozygous translocations prevent random recovery of chromosomes in mitotic haploids from diploids, it is obvious that mitotic analysis of asexual species becomes frustratingly complicated by induced translocations, especially if chromosome numbers are small [e.g., possibly only three in Penicillium (Ball, 1973) 1. Detailed genetic analysis can, therefore, be successful if mutants are obtained spontaneously (e.g., selected by resistance to analogs and inhibitors) or with mutagens that cause no or few aberrations (e.g., chemicals that induce mainly base-pair substitutions). So far few mutagens of this type are known that are effective in fungi [except possibly nitrous acid (Abbondandolo and Bonatti, 1970) ] ; however, low doses of highly mutagenic chemicals, like nitrosoguanidine (NG) , also appear to produce relatively fewer chromosomal aberrations than point mutations [ e.g., permitting fast progress of mitotic mapping in the slime mold Dictyostelium, where mainly NG-induced mutants are being used (Katz and Kao, 1974; Kessin et al., 1974) 1. Although unrecognized aberrations complicate genetic analysis, translocations, once identified by their effects on meiotic and mitotic nondisjunction, can be used to advantage in mitotic mapping of markers and centromeres. This certainly is the case for Aspergillus nidulans, where meiotic recombination also occurs, so that translocations can be combined with suitable markers. In this species, meiotic recombination frequencies are so high that markers of the same chromosome arm often are com-

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

35

pletely unlinked in crosses; such markers can be sequenced either by mitotic crossing-over, or by means of overlapping translocations (Pontecorvo and Kafer, 1958; Kafer, 1958, 1975). I n asexual species, such use of translocations is bound to be very difficult and will probably be restricted to a few favorable cases. One major problem is that in most fungi, mitotic, and even meiotic, chromosomes are too small for recognition of translocations cytologically. However, new techniques might solve this problem, as has recently been the case for mammalian chromosbmes, which has led to fast progress in the mapping of translocations and deletions and to their use for mitotic mapping of genes in human cells (Jacobs et al., 1974; Ruddle, 1973; Shows and Brown, 1975). One great advantage of techniques that use chromosomal aberrations for genetic mapping is the fact that the obtained results are qualitative, and often comlementary to evidence from meiotic mapping [as has been demonstrated in many species of higher plants, e.g., in maize by Kasha and Burnham (1965) and in Drosophila, as in phages and bacteria, even at the microlevel within genes, e.g., by Welshons and Keppy (1975) 1. In addition, chromosomal aberrations may give information on basic processes, e.g., those of pairing and disjunction [as demonstrated in Drosophila by Grell (1962, 1967) ; or in maize by Burnham et al. (1972) 1. This investigation, therefore, had three purposes : 1. T o analyze the effects of one, or two, overlapping, reciprocal translocations on meiotic crossing-over and nondisjunction ; 2. To identify the processes of mitotic recombination in diploids with and without translocations, in triploids, and also in disomics from single and double translocation crosses, as well as the effects of inducing agents on these ; 3. T o assess the various methods of genetic mapping, the uses of translocations for mapping, and the various problems arising from chromosomal aberrations for mapping by the parasexual cycle. All the techniques and information on mutants used in these investigations have been written up in detail and are available from the author on request (origins of most of the translocations are shown in Table 1 ; for other strains, see Barratt et al., 1975). II. Meiotic Crossing-over and Nondisjunction: Effects of Chromosomal Aberrations, Especially Translocations

A. MEIOTIC CROSSING-OVER IN STANDARD CROSSES 1. Homogeneity of Linkage Values

Meiotic recombination in standard strains of Aspergillus nidulans produces typical values between linked markers, which differ no more

TABLE 1 Mitotic and Meiotic Tests for Translocations (T) in Original or Progeny Strains of UV-Induced Mutants'

UVinduced mutant

Residual genotype

FGSC No.*

Linkage group of mutant andarm

ActAl

riboA yA; nicB

23 1

I11 L

adE2O~

biA pabaA biA

50 429

I

adG14 anAl

biA; p A biA

choAla

biA (AcrA; lysB; choA+)

lacAl luAl lysB6

YA; P w A biA biA; smA

lysD2O

biA; sB pabaA y A

35 31d 1 354 58d 5.5 66 395 418

"1

I L I L

R1 VI R I L V L

Mitotic test in diploidsc Recombination between groups Deduced translocation None

T1(ZZ;VZZ) None None

No. of Lowest hap% loids found -

-

-

67

3

51 81

Groups with



proA

+

bjA

AUA

WA

haplold:

FIG.6. Stepwise insertion of selective markers .yA (and simultaneously adE) distal on I R, and suAadE distal on I L, into pro strains by mitotic crossing-over, followed by haploidisation. gives unambiguous results: these again place pro consistently onto the left arm, provided 1% exceptional pro-yellow types are disregarded as possible coincidence of two events of segregation. All other diploids give significant frequencies of “yellow” pro/pro “crossovers,” even though considerably less than “suppressed” pro/pro. These results, therefore, rule out the existence of a pericentric inversion in the original biAl strain but, on the other hand, definitely implicate genetic differences between biA1 and the original wild type ~ b sthe basis of the different results obtained with proA1 (or proA2) compared to pro-94. Since only diploids with “wild type” centromeres give results in agreement with current models of mitotic crossing-over, it looks as if the original biA 1 strain contains a mutation that affects mitotic segregation close to the prod-centromere area. Because no testable hypothesis comes to mind, details of these results are given here as accurately as possible for future consideration: Twelve different derived proAl strains were checked for mitotic crossing over in 18 different diploids. These produced on the average 9.3 k 1.1% of %uppressed” pro/pro crossovers (among 4913 selected ; range 5-15%). From 13 of these diploids, “yellow” crossovers could also be selected (total tested 1880) ; these were homozygous for the proA allele in coupling with yA with an average frequency of 3.4 f 0.5%.However, there appeared to be differences between different proA1-strains ; e.g., the “early” proAy+strain from the published diploid Z gave the highest values of apparent

RECOMBINATION AND TRANSLOCATIONS IN

Aspergil lus

73

crossing over to the left of proA among yA/yA (when tested in three different diploids, including diploid 854, genotype in Table 11): i t showed a range of 5-7% among a total of 452 yA/yA (which all had t o be analyzed for heteroxygosis of proA1 by selection of suA-wA haploids, since in these diploids proA was in replusion to yA). On the other hand, the derived proAl-“control” strain for the translocation work, when tested in four different diploids (one of them being diploid 600, Table 11) showed significantly lower values, ranging from 1.1 to 3.6% (average 1.8%, 539 yA/yA tested; x2 = 10.5, P < 0.01). Most other strains of the pedigree between these two proAl-strains gave, however, intermediate values (average 4.0%, 689 yA/yA tested from 6 diploids). The following procedure was used to check the original proA11 (pabaAl yA2; pyroA4) strain for proA-segregation. T o avoid all crossing, a new fpaA-allele (fpaA91) was induced in this strain by nitrosoguanidine treatment, so that fpaA/fpaA crossover segregants could be isolated on the left arm of group I, as well as yellow ones on I R (diploid 863, Table 12; comparable to the selection in both arms after introduction of suAadE on the left arm, and yA ad23 on the right arm, into proA2 and pro-94 strains, Fig. 6, and Table 11). The general trends emerging from a closer inspection of the data of Table 12 are the following: All diploids which contain suA in coupling with pro-94 of “wild type” origin, give higher frequencies of “suppressed” pro mitotic crossovers on I L (ranging from 13 to 23%, average 15.8%), than the corresponding diploids with proA2 of “biAl” origin (which show a range of 9-1276, average 10.8%). In addition, all the former diploids show lower frequencies of “yellow” pro/pro crossovers on I R (1.0-1.8%) than the latter (range from 2 to 570, average 3.4%). While for any one pair of diploids these differences are barely significant a t the 5% level, the combined values show highly significant differences (x2 > 10, P < 0.01, as expected from the fact that there is no overlap of values). I n addition, the centromere type of the pro+ ‘V.ester” strain also seems to influence results, since less yellow pro exceptional types are produced when pro-94 is combined with a tester having a “wild type” centromere (diploid 865) than when the tester is a “biA”-type strain (diploids 866 and 868). When all data are considered, it seems clear that only diploids with “wild type” centromeres give unambiguous results as expected from reciprocal mitotic crossing-over, while none of the diploids with “biA”-type centromeres conform to these patterns, presumably due to some type of mutation (that may also cause the correlated higher nondisjunction). To speculate on the kind of mutation which would cause the observed results one could postulate that i t creates a weak alternate centromere position (“spindle fiber” attached to the left of proA) which would result

74

ETTA KAFER

in about 1.5-20/0 single crossovers on the “right” arm when the mutation is heteroaygous (diploids 969, 866, 868), and twice that much when it is homoaygous (diploids 854,Z and 867,896, etc.) . At this time it is difficult to visualize how this or any other alternate hypothesis could be tested, since the possibilities of mitotic analysis have been pretty well exhausted, and the cytology of mitosis is obviously too imprecise to be a suitable tool (Robinow and Caten, 1969). The only remaining possibility, namely investigation of the genetic differences between pro-94 and various proA mutants and the anomalous centromere behavior in the latter, by means of meiotic tetrad analysis might, however, be worthwhile. While previously no markers close to any other centromeres were known so that the centromere distances computed from unordered tetrads were, by necessity, very approximate (Strickland, 1958) markers very close to their centromeres have now been identified in both arms of groups 11, 111, and IV, and new potentially linked ones have become known also for all other groups (Fig. 15; Clutterbuck, 1974). Mutations causing changes, e.g., in heterochromatic content, that affect meiotic crossing over as well as nondisjunction, have been observed in several organisms (in maize, by Ward, 1973; or in Drosophila, by Carpenter and Sandler, 1974). I n the above case in Aspergillus, however, extensive crossing of the described strains with “wild type” or “biA” centromere areas to the same tester strains has not revealed any effect of the postulated mutation on meiotic recombination (results are included in Table 2 ) . Whether any other of the reported cases that show reduction of repetitive sequences in the centromere areas correlated with increases of nondisjunction, or diffuse centromeres, are relevant here is hard to guess, even though at least in one case differences between meiosis and mitosis were observed (Comings and Okada, 1972; Laird, 1973). C. MITOTICRECOMBINATION IN TRIPLOIDS Triploids of Aspergillus nidulans have been obtained previously. Provided they were heteroaygous for conidial color markers, it could be seen (Fig. 7) that they are considerably less stable than diploids, and that they produce a high frequency of color segregants, probably by chromosomal segregation (Elliott, 1956; E. Kafer, unpublished). However, i t turned out to be impossible to obtain quantitative information, as long as mainly recessive markers were available, even when almost every homolog contained a marker (as shown for one of these early triploids, triploid 407, Table 13). These triploids were constructed with the aim of recogniaing as many segregants as possible by having two mutant alleles of most

TABLE 13 Genotypes of Triploids Triploid HomoNo. logs

I

Linkage groups :

m

Il

a 407

b C

a

2300

b C

Groups :

Iv a

407

b C

a

2300

b C

+ +

V

GPYYrOA

QymA

---. ---. methG

+

+

methG

+ +

+

QymA

M

VI

==-L- + +

+

nicA

+

nicA

+

VIII

sB

+-

+

+

+

+

+

+

+ +

+

+ + +

+

m'cB

choA

+

-++

choA

+

+

0

+ + +

+

+

nicA

+

+

facA

+ + + + + sbA lacA sB

+

+ +

+ malA

+ +

OIiA

+

choA

+ +

+

-+

+ + i

riboB

+ +

chd ~~

s D f w A +

+ + +

+

nboB

~

chaA

+ +

nboB chaA

76

ETTA KAFER

recessive markers present in the triploid. Crossing-over or nondisjunction would then produce segregants differing in their requirements or color from the original triploid, so that such segregants could fairly easily be recognized. However, it turned out that segregation frequencies are so high, that the problem is not to obtain spontaneous segregants, but to distinguish the various types obtained. To assess the relative frequencies of crossing over vs. chromosomal loss or nondisjunction, crossovers should be distinguishable from nondisjunctionals, and triploids from diploids and haploids. Since it is impossible to prove whether a segregant is homoor hemizygous for all recessive markers of a homolog, such distinctions were difficult or impossible for most segregants from triploid 407. The situation is quite different for the recently constructed triploid 2300 (Table 13). It contains not only more recessive color mutants than triploid 407 (five rather than three), but also a relatively large number of mutants that can be recognized in the heterozygous condition ( f p a B , SulA, suAadE if adE is present, IodA, AcrA, ActA, sB, and OZiA). These can be used to distinguish diploid or triploid segregants from haploid ones, since in the former usually a t least one of them remains heterozygous; also loss of homologs containing these mutants can easily be followed. In addition, mutants are generally present only as a single allele which makes identification of all homologs in haploids possible (except for some white haploids, Table 15; the haploids selected on CM+pfp were isolated to check the genotype of the triploid). Only the mutant rib& of group VIII was present in two homologs, since it served as a balancing marker to obtain the heterokaryon between a diploid containing two of the homologs (b/c) and the haploid strain (a) from which the triploid was selected. I n addition, yA2 and yA91 (a recently obtained olive mutant) are noncomplementing alleles, which, however, can easily be distinguished in haploids (but not in the various diploid or triploid combinations) . Haploids and diploids were also distinguished visually from triploids (Fig. 8 ) . The results obtained with this triploid (2300) demonstrate very clearly that chromosome loss is occurring extremely frequently in triploids of A. nidulans. When conidia from the newly synthesized triploid colonies were plated onto complete medium, very high frequencies of unstable aneuploid colonies of many different types were obtained (30-3576 in two platings with totals of about 200 each). I n addition, most triploid colonies produced one or two color patches or segments large enough to be seen by the naked eye (see Fig, 7b) compared to 1-2 per 100 diploid colonies (Wood and Kafer, 1969) (Fig. 7a), and practically all of these resulted from chromosome loss. To obtain a random sample of segregants, all easily visible color segments from the triploid colonies of the first plating were purified by

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

77

FIG.7. Low-density platings of conidia on complete medium: (a) diploid; (b) triploid. FIG.8. Shape and arrangement of conidial heads permitting visual classification of (a) diploids, (b) triploids, as well as (c) haploid segregants.

78

ETTA KAFER

streaking, and the resulting stable types were tested for markers and for ploidy (285 segregants from 145 triploid-looking colonies, Table 14). Since few segregants remained triploid, chromosome loss is obviously the major process a t work, while nondisjunction is quite rare and mitotic crossing-over even more so. This is also evident from the large frequency of aneuploids found, not only among the plated conidia which resulted in sectoring usually hyperdiploid colonies, but also as precursors of many of the isolated color segregants. Triploids of A . nidulans therefore constitute an excellent source of aneuploids (comparable to triploid meiosis in yeast, Parry and Cox, 1970). a. Random Loss of Chromosomes. That the frequent loss of chromosomes was basically a random process was demonstrated in two ways: (i) All haploids, which constituted about one-third of all color segregants, were classified for loss of homologs for every linkage group. The resulting frequencies then were compared with that expected on the basis of random loss (Table 15). As can be seen from these results, very few cases are found where the observed frequencies differ significantly from the expected ones, and in three of these cases the known reduced viability of the corresponding mutants is likely to be the main cause (homologs Ia, Ic, and I I I b ; only for one, sbA on VIb, does the extremely low recovery remain unexplained). (ii) All diploid yellow segregants (mainly yA2/yA91) were checked for loss of any of the mutants conferring resistance as an indication of loss of one of the homologs of groups 11,111,VI, TABLE 14 Relative Frequencies of Various Types of Spontaneous Color Segregants from Triploid Colonies of 2300

Crossovers Color

Haploids

Yellow-olive

23

Darker or paler green White Fawn Chartreuse Yellow-cha

3 33 16 2 9

Total Nos. and frequencies

86 30 %

Chromosomaltype segregants

Total

2n

3n

2n

3n

No.

%

7

3

151

16

200

12%

25 33

9% 12%

i2

> 10 > 4%

(max. 9%)

> 167 > 60%

285

TABLE 15 Relative Frequencies of Recovery of the Three Homologs a, b, or c in Haploids from Triploid No. 2300

a

F!

Bis

m

Linkage groups and homologs

I a

b

I1

I11

IV

-c

a

b

c

a

b

c

a

b

V

VI

VII

VIII

--c

a

b

c

a

b

c

a

b

c

2el

(a, b a o r c ) b c

(b orc)

Totals (No.)

8 Z

.+ r3

No. from CM So 64 14b 33 19 34 30 22 34 41 lga 26 36 26 24 46 6" 34 33 26 27 29 (1) 23 llb (22) No. from 68c 1 2 24 24 23 48 Oc 23 27 15. 29 29 22 20 37 10 33 28 18 25 21 (8) 21 9' (12) C M pfp Total No. 76c 65 16 57 43 57 78 22c 57 68 34a 55 65 48 44 83 7a 67 61 44 52 50 (9) 44 20 (34)

+

Combined % x2

P

a

4gC 41 10 45.1' 0.25 >.5 1000$ 287." 70h 11190 969s 120018OOU 25307"

* The values in columns 3 to 6 are, in some cases, approximations, or they may be based on incomplete information. Other values may be found in the literature. These are presented only for the purpose of making a general comparison with Neurospora. LM: Measurement with the light microscope of conventionally fixed and stained material. EM: Measurement with the electron microscope of synaptonemal complexes. t Based on 1100 pm double-stranded DNA, measured with the electron microscope. $ Tentative because of high variability in crossing over. For calculations, see Section IV, B, 2, c. 0 Salivary gland polytene chromosome lengths are 2219 pm (entire complement) and 414 pm (X-chromosome) (Lindsley and Grell, 1968). # Euchromatic regions only, except for X-chromosome, which is to nucleolus organizer. The SC shortens as meiosis progresses. 7 The autosomes only. References: C a i r n s (1963). bByers and Goetsch (1978). CBicknell and Douglas (1970). dMortimer and Hawthorne (1975). +3ingleton (1953). 'Gillies (1972). UHorowitz and Macleod (1960). "indsley and Grell (1968). 'Carpenter (1975a). jLaird (1971). kRasch et al. (1971). 'Rhoades (1950). mGillies (1973). "Evans and Rees (1971). ONeuffer and Coe (1974). pBarton (19,50). CKhush and Rick (1968). 'Griffen (1955, 1960). .M. J. Moses (personal communication). 'McCarthy (1969). .Miller and Miller (1975). uHultBn and Lindsten (1973). wA. T. C. Carpenter (personal communication). unique sequence DNA hybridizes with RNA which has been transcribed during log phase mycelial growth. Bhagwat and Mahadevan (1973) have labeled RNA a t 8, 16 and 24 hours after the beginning of germination and growth of conidia. Identifying the mRNA (messenger RNA) component extracted from the hyphae, they find that the mRNA transcribed a t different times after the beginning of conidial germination and growth is different both by amount

THE CYTOGENETICS OF

Neurospora

155

of the total DNA to which it hybridizes and by the amount with which it overlaps the mRNA transcribed at the other times, thus indicating differential activity of genes in the maturing culture. c. Histones. Neurospora crassa appears to have very little histone protein associated with the chromosomes. Dwivedi et al. (1969) and Leighton et al. (1971) were unable to isolate and identify any histones, but Hsiang and Cole (1973) have isolated two “slightly lysine-rich” histones from Neurospora chromatin. Hsiang and Cole find that there is only 25% as much histone protein as in higher eukaryotes; and they believe that the earlier failures may have been due to the small quantity of histones, proteolytic degradation, and other isolation procedures that gave low yields of chromatin and histones. Other basic proteins may be present in Neurospora chromosomes, since there is a requirement for putrescine, which is a precursor of spermidine and other polyamines (Davis et al., 1970; Deters et al., 1974). 3. Techniques for Chromosome Cytology

Conventional techniques developed for eukaryotic plants and animals are not usually successful in fixing and staining Neurospora chromosomes. However, variations have been developed that stain chromosomes eff ectively during meiotic and mitotic divisions in the ascus. McClintock (1945), Singleton (1948, 1953), St. Lawrence (1950, 1953), Barry (19661, Phillips (1967), and Pincheira and Srb (1969) have obtained good results with orcein or carmine, and B. C. Lu (personal communication) and Raju (1976) have used hematoxylin effectively in Neurospora. Hematoxylin and carmine stain the nucleolus and centrosome intensely, while orcein stains the nucleolus only lightly and the centrosome not a t all. Good staining of chromosomes in the asci of numerous other ascomycete genera has also been obtained using Giemsa (Rogers, 1965, and later publications). The procedures we now follow and recommend for Neurospora are described by Barry (1966) for acetoorcein and by Raju (1976) for hematoxylin. Most of the observations reported in the following sections have employed ethanol-acetic acid-lactic acid fixation followed by staining with acetoorcein. A technique for producing giant conidia promises to make vegetative nuclei of Neurospora more accessible to observation. When conidia are suspended in medium containing 3.2 M ethylene glycol, they form spheres that grow without cell division (Bates and Wilson, 1974). N. B. Raju (unpublished observations) has examined these cytologically using hematoxylin. Nuclei divide, and chromosomes can be observed a t different mitotic stages. Some giant nuclei become polyploid.

156

DAVID D. PERKINS AND EDWARD G. BARRY

Particular genotypes also promise to be technically useful in cytology. For example, N. B. Raju (unpublished) has found that a dominant mutant discovered by Newmeyer has as one of its effects the induction of waves of synchronized mitoses in ascogenous hyphae. Thus, large numbers of nuclei in precisely the same stage of division may be observed together, and different stages are characteristic of different groups of hyphae. Attempts to stain Neurospora meiotic chromosomes with Feulgen have apparently not been successful. Weijer and McDonald (1965)have used a modified Feulgen reaction to obtain chromosome staining in the two mitotic divisions in the ascus. The Feulgen reaction has been used successfully for staining somatic nuclei (Horowitz and Macleod, 1960; Somers et al., 1960; Weijer, 1965), but other stains are probably preferred. I n Gillies’ (1972) studies of the synaptonemal complex, he reports difficulty in staining chromosomes and obtaining contrast between chromosomes and nucleoplasm for electron microscope observations of meiosis.

4. Chromosome Identification and Morphology a. Chromosome Number. McClintock (1945)showed the haploid number to be 7 in N . crassa, and all later observations of chromosomes in the ascus have been in agreement. [However, Somers et al. (1960)reported a minute eighth chromosome in somatic nuclei.] The same number has since been found in all the other Neurospora species: N . sitophila (Fincham, 1949; Dodge et al., 1950; Perkins et al., 1976),N . tetrasperma (McClintock and Weaver, reported by McClintock, 1945;Dodge et al., 1950),N . intermedia (Perkins et al., 1976), and the five homothallic species N . terricola (Nelson and Backus, 1968),N . africam, N . dodgei, N . lineolata, and N . toroi (Raju, 1976). [Early counts of n = 6 for N . tetrasperma by Colson (1934) and Cutter (1946) were probably incorrect.] b. Chromosome Morphology at Pachynema i. Idiograms. McClintock (1945) drew up karyotypes numbering the seven chromosomes from longest to shortest and showing relative lengths of the chromosomes measured at pachynema and a t mitotic metaphase stages in the ascus. She tentatively determined the probable centromere position of each chromosome by measurement a t mitotic metaphase in the third division when the chromosomes show a sharp bend a t the centromere. The centromere is not visible in the meiotic stages, although McClintock supposed it to be correlated with the position of the heaviest chromomere of each pachytene chromosome. Singleton (1948,1953) subsequently published more detailed idiograms showing the identifying morphological features of each chromosome. Each chromosome was

THE CYTOGENETICS OF

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Neurospora

distinguished by its chromomere pattern and length a t pachynema, and by its centromere position and length a t mitotic metaphases in the ascus. Singleton's chromosome maps were assembled from observations of about 20 pachytene figures where most or all of the chromosomes could be identified. He regarded the maps as tentative. They are reproduced in the upper part of Fig. 3.

,satel Iite

a1

a

1

2

3

b"

b

a

I

4

a

a

5

b

6

7

I

FIG.3. Pachytene chromosome morphology of Neurosporu crassa. Above: The chromomere pattern as drawn by Singleton (1953). a and b identify the largest chromomeres of each chromosome. Bebw: The patterns that we now regard as most characteristic of each chromosome. Not all chromomeres are included in the diagram (compare with Fig. 4). In many nuclei the chromomeres identified here are not seen and chromosome identification depends on length, or will be in doubt. Even though the patterns of chromosomes 3, 4, and 5 appear to be very distinct, these chromosomes may be easily confused if the conspicuous chromomere does not stain or show clearly. The heteromorphic, double chromomere in 1 is visible in about 50% of nuclei. This may be due to stretching when the chromosome is spread out. The achromatic gap in 2 is not always observed. Singleton's chromosome 7 is our chromosome 6.

158

DAVID D. PERKINS AND EDWARD G. BARRY

The pachytene maps which we feel are more typical of the usual chromomere patterns and configurations are shown in the lower part of Fig. 3. These revised maps are also tentative working maps for our present studies. Probably one may expect to find other typical patterns, depending on the combinations of strains used. Most of our observations are now made on strains from the Oak Ridge wild-type (74-OR23-1A and 74-OR8-la) background. ii. Variability of chromomere patterns. It has been our experience that each chromosome is not always identifiable, and that the chromomere patterns are not distinctive for all the chromosomes, a t least throughout the pachytene stage when aberrations are analyzed and their breakpoints are located most effectively. There are two difficulties that contribute to the problem of correct chromosome identification. The first is that the chromosomes undergo change during pachynema, which is a stage of long duration, and the chromomere patterns shift as the stage progresses. Some chromosomes may also elongate faster than others. The second difficulty is that different strains probably differ slightly in their chromomere patterns. The strains now used as standards are apparently somewhat different in this respect from the combinations used by Singleton and McClintock. Probably for these reasons, Singleton may have omitted or confused some of the chromomere positions in his idiograms. For example, chromosome 1 has a conspicuous, central, heavy chromomere (the a-chromomere) surrounded by a heavily stained region in Singleton’s diagram. This largest chromomere is almost exactly in the middle of the chromosome, and there is a second, less-marked chromomere ( b ) about halfway between the a-chromomere and one end of the chromosome. I n most of our figures, and in some of Singleton’s diagrams reconstructing the figures he photographed (his Figs. 22-23A1 27A and 30A), the a and b chromomeres are about equally intense, and they divide the chromosome into approximate thirds (see Fig. 3). iii. Heterochromatin. Large and numerous chromomeres are a conspicuous feature of the pachytene chromosomes, with the largest and darkest blocks of chromomeres usually appearing in two sets in the midregion of chromosome 1. McClintock (1945) and Singleton (1953) recognized chromosome regions a t second telophase which they called heterochromatic. However, our convention has been simply to refer to the more darkly and intensely stained regions as “heavy” chromomeres. The chromomeres are observed during pachynema and other prophase stages, but variable chromatic regions are not observed a t metaphase of meiosis and mitosis in the ascus. Detecting heterochromatin by a possible differential time of onset of DNA synthesis by chromosome or chromosome region has not been feasible for technical reasons, mostly because of the inability to

THE CYTOGENETICS OF

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159

obtain specific radioisotope labeling of DNA (Fink and Fink, 1962a,b; Baer and St. Lawrence, 1964), but also because the continuous formation of new waves of asci in the perithecium in asynchronous development prevents recognition of specific early or late synthesis by chromosome segments in any one ascus. None of the chromosome ends are marked by knobs of great contrasting size, except the satellite of the nucleolus chromosome. Variations in the size of the terminal chromomeres are seen among nonhomologous chromosomes, however. Nor have any striking heteromorphic chromosomes or chromomeres yet appeared among N . crassa stocks of different background, excepting the satellite and chromosome aberrations. Crosses between wild strains of N . intermedia, and interspecies crosses of N . crassa by N . intermedia have shown some differences in chromomere patterns (Perkins et al., 1976). N . intermedia chromosomes are much less well marked by heavy chromomeres than are N . crassa chromosomes, in the asci of both intraspecific and interspeoific crosses. iv. Spear ends. At pachynema, McClintock, Singleton, and St. Lawrence recognized “spear ends” of chromosomes 1 and &paired homologs appeared to be touching a t the tips while elsewhere the pairs were separated and lying parallel. I n our preparations, this seems not to be a consistent feature of any chromosome pair. Any apparent spear ends can equally well be attributed to a partial twist of the bivalent. I n studies of the synaptonemal complexes of Neurospora, Gillies (1972) found that all homologous ends of pachytene chromosomes are separated at their sites of attachment to the nuclear membrane. v. Appearance of univalent chromosomes. Sometimes unpaired chromosomes (univalents) or portions of unpaired chromosomes can be clearly recognized. At other times their identification is uncertain. The difficulty lies essentially with the small diameter of Neurospora chromosomes. If two paired homologs are closely associated or twisted about one another a t pachynema, i t may be impossible to resolve their double nature. Furthermore, unpaired and paired chromosomes differ in their morphology. The chromatids of an unpaired chromosome are unraveled, and appear to separate somewhat from one another, thus making a fuzzy doubleness and a width sufficient to suggest the diameter and other characteristics of an indistinct bivalent. For a synapsed pair, the chromatids of each chromosome are not visible with the light microscope, and each chromosome appears to be a single strand. Unpaired regions or chromosomes thus often mimic a normal paired region or chromosome. In paired regions, the chromomere patterns are usually distinct and sharp at midpachynema, but unpaired sections often show no such crisp pattern and may be darkly staining yet indistinct in fine detail. vi. Description of individual pachytene chromosomes. Chromosome 1

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DAVID D. P E R S I N S AND EDWARD G. BARRY

can be identified with a high degree of certainty. It is the longest chromosome of the complement by a significant margin (one half again the length of chromosome 2). There are numerous distinctive chromomeres. Confusion regarding the actual position of the heavy a and b chromomeres, discussed above, does not interfere in practice with the identification. The pachytene morphology of an inverted chromosome 1 can be seen in Fig. 4. Chromosome 2 is usually easy to identify because it bears the nucleolus organizer region, and a t pachynema the nucleolus is a very conspicuous body in the nucleus. The region of chromosome 2 near the organizer in a typical nucleus is well marked by large chromomeres displaying a characteristic pattern. In some nuclei, chromosome 2 may be shorter than 3. Some strains of h'eurospora crassa (the St. Lawrence and Oak Ridge wild types and their derivatives) have a small satellite at the end of the nucleolus organizer region which may be identified in well stained, con-

FIQ.4. Pachynema in Neurospora crassa. The photograph is of one focal level of the nucleus, and the accompanying drawing shows the entire nuclear complement of seven chromosomes. (Chromosome 5 has been shifted to the right in the drawing to avoid a confusing overlap with parts of 2 and 4. The overlap in the actual nuclcus was not a problem because the arms were in different focal planes.) Chromomere detail is shown in the drawing, and each chromosome is identified; only 6 is not clear. Chromosome 1 is homosygous for inversion H4250. One parent contained the original inversion, but in the second parent the inversion sequence was derived by mei-3-induced somatic breakdown from an H4250 duplication (see Section V, E, 4, b). (Orcein staining, X4000. Stocks obtained from D. Newmeyer.)

THE CYTOGENETICS OF

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161

trasting figures. At pachynema when the nucleolus is a t its greatest size, the satellites are seen on the surface of the nucleolus, usually in the hemisphere opposite the organizers. Photographs showing the organizers and satellites can be found in Barry and Perkins (1969); see also Fig. 4. Many stocks, both mutant and wild-type, lack the satellite. Chromosomes 3, 4,and 5 are all about the same size, and in the usual orcein-stained nucleus they do not develop constant and distinctive patterns. I n some figures, our comparisons by chromomere patterns match rather well with the McClintock-Singleton diagrams. In others they do not, and our identifications are then very tentative. Since the chromosomes are rarely flattened into two dimensions in our preparations, measurements of true length are not accurate enough to distinguish among the members of the 3-4-5group. Chromosomes 6 and 7 are about equal in size, and usually they are clearly smaller than the intermediate chromosomes 3,4, and 5. I n darkly stained nuclei they are generally not in doubt because the chromomere patterns are distinctive, but in lightly stained nuclei we have found these two chromosomes to be more similar than the McClintock-Singleton descriptions would suggest. Each has a large, shifting, median or submedian chromomere, the a chromomere. Singleton’s (1953) chromosome 7, which other workers (McClintock, St. Lawrence, Barry) call 6, has several additional large chromomeres arranged in a distinctive pattern. Singleton’s chromosome 6 (7 for McClintock, St. Lawrence, and Barry) is less well marked, by comparison with 7. However, in Singleton’s maps of chromosomes 6 and 7, the overall pattern is much the same, with the a and b chromomeres spaced along the chromosome a t about the same ratios from the ends, and with chromomeres a t the tips of each chromosome, although the tip chromomeres differ in size. c. Recognition of Chromosomes at Condensed Stages. Phillips (1967) was able to recognize interchanges involving chromosome 2 a t diakinesis and metaphase I by the rings and chains that indicate aberration pairing in translocation heterozygotes. Our experience has been that the small chromosome size at metaphase I, the possibjlity of overlapping bivalents, and the unsynchronixed anaphase-I disjunction make identification unreliable a t this stage, except for chromosome 2 if the nucleolus is still attached. d. Chromosome Morphology at Later Stages in the Ascus. The number, size, centromere position, and arm ratios of the mitotic chromosome complement have been diagrammed tentatively by McClintock (1945) and Singleton (1953).The mitotic karyotype is obtained a t metaphase of the third nuclear division in the ascus, when the chromosomes may be seen clearly in polar view on the metaphase plate. Each chromosome

162

DAVID D. PERKINS AND EDWARD G. BARRY

has a V or J shape, with the bend presumably located a t the centromere. The following fourth division, after the ascospores have been delimited, is also useful for observing chromosome morphology except that the chromosomes are smaller and more crowded inside the nucleus.

B. MARKERDISTRIBUTION AND GENETICMAPPING 1. The Markers

Over 400 gene loci have been mapped to the seven linkage groups of Neurospora crassa. The broad categories of phenotypes are listed in Table 2. TABLE 2 Numbers of Nonallelic Genes of Various Types That Have Been Mapped to Linkage Groups in Neurospora crassan Linkage group Type of mutant Nutritional Other biochemically defined Morphology Resistance to toxic agents Irreparable temperature-sensitive Suppressor Radiation-sensitive Recombination Meiotic Vegetative incompatibility Carotenoid Melanin Modifier Rhythm Lethal Sterility Growth rate Mating type Total genes

I

I1

I11

34 13

14 10

14 3

31 12

13

7

1

VI

27 5

21 12

10 3

9 11

129

10 4

24 2

18 5

5 5

17 2

118 30

3

3

3

2

1

20

2

2 1

4

17

1 2

1

1 -

-

-

1

-

38 (9%)

2 1

-

-

3 1 1

5 1 1 124 (29%)

Total mapped

V

IV

1 2 1 2

1 1

-

-

-

67

70

-

VII

-

-

2 1 2 -

-

33

2 1

1 1 1 1

-

51 (12%)

57

7

4 3 9 6 5 4 3 3 8 2 1 426

A mutant is listed under only one category, even though it might equally well fall under several categories. Uptake and permeability mutants are not listed separately, but are included under another heading, such as resistance, other biochemically dejined, or irreparable temperature-sensitive.

THE CYTOGENETICS OF

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163

Several types of mutants have not been found, or are rare. There are few autonomous ascospore color mutants compared to Sordaria, Ascobolus or Podospora. This may be because white spores are usually inviable, or because they fail to survive the high temperature routinely used to break dormancy. White ascospores of one mutant type have been shown to germinate spontaneously (Johnson, 1975a). Several other categories of mutants are as yet underrepresented in Neurospora compared to other fungi-for example, radiation-sensitive, super-suppressor, meiotic, and inositol-requiring genes. 2. Genetic Maps and Marker Distribution

All the genes in Table 2 have been assigned to linkage groups, but by no means all are mapped accurately within groups. Useful maps have been published by Fincham and Day (1971), Davis and de Serres (1970), and Radford (1972). Uncertainties of map location are shown by Radford (1972), who has also listed known loci with brief descriptions of phenotypes, including known enzyme phenotypes (Radford, 1976). The maps presented in Fig. 5 have been prepared specifically to show the genes that are referred to in this review and its Appendix. These maps are not complete. Numerous useful, well mapped markers are not included because they do not happen to have been employed in the studies reported here. For special reasons, the positions of a few rearrangement breakpoints are shown, mainly those th at mark linkage-group ends. Gene symbols are identified in the legend to Fig. 5. Neurospora genetic nomenclature follows Drosophila usage more closely than that of maize, yeast, Aspergillus, or bacteria (Barratt and Perkins, 1965; Barratt, 1969). Different genes having the same basic symbol (“mimic genes”) are distinguished by numbers (eg., arg-I, arg-2) as in Drosophila, rather than by capital letters as in bacteria. This usage predates the introduction of the bacterial system and is more suited to an organism having mating types designated A and a. (The only exception in Neurospora is a d 3 A adSB, retained because o f long-standing use.) Neurospora suppressors are also symbolized as in Drosophila, with su for the mutant suppressor, su+ for its wild-type allele. The three-letter gene symbols of bacterial genetics are used for some classes of genes such as amino-acid auxotrophs (Perkins and Barratt, 1973). No scale is shown in Fig. 5, because uncontrolled variability of recombination is so great in Neurospora. Linkage group I is estimated to be about 200 map units (centimorgans) in length. Interval lengths in published maps may be based on recombination values from crosses with either high or low crossing over, or they may represent pooled values, or values felt to be most representative. Whatever their basis, their predictive value for distances is limited to within perhaps one order of magnitude for any strains that are not highly homogeneous. The gene orders, of course, should be invariant.

a. Distribution of Genes and Rearrangements. The distribution of mapped loci and rearrangements among the seven linkage groups is shown in Table 3. Both the number of genes in each group and the involvements

DAVID D. PERKINS AND EDWARD G . BARRY

FIG.5. Genetic map of Neurospora crmsa showing only loci named in this review. Parentheses indicate that order is uncertain

relative to outside markers; where vertical lines do not intersect the map, order within parentheses is uncertain. Distances are only roughly to scale; interval lengths can vary as much as 10-fold in crosses of different parentage. Because of the large number of loci, linkage group IV and the left half of I are both expanded to about 1.5x the scale of the other groups. The second line is a continuation of linkage group I. The following genes are not shown, though they are referred to: Linkage group I : ser-3 is near cys-5 and cys-11; cyt-1 is between ser-3 and leu-3; sn is near centromere, probably left; rg is near his-2; het-5 is right of thi-I; tre and mig are between ud-9 and al-2; uvs-6 is near and left of al-2. Linkage group 11: pi is probably an allele of col-10; pcon is an allele of nuc-2; cpt is between a r g d and pe. Linkage group 111: uvs-4 is near ad-4; het-7 is right of trp-1. Linkage group IV: r i b 4 is right of T(S4342); to1 is near trp-4. Linkage group V: a1-3, trp-5, and Me& are near inl; erg-1 and erg-2 are in VR. Linkage group VI: het-9 is in VIR; lys-5 is allelic with asco. Linkage group VII: thr-1 is near met-7; het-10 is right of for; the centromere position is uncertain relative to qa and met-7. Symbols: A, a-mating-type, ace-acetate, acr-acriflavine resistant, ad-adenine, abalbino, am-amination deficient, urgarginine, aro-aromatic biosynthesis, asco-ascospore color, an-asparagine, at-attenuated, aur-aurescent, bal-balloon, bis-biscuit, chol-choline, col-colonial, cot-colonial-temperature-sensitive, cpt-carpet, cr-crisp, c u t - c u t , cyh-cyloheximide (actidione) resistant, cys-cysteine, cyt-cytochrome, deddelicate, dow-downy, erg-ergosterol, eth-ethionine resistant, $-fissure, fl-fluffy, for-formate, fr-frost, het-heterokaryon (vegetative) incompatibility, his-histidine, hom-homoserine, ilv-isoleucine valine, In-inversion breakpoint, inl-inositol, leu-leucine, ly 3-lysine, m a t m a t , mei-meiotic, met-methionine, mig-migration of trehalase, mo-morphological, mt-mating type, &-niacin, nit-nitrate nonutilizer, nt-niacin or tryptophan, nuc-nuclease, 0s-osmotic, pub-p-aminobenzoate, pan-pantothenate, pcon-phosphatase control, pdz-pyridoxine, pe-peach, per-perithecial color, phe-phenylalanine, pi-pile, pk-peak, preg-phosphatase regulation, ptphenylalanine or tyrosine, pyr-pyrimidine, qaquinate catabolism, R-Round spore, rg-ragged, rib-riboflavin, ro-ropy, satsatelliteless, sc-scumbo, ser-rine, sk-skin, sn-snowflake, so-soft, sor-sorbose resistance, sp-spray, spco-spreading colonial, suc-succinate, T-translocation breakpoint, tatufted aerial, thi-thiamine, thr-threonine, todtolerant, tre-trehalase, trp-tryptophan, tyr-tyrosine, un-unknown heat-sensitive, uvs-ultraviolet-sensitive, vel-velvet, wc-white collar, ws-white spore, ylo-yellow. For full symbols of the translocations and inversions, see the Appendix.

e m M

c)

2

o

0

m

3 c)

rn

$ 2: 0

52

+

c-L Q,

01

166

DAVID D. PERKINS AND EDWARD G. BARRY

TABLE 3 Comparison of Cytological Lengths of the Seven Neurosporu Chromosomes with the Numbers of Genes and Rearrangements Mapped in Each Linkage group Corresponding chromosome

N0.a

Mapped genes ( N = 426)* Number of rearrangements in which involved ( N = 3 3 6 ) ~ Genes plus rearrangements ( N = 762) Measured length of synaptonemal complex (total = 45.5 pm)d

I

v

111

IV

VI

I1

VII

1

2

(3)

(4)

(5)

6

7

29% 27%

16% 12%

9% 11%

16% 17%

8% 12%

10% 12%

12% 9%

28%

15%

10%

16%

10%

11%

11 %

22%

16%

15%

13%

12%

10%

10%

a See Section IV, C and Table 4 for basis of linkage group-chromosome assignments. Chromosomes were numbered in the order of cytological length (McClintock, 1945; Singleton, 1953). b Includes all genes that have been mapped to linkage groups and that are thought t,o be nonallelic. See Table 2 for the distribution of genes by phenotype category and linkage group. c Based on the 167 rearrangements summarized in Table 5 and described in the Appendix. Values are not based on number of breakpoints; e.g., inversions are counted only once, and an insertional translocation is counted once for donor chromosome and once for recipient. d Gillies (1972). Average for three nuclei reconstructed from thin sections.

in gross rearrangements are approximately proportional to the cytological length of the corresponding chromosome a t pachynema. (The slight excess in I might be expected because this linkage group contains the mating-type alleles and other markers favorable for detecting linkage.) There is no obvious concentration of any phenotypic category of mutant in a particular linkage group. Rearrangement breakpoints do not appear to be distributed randomly within individual linkage groups. Centromere regions and chromosome tips seem more likely to be involved, and there is a suggestion of excess breaks in IVR. The distribution of genes within linkage groups can be seen from the genetic maps. Identified loci are the most crowded in a portion of I that includes mating type and the centromere, and in the proximal half of IVR. There is a n unexplained lack of markers in the left arms of I11 and V. Cytological observations indicate that the centromere is not near the end of 111,and the genetic map would thus be expected to have two arms. Nothing is known about recombination in IIIL, but crossing over is known not to be suppressed in VL, where 35% recombination occurs between a centromere marker, Zys-1, and the terminal cytological marker

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sat, satelliteless (Barry and Perkins, 1969). There is thus no reason to suspect that the arm is genetically inert. VL bears the nucleolus organizer near its tip. There is no evidence of chromosome breakage from entanglement of a terminalized chiasma in the nucleolus. (In Neurospora, the nucleolus is usually detached from the chromosomes before anaphase I.) Rick (1971) summarized information on silent regions in the chromosomes of several organisms and noted that when the two arms of the nucleolus organizer chromosome are compared, mapped genes are preferentially more abundant in the arm opposite the nucleolus, in both tomato and maize. Neurospora apparently conforms to the same pattern. b. Clustering of Functionally Related Mutations. When nutritional markers originally became available in Neurospora, one of the first questions to be asked was whether functionally related genes would be linked or clustered. The answer was clearly no clustering, for one biosynthetic pathway after another (see Horowitz, 1965). However, there are several exceptions. Some of these appear to be single genes with multiple functions, others clearly involve separate genes. Where the products have been studied, enzyme aggregates or multifunctional enzymes are usually found. Histidine biosynthesis is specified by nine genes in the histidine operon of Salmonella. In Neurospora, the corresponding genes are scattered through four linkage groups. However, three of the enzyme activities are specified by a single region ( h i s s ) in Neurospora, and an enzyme or aggregate has been purified that possesses all three activities (see Ahmed et al., 1964; Ahmed, 1968; Catcheside, 1965; Catcheside and Angel, 1974; Webber, 1965; Minson and Creaser, 1969). Two distinct activities are both specified by the p y r - 3 locus (Davis and Woodward, 1962; Williams and Davis, 1968). Multiple enzymic functions are also specified by trp-3 (see Crawford, 1975) and trp-1 (DeMoss et al., 1967). The mutants mac and met-6 behave recombinationally as though they were a t different sites within a single gene locus (Murray, 1969). However, they differ in response when adenine as well as methionine is added, they complement one another (Murray, 1969), and they differ in the activity of two different enzymes related to folyl polyglutamate biosynthesis (Ritari et al., 1973). Other examples in Neurospora involve clustered genes that govern related functions. The aro cluster consists of five genes governing steps in the aromatic synthetic pathway (Gross and Fein, 1960; Giles et al., 1967; Rines et al., 1969). The aro genes are transcribed together and their products form an aggregate. The qa (quinate) cluster consists of one regulatory and three structural genes governing the aromatic catabolic pathway (Chaleff, 1974a,b; Case and Giles, 1975). The products are not aggregated.

168

DAVID D. PERKINS AND EDWARD G. BARRY

met-7 and met-9 appear to be adjacent genes (Murray, 1970). They specify different enzymes (Kerr and Flavin, 1970), yet mutant sites in the two loci are so close that they undergo coconversion. The Neurospora maps also reveal several other apparently adjacent pairs of genes with functionally related phenotypes-cys-1, cys-2 (Murray, 1965) ; thr-2, -3 (Perkins et al., 1962; Emerson, 1950) ; ilv-1, -2 (Kiritani, 1962) ; arg-10, -11 (Newmeyer, 1957) ; cys-5, -11 (Murray, 1965, 1968b) ; erg-1, -2 (Grindle, 1974) ; tre, mig (Sussman et al., 1971) ; pcon, p e g (Littlewood et al., 1975) ; and pyr-3, arg-2 (Reissig, 1960, 1963). Other related gene pairs are close but not contiguous-ad-3A, ad-3B (de Serres, 1969) and d-1, al-2 (Hungate, 1945; Subden and Threlkeld, 1970; Perkins, 1971b). It is not known whether there is any functional relation between the two distinct roles of the mating-type alleles-in sexual crossing and in vegetative incompatibility (Section 11). A functional relation has been suggested between mating type and the closely linked locus un-3, which is known to affect membranes (Kappy and Meteenberg, 1967). For a general review of genetic clustering of biochemically related functions in fungi, see Fink (1971). c. Estimates of Total Map Length. Because crossover frequencies are extremely variable in Neurospora (Section IV, B, 3), any estimate of overall genetic map length will be unreliable within wide limits. Nevertheless, rough estimates can be based either on cytological chiasma counts or on genetic recombination frequencies. An average of a t least two chiasmata are observed per bivalent (Singleton, 1953; E. G. Barry, unpublished observations), indicating that the total map length is a t least 700 units. Maps compiled from genetic data by Fincham and Day (1971) total about 750 units. Several linkage groups have since been extended by means of newly discovered genes or rearrangement breakpoints, bringing the total length to perhaps 900 units. This is in reasonable agreement with an independent estimate based on exchange frequencies in a long segment of I R which was multiply marked so that all exchanges could be detected (Perkins, 1962a). On this basis, the longest linkage group is estimated to be 200 units, and it (chromosome 1) represents 22-25% of the total genome, based on cytological measurement of synaptonemal complexes and pachytene chromosomes (see Gillies, 1972). Thus the total length in Neurospora is probably a t least 1000 genetic map units. How this value compares with genetic maps in other organisms can be seen in Table 1. 3. Crossing-over Variability and Its Basis Recombination between the same two markers may vary 2-fold, 4-fold, or even 10-fold in crosses of different parentage (see, for example, Stadler,

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1956; Nakamura, 1966; Landner, 1971 ; Catcheside and Corcoran, 1973). Yet recombination values are reproducible within close limits in repeated crosses between the same two strains. It is not known whether an increase in crossing over in one region is accompanied by a compensating decrease elsewhere in the genome, except that chiasma interference is positive (see, for example, Perkins, 1962a). Structural heterozygosity and genic regulation have both been considered as possible causes of the variability of recombination. Abnormal crossing over and changed linkage relations have several times led to the recognition that chromosome rearrangements were present in heterozygous condition in particular crosses (for example, by Houlahan et al., 1949; de Serres, 1971). It may also be that paracentric inversions are present in some stocks and have gone undetected. (The problem of “silent” paracentrics will be discussed in Section V, C, 3.) However, most cases of crossing-over variability appear to be due to genic control. Recombination (rec) genes have been shown to regulate both intragenic and intergenic recombination in specific regions located a t a distance from the controlling gene (Jessup and Catcheside, 1965). Three rec systems have been described in studies by D. G. Catcheside and his associates B. R. Smith, K. K. Jha, D. E. A. Catcheside, D. R. Smyth, P. L. Thomas, T. Angel, B. Austin, and D. Corcoran. The experimental results and interpretations have been reviewed by D. G. Catcheside (1974) ; see also Catcheside and Corcoran (1973) and D. E. A. Catcheside (1974). Differences a t all three rec loci are present among commonly used laboratory wild-type stocks (Catcheside, 1975). More than one region may be under control by the same rec gene. High recombination is recessive to low for all the rec genes studied. One example is known of a different class of element, cog (recognition), which is closely linked to his-3. The dominant cog+ allele must be present in order for high recombination to occur in or near his-3 when the appropriate rec gene is homozygous recessive. The existence is inferred of a third class of genes with local effects on recombination. Other approaches have also been used in attempts t o clarify the genetic basis of crossing-over variability (Frost, 1961 ; Lavigne and Frost, 1964; Towe, 1958; Stadler and Towe, 1962; Nakamura, 1966; Landner, 1971, 1974; de Serres, 1971). These include selection and inbreeding. It was shown by Towe (1958), Stadler and Towe (1962), and Cameron et al. (1966) that inbreeding tends to increase crossing over. Other experiments showed that reciprocal recombination between the closely linked genes ad-3A and ad-3B was greater when markers originated in different genetic backgrounds than when they arose in the same background (de Serres, 1971). These results must now be evaluated in light of the findings on

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DAVID D. PERKINS AND EDWARD G . BARRY

regulation of recombination. If high recombination requires that recessive rec genes be homozygous, inbreeding may be expected sometimes but not always to increase recombination, depending on what regions are being monitored, what rec alleles are initially present, and which of them are retained during inbreeding.

4. Mapping Methodology Genetic mapping consists of three phases-detection of linkage, determination of gene order, and estimation of interval length. In choosing methods to solve a practical mapping problem a t any of these stages, it is important not to confuse the objectives of mapping with investigations into the mechanism of recombination. a. Linkage Detection. I n terms of labor, random isolates are more efficient than tetrads for detecting linkage (Perkins, 1953), and unordered tetrads are more efficient than ordered tetrads. Effort can be minimized by using multiply marked tester strains such as alcoy (Perkins et al., 1969), mu2ticent (Perkins, 1972c) and related follow-up testers ( Perkins, 197213, 1973). Markers in these strains have been selected for ease and reliability of scoring. The multicent tester contains proximal markers for all seven linkage groups, in normal sequence: mt, bal, acr-2, pdx, at, ylo-1, wc. alcoy contains three independent reciprocal translocations, each associated with a marker: aZ-1 ( I ; I I ) , cot-1 (IV;V), ylo-1 (1II;VI). Other less complete multiple-group testers are also available from FGSC. If tetrad data are available, gene-to-gene linkage is indicated by an excess of Parental Ditype over Nonparental Ditype tetrads (Perkins, 1953). b. Determining Gene Order. The method of preference is to use random isolates from 3-point crosses, where gene order is established not from absolute recombination frequencies, but from the relative frequencies of single- and double-crossover classes. Chiasma interference is positive in Neurosporu (see, e.g., Perkins, 1962a), and this increases the efficiency of determining gene order by reducing double crossovers relative to singles. Because crossing over is so highly variable, map sequences are likely to be incorrect if gene order is determined solely by combining the recombination values of single intervals from different crosses. Many errors have resulted from using 2-point data in this way. Maps with completely reliable sequences can be built up by basing gene order on a series of overlapping 3-point crosses that have intervals in common. In special situations, gene order can be resolved most easily by testing for duplication-coverage (Perkins et ul., 1969). That is, a right-left test is made, using the breakpoint of a chromosome rearrangement as a reference point. Duplication-generating rearrangements such as insertional

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translocations are employed. If rearrangements with appropriate breakpoints are available, determining the order of closely linked genes by duplication coverage can be far quicker and easier than by conventional 3-point tests. Centromere mapping is a special case. Location of the centromere is already known with varying degrees of certainty for each of the seven linkage groups. Historically, the centromere positions were based on critical crossovers in ordered tetrads. Only rarely now does the need arise to determine a gene-centromere sequence, and that occurs when a gene is found to map between the most proximal loci that are reliably positioned in opposite arms. When this occurs, the gene-centromere order can be established in any of three ways-by ordered tetrads, by unordered tetrads, or by random ascospores where an appropriate chromosome rearrangement makes mapping possible by duplication coverage. The use of ordered tetrads has been familiar since Lindegren’s work in the 1930s. Unordered tetrads are the usual method of centromere mapping in organisms such as yeast and Chlamydomonas, and they might well be used to advantage for this purpose in Neurospora, where the presence in a cross of gene markers at two or more known centromeres would enable ejected groups of eight ascospores to be treated as though they had been isolated in order. See Mortimer and Hawthorne (1975) for this and other aspects of mapping with unordered tetrads. The ability to determine centromere location by duplication-coverage of nearby genes depends upon availability of a duplication-generating rearrangement with one breakpoint near the centromere in question. Since centromere regions seem prone to be involved in rearrangements, a number of such strains are available (Fig. 19). (Combinations of overlapping reciprocal translocations can also be used, where one breakpoint adjoins the centromere.) The method is best illustrated in linkage group I, where the centromere has been shown to lie between the left-arm duplication 39311 and the right-arm duplication ARl73, hence between mei-3 (covered by 39311) which must be in the left arm, and wn-2 (covered by ARl7S) which must be in the right arm. This method is far less laborious than isolating and scoring the large number of ordered asci that would be required for conventional mapping. i. Appropriate uses of tetrad analysis. Tetrads have unquestionably played an important role in research on the mechanism of recombination, providing basic information on gene conversion, crossing over, and interference. Fungal tetrads have contributed in an essential way to molecular models of eukaryote recombination. Ascus analysis has also been very useful for distinguishing whether a variant was cytoplasmic or chromosomal, for constructing double-mutant stocks involving mimic genes or suppressors, and for identifying and characterizing chromosome rearrangements.

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DAVID D. PERKINS AND EDWARD G. BARRY

The most familiar use of ordered tetrads has been to determine gene-centromere distances from second-division segregation frequencies. This was a novel and fascinating feature in the early years of Neurospora genetics, enabling a direct determination of what could be arrived a t only indirectly in higher organisms. Consequently, ordered-ascus analysis seems to have become associated with Neurospora as though it were a fundamental cornerstone of all genetic analysis with the organism. (This is the impression given by many textbooks.) Ordered asci have been a mixed blessing, however, because they have often been used unnecessarily where the needed information could have been obtained by a less laborious method, as has been shown for routine mapping. Tetrad analysis is certainly not the best or most economical method for gene-gene mapping, and gene-centromere mapping that would require tetrads is rarely necessary any longer.

ii. Estimation of interval length. Recombination frequencies are highly variable in different genetic backgrounds in Neurospora (Section IV, B, 3), and the marker genes used for mapping are of mixed ancestry. Thus there is usually little point to refining the statistical analyses of mapping data, or to applying corrections for undetected multiples, or to enlarging the data beyond a modest level, because the uncontrolled background variability of recombination is so large. The idea of a genetic map of Neurospora with standard interval lengths is illusory. For organisms or strains in which markers are all in the same background, so that recombination frequencies are more nearly constant, cumulative map lengths would ideally be built up using recombination frequencies in marked intervals short enough to preclude the occurrence of double crossovers (probably 10 or 15 units). I n an organism like Neurospora, this would be most economically and reliably accomplished using random isolates in sufficient numbers to minimize statistical error (Fig. 8 of Barratt et al., 1954, can be adapted for this purpose). The eight spores of an ascus yield far less information than eight ascospores isolated a t random (Mather and Beale, 1942). If tetrad data are already available for use, the best estimate of the map length of an interval between two genes is given by the formula map distance 50 (T 6 N P D ) / ( P D N P D T),where PD, N P D , and T are frequencies of Parental Ditype, Nonparental Ditype, and Tetratype asci (Perkins, 1949). This corrects for undetected 2-strand and 3-strand double exchanges on the basis of 4-strand doubles, which are manifested as Nonparental Ditypes. The validity of this formula across long distances has been shown in experiments where closely spaced intermediate markers enabled all multiple exchanges to be detected between the distant markers to which it was applied (Perkins, 1962a).

+

+

-

+

C. LINKAGE GROUP-CHROMOSOME CORREL,ATIONS Chromosomal assignment of particular linkage groups was first attempted by Singleton (1948) and by St. Lawrence (1953), a t a time when the genetic maps were still rudimentary and the number of chromosome structural variants was small. Now, 28 years later, assignment of the seven linkage groups to specific chromosomes is for all practical pur-

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Neurospora

poses complete. Only a beginning has been made, however, toward locating genes within individual chromosomes. The present status of linkage group-chromosome correlations is shown in Table 4. Linkage groups are designated with Roman numerals, and chromosomes with Arabic numbers. General Methodology. The assignment of linkage groups to chromosomes has depended on chromosome aberrations, which can be recognized and localized both genetically and cytologically. An aberration is mapped genetically t o a linkage group (or groups) and the aberrant chromosome TABLE 4 Relation of Genetically Defined Linkage Groups to Cytologically Defined Chromosomes in Neurospora crassa ChromoLinkage some assogroupo ciationb

Aberrationc

I

1

R.55, 4637 4637 4637 4637, S1007, 17084, H42.50 AR190, S1325, 36703 39311

I1

6

R55, 4637 4637 4637 4637 NM149, 39311

I11 IV V

(3) (4) 2

VI

(5) 7

VI I

45502, AR211, S4342 C-1670, R2355, 36703, 46802 NM149, AR190, S1325, 36703, sat 46802 45502, 46802 S1229 S1007, 17084 5936

Referenced St. Lawrence (1953) McClintock (1955) Singleton (1948) Barry (1967) Barry and Perkins (1969) Barry (1 972) ; Perkins (1972a) St. Lawrence (1953) McClintock (1955) Singleton (1948) Barry (1967) E. G. Barry (unpublished; 1972)

E. G. Barry (unpublished) Phillips (1967) Barry and Perkins (1969) E. G. Barry (unpublished) E. G. Barry (unpublished) Barry (1960a) Barry (1967) E. G. Barry (unpublished)

Linkage group numbers (Roman) were assigned in chronological order of discovery (see Barratt et al., 1954). Chromosome numbers (Arabic) were assigned in descending order of length (McClintock, 1945). Parentheses indicate that the association is probably correct but is subject to some uncertainty. Or heteromorphic satellite (sat). d References are omitted to studies that do not support the assignments made in this table; all contrary findings are discussed in the text, however.

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DAVID D. PERKINS AND EDWARD G . BARRY

(or chromosomes) is identified cytologically. Two rearrangements are examined cytologically which have one genetic linkage group in common and a second linkage group that is not in common. The chromosome altered in both strains must be the counterpart of the shared linkage group. Examples of this type of analysis will be given below. Aberrations are also the best means of determining the location of specific genes within a chromosome. This is because rearrangement break points can be placed precisely on linkage maps by genetic analysis (see Section V, A, 3 ) . However, attempts to use rearrangements for intrachromosomal correlations have not been very productive so far. There is often uncertainty in recognizing chromomere patterns of the aberrant chromosomes. Sometimes there may be mispairing in the region of the breakpoints or even “slippage” where the paired regions are nonhomologous. Consequently, only tentative intrachromosomal correlations have been possible using the usual structural rearrangements. One unambiguous intrachromosomal localization has been made, however, and this employed a heteromorphic chromosome deficient in the small satellite that lies distal to the nucleolus organizer in chromosome 2. The satellite is clearly located a t the left end of linkage group V (Barry and Perkins, 1969). Steps in Establishing the Linkage Group-Chromosome Correlations. Singleton

(1948) made a brief attempt to associate linkage groups and chromosomes using

four chromosome aberrations that had been only partially mapped genetically. He very tentatively suggested that chromosome 2, the nucleolus-organizer chromosome, is linkage group I. I n view of his limited observations, it is not surprising that his suggested correlation proved to be wrong. The first detailed effort to correlate linkage groups with chromosomes was made by St. Lawrence (1953). She concluded from cytogenetic studies that chromosome 1 is linkage group 11, 2 is IV, and 6 is I. (These are not quite right, as will be seen.) St. Lawrence’s correlations were based on examination of a complex aberration, R66, which had major rearrangements in three chromosomes (1, 2, 6) for which she located breakpoints in three linkage groups (I, 11, IV). Her determination that I was 6 was based on the argument that the R66 breakpoints found in chromosome 6 and linkage group I were both far out in their respective arms, while the breakpoints of chromosomes 1 and 2 and linkage groups I1 and IV were all near their respective centromeres. Observations on two other translocations supported and extended this conclusion. Her correlation of linkage group IV with chromosome 2 was based on an analysis of T(ZV;VZ)46602. This translocation is linked in groups IV and VI (Houlahan et al., 1949). B. McClintock (personal communication to St. Lawrence) identified an exchange between chromosome 2 and another, smaller chromosome, possibly 4. Since 46602 and R66 both involved only chromosome 2 and linkage group IV in common (R66 was shown not to be linked to ad-f in VI), St. Lawrence concluded that linkage group IV equals chromosome 2. Study of T(Z;IZ)4637 completed St. Lawrence’s analysis. This translocation has breakpoints in IR (Houlahan et a!., 1949) and IIR (St. Lawrence, 1953; Hagerty, 1952). B. McClintock (personal communication to St. Lawrence) determined that

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the translocation was between chromosomes 1 and 6. Since translocations R66 and 4637 both showed involvement of chromosomes 1 and 6 and linkage groups I and 11, and since chromosome 6 by other evidence seemed to be linkage group I, St. Lawrence concluded that chromosome 1 should be linkage group 11. Barry (1962, 19671, however, proposed that the St. Lawrence correlations of I = 6 and I1 = 1 should be reversed with I = 1 and I1 = 6. This amendment was based on studies of translocations T(I;VII)17084 t h i l and T(I;VII)S1007 which

both involve linkage groups I and VII. Neither translocation has an abnormality of chromosome 6, whereas chromosome 1 is involved in both. Also, inversion strain In(IL+IR)H4260 involves both group I (Newmeyer and Taylor, 1967) and chromosome 1. Further confirmation that chromosome 1 is linkage group I was found in other studies (Barry and Perkins, 1969; Barry, 1973). Barry (1960a) proposed that VII = 7. A complex chromosome rearrangement in 51229 is linked in I, 11, IV, and VII, and it appeared to involve chromosomes 1, 2, 6, and 7. The involvement of a chromosome (7) and a linkage group (VII), in addition to those detected by St. Lawrence in R66, indicated that VII = 7. Supporting observations were reported by Barry (1967) on T(I;VII)S1007 and T(I;VII)17084 thi-1. Phillips (1967) examined four translocations that showed interchanges involving chromosome 2. The translocations (36‘703, R2366, 46802, and C-16‘70) all involved linkage group V, but only one of them involved IV. He therefore proposed that chromosome 2 corresponds to V rather than to IV. This was confirmed (Barry and Perkins, 1969) by using other translocations and mapping the satellite of chromosome 2 to linkage group V. The earlier errors can now be explained as follows. St. Lawrence did not test R66 for V linkage, presumably because she had already located a linkage group involvement for each of the chromosomes known to be aberrant. McClintock’s determination that translocation 46602 involved chromosome 2 was probably in error because of stock problems. The aberration is separable from a closely linked pvr-3 mutant which was present in the stock with the original aberration, but this may not have been realized a t first, and derived stocks with the mutant may have been thought to contain the aberration also. McClintock made several observations of supposed 46602 stocks. In 1945 she reported that the translocation “involved a very unequal exchange of segments of two nonhomologous chromosomes. The breaks appear to have occurred close to the end of the long arm of chromosome 1 and close to the centromere in the long arm of one of the chromosomes with a subterminal centromere, possibly 4.” In 1952, McClintock’s observations were as described by St. Lawrence (1953). In 1955 she again examined stocks derived from the original 46602, but found no gross structural rearrangements. It now seems probable that different aberration stocks, or suspected aberrations, were examined in the three studies. (It is not unusual for more than one rearrangement to be found in the same strain following mutagenesis, or for a second aberration to occur spontaneously. Examples of both are documented in the Appendix.) Singleton (1948) also examined a 46602 strain and interpreted i t as a reciprocal translocation between chromosomes 1 and 6. E. G. Barry (unpublished) has examined available strains of 46602 known genetically to involve IV and VI, and believes them to contain a translocation between chromosomes 4 and 5. Barry’s (1960a) initial analysis of 51229, which appeared to support the St. Lawrence correlation of 2 with IV, was rechecked, and the conclusion that 2 = IV was withdrawn (Barry and Perkins, 1969).

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DAVID D. PERKINS AND EDWARD G . BARRY

This completes the chromosome assignments for four linkage groupsI, 11,V and VII. These are considered to be firmly established. The remaining three linkage groups, 111, IV and VI, are clearly associated with the three middle-sized chromosomes, 3,4, and 5. The tentative assignments shown in Table 4 are probably correct, although an error may possibly have occurred because the three chromosomes are similar in size and appearance, and their chromomere patterns tend to be inconsistent. It is doubtful if such an error would ever be of any practical consequence, because the experimental usefulness of the three is severely limited because of their similarity. It is proposed to designate the chromosome numbers permanently as shown in Table 4. Number 3 is the chromosome bearing linkage group 111, 4 carries IV, and 5 carries VI. If further refinement of techniques makes morphological distinctions practical, it is proposed that these numbers be retained, even if the chromosome lengths prove to be out of order. In order for all linkage groups to correspond exactly with chromosome numbers, three changes would now be required (11, V, and VII). However, the present linkage group numbers are so firmly established in general use that we do not propose to make any changes. To do so would only introduce confusion. Bask of the Tentative IIZ, IV, V I Assignments. From our cytological analysis of T(ZV;VI)46602, one breakpoint is probably near centromere in the long arm of chromosome 4, and the other is probably near the tip of 5. The resulting exchange has produced one very long chromosome (most of 5 plus three-fourths of 41, which is about the length of chromosome 1, and one very short chromosome (the centromere and short arm of 4, and the tip of 5 ) . In the genetic map, the positions of &602 breakpoints are close to centromere in IVR and far distal in VIR. I t can be concluded that IV = 4 and VI = 5 from the position of genetic and cytological breakpoints. This conclusion is based on the assumption that cytologically and genetically determined distances correspond. If IV = 4 and VI = 5, then chromosome 3 and linkage group I11 are associated by default, being the last chromosome and linkage group not otherwise designated. However, Griffiths, et al. (1974) tentatively proposed a correlation of chromosome 3 with linkage group IV. Their evidence is based on a cytogenetic study of T ( I ~ Z V ) Y l l B M l a6d d , which involves I and IV. They state that in one nucleus one of the chromosomes involved corresponds cytologically to McClintock’s description of 3.

I n no other fungus or eukaryotic microorganism has there been a well established correlation between a linkage group and a specific cytologically recognizable chromosome. However, Mu’Azu (1973) has tentatively suggested four correlations of chromosomes and linkage groups of Sordaria brevicollis, based on cytogenetic analyses of six translocations.

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D. CYTOPLASMIC GENES Mutant strains of Neurospora are known whose differences from wild type are transmitted independently of the nuclear chromosomes, as first shown by Mitchell and Mitchell (1952) and Mitchell et al. (1953). The mutants show slow or abnormal growth, abnormal cytochromes, and respiratory defects. No drug-resistant cytoplasmic mutants have been reported. With one exception (Srb, 1963) transmission of the cytoplasmic determinant is strictly maternal, i.e., through the protoperithecial parent. The cytoplasmic factor carried by the paternal (fertilizing) parent is excluded or eliminated. Abnormal cytoplasm has been transmitted by microinjection, using techniques developed by Wilson (1961). The cellular locus of the cytoplasmic genes is probably, but not certainly, the mitochondria (Diakumakos et al., 1965). Measurements of individual molecules by electron microscopy (Agsteribbe et al., 1972) or by reassociation kinetics (Wood and Luck, 1969) show that the mitochondria1 DNA of Neurospora is long enough to contain a t least 50 genes. No evidence has been obtained of recombination between different cytoplasmic determinants. For a review and critique of cytoplasmic inheritance in Neurospora and other fungi, see Sager (1972, Chapter 5). V. Chromosome Rearrangements

Structural rearrangements occur frequently in Neurospora, are readily recognized, and can be used quite effectively. This is fortunate because the possibility of cytogenetic manipulation by means of numerical variants is extremely limited in Neurospora, as disomics and diploids are highly unstable and only one useful heteromorphic chromosome variant is known. Most cytogenetic experiments must depend therefore on chromosome aberrations. Each of the 167 rearrangements that have been characterized in Neurospora crassa is described in the Appendix. A sizable number of these rearrangements can be used to produce nontandem duplications-aneuploids of recombinational origin equivalent to partial diploids. These are valuable research tools, resembling the tertiary or telo-trisomics of plant cytogenetics in some of their applications. Appendix Table 1 gives a complete list of the reciprocal translocations, arranged according to linkage groups. All possible combinations of the linkage groups are represented among the 123 interchanges. Two mutual

178

DAVID D. PERKINS AND EDWARD

G.

BARRY

insertions are also listed. Appendix Table 2 lists the identified duplicationproducing rearrangements, 42 in all. These fall into three categoriesinsertions, quasiterminal rearrangements, and those still uncertain whether interstitial or terminal. The two Appendix tables are intended to serve as indexes, in which a rearrangement having desired specifications can be identified by isolation number. Information on it can then be found in the main part of the Appendix, where the aberrations are all arranged in a single sequence according to isolation numbers, without regard to aberration type. The main Appendix entry summarizes information on each rearrangement, describing its type, map relations, phenotype, fertility, genetic behavior, cytology, and origin. Rearrangements discovered and analyzed by other workers are included. Secondarily derived rearrangements (such as the duplications generated by meiotic recombination from each of the known duplication-generating aberrations) are not listed as separate entries in the Appendix or the Appendix tables. A. METHODSOF IDENTIFYING AND MAPPINGREARRANGEMENTS 1 . Detection

Almost all the Neurospora rearrangements were detected initially by noting that defective, white ascospores were produced in crosses heterozygous for the rearrangement, a situation exactly comparable to pollen abortion in flowering plants (Belling, 1914). Pollen abortion that originates from heterozygous rearrangements is termed semi-sterility or partialsterility in plant cytogenetics. These terms are best avoided in Neurospora because they would be confused with sterility of other types, such as barrenness of perithecia. Only a handful of the known Neurospora rearrangements were discovered because of altered linkage (e.g., T (I;VZI) 17084 thi-1 ) , and only one translocation was first recognized cytologically, by abnormal meiotic pairing ( T (V + V I I ) E B 4 ) . Standard methods for identifying and analyzing rearrangements have been described by Perkins (1974). The procedure will be summarized here. About 90 or 95% of ascospores from structurally homozygous crosses are viable and develop normal black pigment. I n contrast, heterozygous reciprocal translocations usually produce 50% defective, inviable spores, due to deficiencies, while insertional translocations and other aberration types that generate viable duplications typically produce 25% defective ascospores. The deficiency ascospores remain unpigmented or pale ; for simplicity they will be called white. (Point mutants that result in white ascospores are rare in Neurospora, compared to rearrangements.) I n

THE CYTOGENETICS OF

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179

searching for new rearrangements, each strain to be tested is crossed to a normal-sequence tester strain on agar medium in a small (12 x 75 mm) tube. Ten days later, when ascospores have matured and been shot from the perithecia, the glass wall of each tube is examined, and the proportion of black and white spores is estimated. Strains producing less than 90% black spores are saved for further testing. Putative rearrangement strains are then subjected to a second step of analysis that employs unordered asci, which can be obtained in large numbers by the method of Strickland (1960). Each strain t o be tested is crossed on a petri dish to the same normal-sequence tester as before. After 10 days, unordered asci are collected as spontaneously shot groups of eight ascospores on an agar slab under the inverted cross plate. The collecting slab is exposed for a period ranging from a few seconds t o several minutes, depending on the rate of shooting, and is then scanned, classifying each well separated group according to number of black and white spores. The major classes are 8:0, 6:2, 4:4, 2:6, 0:8 (Black: White). 2. Diagnosis

Frequencies of the unordered ascus classes in crosses of Normal X Rearrangement immediately enable us to classify most rearrangements into two main categories, according to whether they do or do not generate viable duplication progeny. The frequencies also provide information on the positions of break points relative to centromeres. Frequencies of the unordered ascus types from structurally homozygous crosses are shown in Fig. 6. I n contrast, Fig. 7 shows the frequencies for heterozygous translocations. The top row is typical of translocations that do not produce viable duplications. Two examples are given, with breakpoints far from centromere (A), and close to centromere ( B ) . Examples in the bottom row are typical of duplication-producing translocations whose breakpoints are far from centromere (C) and close to centromere (D). The ascus frequencies from translocations that do not produce viable duplications are typically symmetrical around 4B:4W, with 8:O = 0:8. The ascus frequencies from duplication-producing translocations are usually symmetrical around 6B:2W, with 8:O = 4:4. The occurrence of 6B :2W asci in significant numbers usually indicates that viable duplications are being formed. The meiotic basis for these distributions will be described later for each specific type of rearrangement. Frequencies of unordered tetrad types which correspond to the histograms in Fig. 7 are given as part of the description of each aberration in the main part of the Appendix.

180

DAVID D. P E R K I N S AND EDWARD G . BARRY

l

W

I Tll;Vl36703 X Tll;V136703 IN = 1351

Normal lfIPl Normtl (fI’I

IN = 1391

75 -

75 I

_

5

K :

Di

8 -

8

5 50-

50-

E -

c

%4! 8

8 :

-

25 -

O

1

f 25 -

L

~

8:O

6:2

4:4

2:6

A5mr class (8lack:Whitel

0:8

0-

II 62

4:4

2:6

0:8

A r u r class 18lack:Whitel

Fro. 6. Results of structurally homozygous crosses, when the parents are both wild types (left), and when they both contain the same reciprocal translocation (right). In this and succeeding figures, histograms show the frequencies of unordered asci having various members of black (viable) and white (inviable) ascospores. There are five major classes, ranging from all black (leftmost histogram) through 6B:2W, 4B:4W, 2B:6W, to all white (rightmost). N is the observed number of asci. Rare asci with odd numbers of defective spores (5:3, 7:1, etc.) are not shown. Reproduced from Perkins (1974).

The rearrangements that do not produce viable duplications are predominantly reciprocal translocations. The rearrangements that are capable of making viable duplications are predominantly insertional and quasiterminal translocations. Other tests in addition to the ascus patterns are required in order to distinguish one type of duplication-producing rearrangement from another. Advantages and Limitations oj Unordered Tetrads jar Diagnosing Rearrangements. The examples given in Fig. 7 conform well to theoretical expectations. The arrays of ascus types are distributed symmetrically around the 4B:4W or the 6B:2W class. From such a result it can be confidently inferred that a strain is a rearrangement, and the type of rearrangement can be predicted with reasonable confidence. Because the contents of unripe asci are usually not ejected, the problem of distinguishing true 0:s asci from unripe asci of other types is avoided when the analysis is based on unordered asci that have been shot out of the perithecium. If a similar analysis is made by opcning perithecia, unripe as well as ripe asci are seen and may not readily be distinguished from one another, leading to an excess of asci in the OB :8W class.

THE CYTOGENETICS OF

Neurospora 70

67 %

IBI L

Tlf: Vl36703 X Normal IN=2451

a

181

a >

.

50-

TII: IVlNM137

X

Normal IN = 116)

E

D

i

I

r ’ 25.

7

-I 8

6

2:6

-

-

9%

08

A r u r class 1Biack:Whitel

,-

70 1

64% ICI

-c

-

a >

-

{ 50-

TI1 -I I 114540 X Normal IN = 941

1 T(l-11)39311

-

g

50

Normal IN = 3921

D

7 c

25-

m 8 0

8:0

6:2 4:4 26 Axus class l8lack:Whitel

I

0:8

Arcus claw (8lack:Whitel

FIG.7. Frequencies of unordered ascus types in crosses of Reciprocnl Translocation x Normal (A and B), and Insertioncil Translocation x Normal (C and D).

The rearrangements on the left have breakpoints far from centromere, while those on the right, have both breakpoints close to centromerc. Note that with reciprocal translocations, which produce no viable duplications, the frequencies are symmetrical around the 4B:4W class and effectively no 6B:2W asci are seen. With insertional translocations, which do produce viable duplications, symmetry is around the 6B:2W class, and effectively no OB:8W asci are seen. Reproduced from Perkins (1974). I n some situations, errors of diagnosis may occur when unordered tetrads are used. Nonblack ascospores may result from mutant ascospore-color genes rather than deficiencies. Conversely, lethal deficiencies from some rearrangements do not prevent the ascospores that contain them from becoming black. Asci of the 0:8 type mag be underrepresented because they disintegrate. Most of these exceptions are rarely encountered. The most common departure from ideal expectations occurs when one of the inviable duplication-deficiency classes from a nonterminal reciprocal translocation becomes pigmented. The resulting ascus array, and the patterns of black and white ascospores in individual asci, misleadingly resemble those of a n insertional or quasiterminal translocation. Fifteen of the 123 identified nonterminal reciprocal translocations are clearly of this typc (see T ( I I I ; V ) A R l 7 7 as an example). Unordered tetrads are so informative in analyzing aberrations that ordered tetrads are not required even to determine centromere positions. A few special applications

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where ordered asci are necessary or useful have been listed by Perkins (1974, p. 485). Anomalies that can lead to misdiagnosis are discussed and examples given by Perkins (1974, pp. 474-477). It is clear that the preliminary diagnosis based on ascus t.ypes should be confirmed by other methods.

3. Genetic Verification and Mapping

Following such a preliminary diagnosis based on abortion patterns in the asci, each rearrangement is then verified and analyzed genetically, using appropriately marked tester strains. First the aberration breakpoints are mapped to linkage groups. For this purpose a normal-sequence tester such as multicent is preferable to alcoy, which itself contains translocations (Section IV, B, 4, a ) . In mapping a rearrangement, segregants are scored for Rearrangement us. Normal by the incidence of white spores in test crosses. The breakpoint of a translocation is treated as though it were a mutant allele with the phenotype “50% black spores in test cross.” The “allelic” normal sequence has the contrasting phenotype “90% black spores in test cross.” If few or no ascospores are produced, the test cross is classed as barren. Usually the necks of barren perithecia are rudimentary or absent. It is characteristic of most or all duplications in Neurospora that crosses involving them are barren. In this way, the rearrangement is confirmed, linkage groups are identified, breakpoints are located, and if duplications are produced they are verified. For details of the identification and verification procedures, see Perkins (1974).

4. Cytological Verification Genetic analysis has usually been complemented by cytological examination only when a rearrangement is of special interest. Each type of chromosome rearrangement is recognized cytologically by its heterozygous meiotic pairing with standard-sequence chromosomes, or by the production of bridges and acentric fragments. Identification of Neurospora aberrations is very similar to the meiotic analyses developed in maize, with allowances for the smaller chromosome size. Pachynema is the most favorable stage for diagnosing Neurospora aberrations. 6. Simulation of Synthetic LethaE Genes b y Rearrangement Breakpoints The breakpoints in simple (nonterminal) reciprocal translocations and inversions are in fact formally equivalent to synthetic lethal genes (Dobzhansky, 1946) in normal sequence. Most Neurospora rearrangements clearly cannot be attributed to synthetic lethals. Either they are known to generate viable duplications, or they have been confirmed cytologically, or markers show new linkage relations in homoaygous aberration sequence. Where none of these tests has been made, there remains a remote possibility that a pair of synthetic lethal genes could be responsible for the properties attributed to a rearrangement. It might be especially difficult to dia-

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tinguish short mutual insertions from synthetic-lethal point-mutants. However, this is no problem for the two Neurospora aberrations believed to be mutual insertional translocations-T(I V)S1326 and T ( I ZV)Y112M16 ad-3A. Genetic and cytological evidence shows that they are both chromosome rearrangements.

+

+

6. The Need for Quantitative Data on Induced Rearrangements

A committee of the Environmental Mutagenesis Society has listed quantitative studies of chromosome aberrations in microbial eukaryotes among major recommendations for future research : “Cytogenetic tests are now feasible only with the higher eukaryotes. It would be of great significance for determining comparative rates for point versus chromosomal mutation to possess a system capable of detecting chromosomal aberrations in a microbial eukaryote” (Drake, 1975). Among eukaryotes with small genomes, Neurospora seems the most likely practical source for both types of information. Point mutations can already be measured and characterized very precisely (see, for example, de Serres and Malling, 1971; Malling and de Serres, 1973). If Neurospora is to be considered as a test organism for chromosome aberrations, methods must now be developed for obtaining reliable quantitative measures of new rearrangements. Availability of such methods would also enable rearrangement frequencies to be compared in genotypes suspected of affecting chromosome stability and DNA repair. The rationale behind the quoted recommendation is as follows: When organisms in a phylogenetic series are examined, their sensitivity to radiation-induced forward mutation per rad per locus is directly proportional to DNA content per haploid genome (Abrahamson et al., 1973). Likewise cellular radiosensitivity is proportional to chromosome volume and nucleotide content (see Sparrow et al., 1967; Underbrink et al., 1968). This correspondence between sensitivity and DNA content allows extrapolations to be made with increased confidence from experimental organisms to man. I t is still not known whether a similar relation holds for induced chromosome rearrangements. Such information is needed in order to evaluate whether point mutations or chromosome rearrangements are the more hazardous in relation to environmental mutagens.

B. THEIDENTIFIED REARRANGEMENTS : TYPES, FREQUENCIES, ORIGINS,AND PHENOTYPES

I. T y p e s The numbers of rearrangements of various types that have been identified are summarized in Table 5 . Reciprocal translocations are the most frequent, followed by insertional translocations and quasiterminal (tip) translocations. There are no paracentric inversions and only three pericentric inversions, all of which involve a chromosome tip. The 167 rearrangements are probably a representative sample of those newly arisen aberrations that result in ascospore abortion. Some bias doubtless exists against aberrations that have very drastic effects on ascospore abortion; these may have been rejected as too complex for analysis. At the other extreme, a bias must exist against aberrations with slight or borderline effects. These may not have been recognized in the

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TABLE 5 Identified Rearrangements in Neurospora crassa, Summarized According to Type of Aberration Type

Number.

I. Rearrangements that do not generate viable duplications b y meiotic recombination i n crosses by Normal Reciprocal translocations 123 Mutual insertions 2 Inversions 0 125 (75%) 11. Rearrangements that regularly generate viable duplications b y meiotic recombination in crosses b y Normal Insertional translocations 19b Intrachromosomal transpositions 1 Quasiterminal translocations 12 Quasiterminal pericentric inversions 3 Type uncertain 7 42 (25%) Total rearrangements 167 Similar rearrangements from the same experiment, that could have had a common origin, are counted only once. Duplications or other secondarily derived aberrations are not included. * Including some that are complex.

first place, or they may have been abandoned because of scoring difficulties. Short intrachromosomal transpositions with small displacements would not be detected by our methods, nor would short inversions. Tandem or nontandom duplications that arose directly as deviants from wild type would escape detection. Deficiencies would be lethal in a haploid organism unless sheltered, and would not be transmitted. Deficiencies of considerable length have, in fact, been obtained somatically by de Serres (1969) using techniques that allowed them to be rescued in heterokaryotic condition. The properties of each of the rearrangement types will be described and discussed in separate sections to follow. The basis for recognition and diagnosis of each type will be shown, and a possible explanation will be suggested for the absence of paracentric inversions. 2. Origin and Frequency

The aberrations have come from a variety of sources. Some were encountered as newly arisen variants in routine crosses. Others were recognized as present in various laboratory stocks. A majority were obtained by screening the survivors of experiments primarily designed to recover

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point mutants. Most of these experiments involved mild UV-irradiation followed by a procedure to enrich for particular mutant types. Less commonly, X-rays or chemical mutagens were used. The frequency of recovered rearrangements has not been determined as a function of treatment. It is our impression, based on several UV experiments where both point mutants and rearrangements have been sought, that the frequency of recovery is in the same order of magnitude for new gross rearrangements as for gene mutations. With UV doses giving 10 to 50% survival of treated conidia, from 5 to 20% of survivors have been found to contain new gross rearrangements. For representative recovery frequencies see Table 4 in Perkins (1974). Tector and Kafer (1962) have found new rearrangements in a high proportion of Aspergillus nuclei following commonly used radiation dosages. Neurospora strains have been collected from nature in such a way as to sample local populations from many geographical regions (Perkins et al., 1976). These were examined for structural differences within populations, and only two rearrangements have been found and confirmed. Both of them were reciprocal translocations, recovered as single isolates.

3. Fertility and Vegetative Phenotype

A vast majority of the 167 rearrangements are homozygous fertile and have no obvious mutant phenotype. I n Aspergillus also, most balanced rearrangements are morphologically wild type (Kafer, 1965 ; Nga, 1968). Eight of the rearrangements are barren in crosses where the rearrangement is homozygous-i.e., perithecia are formed that produce no ascospores, or few. Ascospore production is somewhat reduced in an additional five cases when rearrangement sequence is homozygous. Fourteen rearrangements have not been tested as homozygotes because of femalesterility (nine) or for lack of a recombinant with mating type (five). Twelve rearrangements acquired a t their origin a mutant phenotype that is allelic with a known gene locus and inseparable by recombination from the arrangement: HK5S cut, K79 me-7, Y l l 2 M 4 i ad-SB, Y l l b M 1 5 ad-SA, C161 aro, TM429 his-3, S1325 nic-2, C-1670 pk-1 (bis),4540 nic-2, 46S7 al-1, 17084 thi-I, and 46702 inl. Several other rearrangements are inseparable from a mutant phenotype that is not known to be allelic with a previously recognized gene locus-NM139 bs, 81229 arg, and T51M156 un are examples. To this list should be added 17 other rearrangements classified as morphologically mutant and nine as near-wild. These have not been tested for allelism with known morphological mutants. They include eight of the nine female-sterile rearrangements. At least 18 rearrangements arose simultaneously with a linked but separable point mutation: T54M94 (zcn-18), NM136 (arg-S), NM160 ( p h e - I ) ,A R l 7 4 ( p e r ) ,NM177 ( m o ) , AR216 (al-1 or - 2 ) , AR2dl (ilv),

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Y234M419 ( a d - 1 ) , 0 3 0 4 (morph), A420 ( t r p - 5 ) , H4250 (arg-1), S4342 ( p t ) , 5936 (leu-3), Y16329 (phe-2), 36703 ( a r g - l ) , 39311 ( S U C ) , 45502 ( p y r - S ) , 47711 (ilw). Also, five examples are known where two linked but separable rearrangements appear to have arisen simultaneously : T54M140b originated with another still unanalyzed rearrangement, Y l l 2 M 4 i originated with Y l l g M d r , AR180b with ARMOr, B362i with B362r, and STL384b with STL384r.

4. Position Efject No observations in Neurospora suggest a variegated-type position effect, but the growth habit might make a variegated phenotype difficult to detect. Rearrangements that originated simultaneously with mutant phenotypes have either been separable by recombination, or can be explained in terms of a breakpoint within a gene or within a single transcriptional unit. Several closely linked genes are simultaneously inactivated in T(ZZ;ZZZ)C161 aro (Gross and Fein, 1960). However, this is the result expected of a breakpoint within a cluster of genes that are coordinately transcribed (see Section IV, B, 2, b). All three functions specified by his-3 are missing in T (Z;VZZ)TM429 his-3, which has one break in the locus and no detectable deletion (Catcheside and Angel, 1974). Position effects might be anticipated when closely linked genes with coordinate functions are separated from each other by rearrangement. al-1 is separated from al-2 by T p ( I R + ZR)T54M94 and arg-2 is separated from pyr-3 in two translocations, Sl229 and S4342. The relevant phenotypes are not noticeably altered in any of the three rearrangements, however. A translocation in Aspergillus (VIII +=111) is characterized by a variegated morphological phenotype which is allelic with a known gene located a t or near one of the breakpoints (Clutterbuck, 1970). Evidence suggests but does not prove that the wild-type allele can be recovered from the rearrangement by crossing over; if so, i t would be the first example of a variegated-type position effect in fungi. C. REARRANGEMENTS THATDo NOTMAKEVIABLEDUPLICATIONS Within this category, no duplication progeny survive from crosses of Rearrangement X Normal, because whenever aneuploid segregants are duplicated for one chromosome segment they are deficient for another. Most of the Neurospora rearrangements that behave in this way are reciprocal translocations. Two are probably mutual insertions. Inversions behaving in this way would also be expected, but none has been found.

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1 . Reciprocal Translocations

Simple reciprocal translocations are the most readily identified and the most commonly occurring rearrangements in Neurospora (Table 5 ) . Appendix Table 1 lists the 123 mapped reciprocals according to linkage groups, and they are described individually in the Appendix. a. Ascus Effects and Their Meiotic Basis. Unordered asci from crosses heterozygous for typical reciprocal translocations show a distinctive distribution of the frequencies of ascus types (Fig. 7A, B ) . The meiotic origin of these ascus types is shown in Fig. 8. Normal disjunction of the centromeres of the two chromosomes involved in an interchange should produce 8 : 0 and 0:8 asci with equal frequencies when there is no crossing over in the interstitial centromere-breakpoint intervals (top half of Fig. 8 ) . Occurrence of interstitial crossing over is expected to result in 4:4 asci (bottom half). 4:4 asci will thus be rare if interchange points are both close to their respective centromeres, but frequent if one or both interchange points are far out in a chromosome arm. The breakpoints of many translocations have been mapped genetically, and they conform generally to these expectations. The examples in Fig. 7 were selected to show the two extremes-T (I;V)36703, with breakpoints far out from centromere, and T ( I ; I V ) N M l S 7 ,with breakpoints close in to centromere. For additional examples showing intermediate amounts of interstitial crossing over, see Fig. 3 in Perkins (1974). b. Cytology of Reciprocal Translocations. Pachynema is by far the most reliable and informative stage for verifying and diagnosing interchanges. Completely paired heterozygotes show a typical cross-shaped arrangement of the four bivalent arms (for photographs, see Barry, 1967, Figs. 2, 5, 7 ; Barry and Perkins, 1969, Fig. 1 ; Fincham and Day, 1971, Fig. 26). Pachytene associations have been used to confirm the genetic analysis of numerous translocations and to relate linkage groups to specific chromosomes (Section IV, C ) . I n theory, the contracted stages following pachynema should be useful for verifying and diagnosing interchanges, which should appear as rings or chains of four chromosomes. I n practice in Neurospora, however, diakinesis and metaphase I are very limited in their usefulness. The chromosomes are so small that recognition of a quadrivalent may be uncertain, and overlying bivalents can easily be mistaken for an interchange complex (see, for example, Fig. 4 in Barry, 1967). I n spite of these difficulties, contracted stages have been used successfully t o study interchanges that involve the easily recognized nucleolus organizer chromosome (Section IV, A, 4, c). McClintock and Singleton (Singleton, 1948) attempted to analyze

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Ascus

PROPHASE I ORIENTATION AND CROSSING OVER

ADJACENT CENTROMERES TO SAMEPOLE

-

CONSTITUTION

...:::::r::: I

Q”’ 4 . I .;:.”””. ;””’+”’ .

f

0:8

.. .. .. .. ....

f >

l

Dup, Def N T

- 4 >

x

Def, Dup

H

CEN TROMERES TO SAME POLE

VIABLE: DEFICIENT ASCOSPORES

‘ 4 ‘ 4

.. .,. .....,... .. .. : :

H

f

4:4

FIG.8. The origin and constitution of asci containing various numbers of deficient spores, from crosses of Reciprocnl Trnnslocntion (black centromeres) x Normal (white centromeres). Segments originally in one of the Normal chromosomes are shown as solid lines, those in the other Normal chromosome as dotted lines. The consequences of segregation without crossing over are shown in the two top diagrams. Crossing over between either breakpoint and centromere is expected to produce 4:4 asci, as shown in the bottom two diagrams. Other linear orders of black and white spore-pairs are possible, depending on which chromatids were involved in crossing over; the resulting unordered tetrad is classed as 4:4 regardless of its original order. The defective spores are of two types, representing complementary duplicationdeficiency classes. These may or may not he recognizably different, depending on the particular translocation. If adjacent-2 segrcgations occurred (where homologous centromeres failed to disjoin), 0:s asci would result, with all spores deficient (not shown in the figure). Reproduced from Perkins (1974).

trans’locations by determining changes in chromosome lengths and arm ratios a t metaphase of the third division in the ascus. In our experience this stage is of doubtful practical value because of small chromosome

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size and possible inaccuracies of measurement if a chromosome is tilted in the polar plane.

c. Some Research Applications of Reciprocal Translocations i. Mapping genes lo linkage group. The efficiency of assigning unmapped point mutants has been greatly increased by a tester strain (acronym alcoy) containing three independent translocations tagged by markers which can be scored visually (Section IV, B, 4, a ) . A majority of new point mutants readily show linkage to one of the markers, and linkage group assignment is then completed with a single follow-up cross (Perkins et a/., 1969; Perkins, 197213). ii. Production of duplications. 50% black; unordered asci 49:5:35:2:9 (N = 162). Origin: 74A, W . Detected and analyzed by Perkins. No barren duplications are produced. Cryptic aneuploids have been obtained from several crosses. These segregated acr-2, thi-4, or bal and are thought to originate as disomics, from 3 : l segregations. Black ascospores show good viability. Stock of origin also contained another probable rearrangement, tentatively T ( I + 111)T54M150 un ; this has probably been separated, but is inadequately analyzed. FGSC 2941, 2942. T ( I;II;IV)R55 A complex translocation showing linkage in I R near Zys-3, in I1 near pe, and in IVR. (Not tested for linkage in group V.) Wild morphology, but slower growing. Homozygous-fertile. Cytology: Chromosomes 1, 2 and 6 are involved in the translocation. Breaks in 1 and 2 near centromere with arms exchanged, and in 2 through the nucleolus organizer with the tip of the organizer translocated to the long arm of 6. Origin: pe fl, X-rays. Detected and analyzed cytologically and genetically by P. St. Lawrence. Stock now lost. Reference: St. Lawrence (1953).

Duplications: Two types of probable duplications observed, called abnormal ( a b n ) and abnormalsterile (abn-s). The abn type crossed by standard chromosome sequence, R55 sequence, or other abn’s produced asci developing to late meiotic prophase stages, but chromosome pairing appeared to be abnormal. Few asci completed the third division, and spore formation was rare and irregular. ablzrs crossed to standard was barren, with perithecial development arrested prior to ascus formation.

T ( 1I;III ) AR62 Reciprocal translocation. I1 (linked a r g d ) interchanged with IIIR (linked trp-1) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 36:1:40:1:22 (N = 306). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross with

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T(II;IIIR) NM161. Strain of origin also contained an unlinked ascospore-color gene bs (AR62, in IR). FGSC 1545, 1546. T( I;II)B66 Reciprocal translocation. I (linked mt) interchanged with I1 (linked p e ) . Wild phenotype. Homozygous-fertile. T x N ascospores 5070 black ; unordered asci 14:9:52:7:18 ( N = 88). Origin: 74A, UV. Detected and analyzed by Perkins. FGSC 1464, 1465. T( IV;VI)P73B12 Probably reciprocal translocation. IV (near p d z ) interchanged with VIL (near choZ-2). Wild phenotype. Homozygous-fertile. .Tx N ascospores > 50% black; unordered asci 16:13:56:6:9 (N = 111). (Interpreted 16:0:69:0:15.) Origin: sn cr; aG3 inl, EMS. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores become black. FGSC 2623, 2624. T( I;III)P73BlOl Reciprocal translocation. I R (linked aur) interchanged with IIIR (linked trp-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 18:25:41:11:6 ( N = 175). Origin: sn cr; a M in!, EMS. Detected and analyzed by Perkins. FGSC 2645, 2646. T( II;VII)P73B169 Reciprocal translocation. I I R (right of p e ) interchanged with VIIR (right of nt). Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 21 :49:27:2:1 (N = 182). No viable duplications recovered. Origin: sn c r ; a14 inl, EMS. Detected and analyzed by Perkins. Evidently some inviable duplicatios-deficiency ascospores become black. Less than two-thirds of black ascospores are viable. FGSC 2625, 2626. T ( 1;II ) STC76 Reciprocal translocation. IR (between cyh-1 and p e l ) interchanged with IIR (between p e and ace-1). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black, unordered asci 29:8:32:6:24 ( N 139). Origin: 76A, spontaneous. Aberration detected by St. Lawrence, analyzed by Perkins. Generates viable duplications from intercross with T ( I R ;ZIR)4837 d-1. Markers shown covered by progeny test of duplications: os-6, un-7, al-2, arg-6, p e . Loci shown not covered because duplications from intercross are of recessive marker phenotype : ace-1, trp-3. FGSC 2096, 2097.

-

T( I;VII)K79 met-7 Reciprocal translocation. I (linked mt) interchanged with VII (at met-7). Phenotype met-7, wild morphology. Homozyqous-fertile. T x N ascospores 50% black; unordered asci 47:4:7:2:41 (N = 189). Origin: E m a. Translocation detected and linkage groups identified by N. E. Murray. Generates viable duplications from intercross with T(I;VIZ)S1007.FGSC 2297,2298. T( 1II;V)NMIOL Reciprocal translocation. I11 (linked acr-2, trp-1) interchanged with VR (linked id). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; un-

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ordered asci 31:9:44:4:13 (N = 231). Origin: E m a, W. Detected and analyzed by Perkins. Intercrosses indicate probably identical to I11;V translocations NMlO.3, 10.4, 111, 112, 114, 116 isolated from the same experiment. FGSC 1879, 1880.

T(III;V)NM102

See T (111; V )NMlOl .

T(I + V1)NMlOJ

Quasiterminal translocation. A segment of IR including met-6 and distal markers is translocated to the right end of VI beyond trp-g. Wild phenotype. Homozygousfertile. T x N ascospores 75% black, or more; unordered asci 26:42:27:4:1 (N = 219). Origin: Em a. W. Detected by Perkins. Analyzed by B. C. Turner. Used as het-6 tester to study vegetative incompatibility. FGSC 2137, 2138 ( h e t b o R ) . References: Turner, 1975, 1976; Mylyk, 1975, 1976. Duplications: D p ( 1 R + V1R)NMlOS. In one-third of surviving progeny from T x N . Duplications make relatively large, diffuse colonies on sorbose medium. In tubes, duplication resembles a slow wild type a t 25°C but resembles fZ: fluffy at 34". Sectoring (uncovering of recessive heterozygous markers) is apparent in some backgrounds. Initially highly barren. Marked crosses give no recovery of intact duplications. However, in many crosses a few perithecia are fully fertile through loss of one duplicated segment. Either segment-that in N or in T sequencemay be lost with equal probability. Euploid derivatives are always found in platings of such partially fertile duplications. There is no evidence of delayed fertility, which might be expected if loss were stepwise. Markers shown covered: met-6, ad-9, nit-1, cyh-1, al-2, arg-13, R , nn-18, het-6. Markers shown not covered: thi-1, un-1, cr, his-3, mt, fr.

T(III;V)NM104

See T ( I I I ; V ) N M l O l ,

T(I;III)NM107

Reciprocal translocation. I (linked m t ) interchanged with I11 (linked acr-2). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 20:1:55:1:23 (N = 82). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T(I;III)NMlOS, NMlgY, NM136, or N M l 4 6 . FGSC 2058, 2059.

T(I;III)NM109

Reciprocal translocation. I L (between un-6 and m t ) interchanged with IIIR (between acr-2 and dow). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 20:0:63:2:15 (N = 54). Origin: Em a, W. Detected and analyzed by Perkins. Intercrosses show not identical with T ( 1 ; I I I )NMlO7, NM127, NM136, NM146. FGSC 2627, 2628.

T(1;V)ALSlll

Reciprocal translocation. I R (near cr) interchanged with VR (between al-3 and his-6). Wild phenotype. Homozygous-fertile. T x N ascospores 70% black; unordered asci 39:16:35:3:6 ( N = 148). Origin: rg cr a, UV. Detected and analyzed

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by Perkins. No viable duplications are produced in crosses by Normal. Frequencies of ascus types suggest the one inviable duplication-deficiency class darkens. However, germination is good among black ascospores. Possibly white spores degenerate. Produces viable duplications from intercrosses with T(1;V)SSrOS and C-1670 pk-1. FGSC 2629, 2630. T ( III;V)NMlll, T ( III;V)NM112 See T ( l 1 1;V)N M l O l . T ( I + 111)Y112M4i adSB Insertional translocation. I R segment including nic-2 is inserted in IIIR near vel. a d S B phenotype: requires adenine, accumulates purple pigment. T x T crosses infertile, perhaps because of adenine requirement. T x N ascospores 7570 black; unordered asci 15:50:26:6:3 ( N = 185). Origin: 74A, X-rays. Discovered and first analysis by de Serres. I11 linkage identified by P. St. Lawrence. Separated from T ( Z l l ; V l l )Y112M4r which was also present in original strain. FGSC 2637, 2638. Reference: de Serres, 1957. (This is the first published account of an insertional translocation in Neurospora.)

Duplications: D p ( l R + I I I R ) Y112M4i. One-third of surviving progeny from T x N . ad+ phenotype. Highly barren in crosses by nonduplication. Markers shown covered: nic-2. Markers shown not covercd: his-2, ad-3A, cr, thi-1, at-2. T ( 1II;VII) Y 112M4.r Reciprocal translocation. I11 (near acr-2) interchanged with VII (linked W C ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 25:3:40:3:29 ( N = 177). Origin: 74A, X-rays (de Serres, 1957). Strain of origin also contained T ( I + I l l ) Y l l d M 4 i ad-3B. Extracted, mapped and analyzed by Perkins. FGSC 2631, 2632. T(I IV)Y112M15 ad-3A Translocation with breakpoints a t or near ad-SA in IR and near pdx in IV. Probably an inverted insertional I + IV, with mutual IV + I insertion postulated to explain absence of viable duplications among progeny of T x N . Phenotype n&SA : requires adenine and makes purple pigment. Cytology: Chromosomes 1 and 3 reported aberrant by Griffiths et nl. (but see Section IV, C). Acentric chromosome fragments are formed, which persist in micronuclei and replicate (Barry). Origin: 74A, X-rays. Aberrant recombination noted by de Serres. Aberration analyzed by Griffiths. References : de Serres, 1971 ; Griffiths, 1970, 1972; Griffiths, et nl., 1974. T ( IV;VII)NM113 Reciprocal translocation. IV (linked pdx) interchanged with VII (linked met-7). Sub-wild phenotype, pale pigment. Lysis and exudate at top of agar slant. Homozygous-fertile. T x N ascospores 50% black; unordered asci 31:13:20:1:34 ( N = 70). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T(IV;VII)NM16G or NM158, which originated from a different experiment. FGSC 1917, 1918. T ( III;V)NMI14, T(III;V)NM115 See T ( I I I ; V ) N M l O l .

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T ( III;IV)NM118 Reciprocal translocation. 111 (near ncr-2) interchanged with IV (near p d z ) . Slow to conidiate. Homozygous-fertile. T x N ascospores 50% black; unordered asci 31:5:35:2:27 ( N = 213). Origin: Em a, W. Detected and analyzed by Perkins. Intercross shows not identical with T(I1l;ZV)N M i 3 1 . FGSC 2403, 2404. T ( 1;IV) NMl19 Reciprocal translocation. Probably identical structure with T(I;ZV)NMI72, q.v. Morphology is ropylike, variable. Homozygous fertility reduced. Cytology: Chromosome 2 is not part of the rearrangement. Generates viable duplications in crosses with T(IR;ZVR)NM140. FGSC 1447. 1334. References: Barry and Perkins, 1969; Kowles, 1972, 1973. T ( VI;VII)NM124 Reciprocal translocation. VI (linked ylo-1) interchanged with VII (near met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 46:3:5:1:45 ( N = 361). Origin: Em a, UV. Detected by Perkins. Analyzed and linkages established by Anna Kruszewska. Shot asci shift with time from excess OB:8W to excess 8B:OW. FGSC 2214, 1472. T(IV;V)NM125 Reciprocal translocation. IVR (linked pdx) interchanged with VR (linked a t ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 13:6:41:3:36 (N = 63). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross by T ( I V R ; V R )A R l l r . Arm assignments were made on this basis. FGSC 2447,2448. T(II;IV)NM126 Reciprocal translocation. IIR (near tip-3, probably distal) interchanged with IVR (near col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50-75% black; unordered asci 21:22:49:7:1 (N = 295). Origin: Em a, UV. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores become black. Germination of black ascospores is low. No viable duplications are recovered. cot-1 and 11 are linked in T x T cross. FGSC 1611, 1612. T ( 1;IJ.I )NM127 Reciprocal translocation. I (near m t ) interchanged with 111 (probably R ; loosely linked ncr-2). Wild phenotype. Homozygous-fertile. T X N ascospores 50% black; unordered asci 17:1:53:3:25 ( N = 217). Origin: E m a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(I;IIZ)NMlS(S. Arm assignments are based on this fact. Intercrosses show not identical with T(I;ZII)NMlO7,NM109 or NM146. FGSC 2405,2406. T ( I;IV)NM128 Reciprocal translocation. I (linked ml) interchanged with IVR (near p t ) . pe-like morphology. T x T crosses nearly infertile. T x N ascospores 50% black; unordered asci 50:8:25:3:15 ( N = 93). Origin: Em a, UV. Detected and analyzed by Perkins. Not tested for identity by crosses with NMl4O or other pe-like I ;IV translocations from the same source.

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T(I;II)NM129

Reciprocal translocation. I (near his-2, probably right) interchanged with I1 (near

arg-5). Morphology not wild: flat growth habit. Homozygous-barren (empty perithecia). T X N ascospores 50% black; unordered asci 30:5:9:6:51 ( N = 661). Origin: Em a, UV. Detected and analyzed by Perkins. Progeny from

T x N include a few percent of barrens, “Dark Agar” phenotypes (attributed to A / a heterozygosity), and in some crosses “Brown-flats” (attributed to het-Clhet-c) . These are thought to arise from 3 : l segregations. FGSC 2330, 2331. T(1;V)NMlSO

Reciprocal translocation. I (linked arg-1) interchanged with VL (26 units left of a t ) . Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 36:16:30:4:15 ( N = 230). Origin: Em a, UV. Detected and analyzed by Perkins. Not overlapping with T ( I R += VL)ARISO, because no viable duplications are recovered from intercrosses. This favors IL location of NMiSO break point. Evidently some duplication-deficiency ascospores darken. FGSC 2407, 2408.

T(III;IV)NM131

.

Reciprocal translocation. I11 (linked acr-2) interchanged with IV (linked pdz) Wild phenotype. Homozygous-fertile. T X N ascospores 50% black; unordered asci 17:3:69:0:11 (N = 36). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T(IZI;IV)N M i 1 8 . FGSC 2409, 2410.

T(I;IV)NM132

Reciprocal translocation. I R (right of aG2) interchanged with IV (near p d z ) . “Creamy” morphology. Female-sterile. T x N ascospores 50% black; unordered asci 17:10:30:10:34 (N = 95). Origin: Em a, UV. Detected and analyzed by Perkins. Not tested for identity by intercrossing with similar 1;IV translocations from. the same source.

T(II;VII)NM134

Reciprocal translocation. I1 (near arg-6) interchanged with VII (linked met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 46:2:11:2:39 (N = 54). Origin: E m a, UV. Detected and analyzed by Perkins. FGSC 1919, 1920.

T ( 1;II) NM135 Reciprocal translocation. I (linked m t ) interchanged with I1 (linked arg-5). Slightly flat, pe-like morphology. Homozygous-fertile. T x N ascospores 50% black; unordered asci 43:6:30:3:17 ( N = 178). Origin: Em a, UV. Detected and analyzed by Perkins. FGSC 2023, 2024.

T(I;III)NM136

Reciprocal translocation. I (near m t ) interchanged with I11 (probably R ; linked t r p - f ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 29:4:46:2:19 ( N = 94). Origin: Em a, UV. Detected and analyzed by Perkins. Original isolate contained a linked but separable arg-3 mutation. Generates viable but morphologically distinct duplications from intercross with

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DAVID D. PERKINS AND EDWARD G. BARRY

T ( I ; I I I ) N M l 2 7 . Intercrosses show not identical with T(I;III)NMlOY, NM109, or NM146. FGSC 2639,2588.

T ( I;IV)NM137 Reciprocal translocation. I (linked m t ) interchanged with IV (linked col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 41:5:9:3:42 ( N = 116). Origin: Em a, W. Detected and analyzed by Perkins. FGSC 1874. 1875.

T( I;IV)NM139 bs

Reciprocal translocation. I R (near nl-2 and proximal) interchanged with IVR (near p d z and distal). Wild vegetative morphology. T ascospores are brown, yet viable. Homozygous-fertile. T x N ascospores 2570 black; 25% viable brown, 50% white. Unordefed asci 22:0:53:0:25 ( N = 271), when brown ascospores are counted with black. Origin: Em a, UV. Detected and analyzed by Perkins. Allelic and identical with T(I;IV)NM147 and NMlBY, both of which show the same brown-ascospore phenotype. Not allelic with I R point mutant bs-1 : brown ascospore (AR62). Generates viable duplications from intercrosses with T ( I R ; IVR)NMl4O and NMlY2. Intercross shows not structurally identical with T ( I R ; I V R ) N M 1 6 0 . FGSC 1565, 1566.

T(I;IV)NM140

Reciprocal translocation. I R (near 08-1, distal) interchanged with IVR (proximal to t i p - 4 ) . Smooth pe-like morphology. Female-sterile. T X N ascospores 50% black; unordered asci 20:3:55:4:18 ( N 383). (Values in Fig. 11 of Perkins, 1974, are incorrect.) Also 29:2:45:2:22 ( N = 121-Kowles). Cytology: Chromosome 2 is not part of the rearrangement (Barry). Origin: E m a, UV. Detected and linkage groups identified by Perkins; further data by Kowles. Generates viable duplications from intercrosses with T ( I R ; I V R ) N M l l 9 , NM139, NM160, and NM172. FGSC 1759, 1548. References: Barry and Perkins, 1969; Perkins, 1971a; Kowles, 1972, 1973.

-

T(IV;V)NM141 Reciprocal translocation. IVR (linked p d z ) interchanged VR (near al-3). “Creamy” morphology. Homozygous-fertile, T x N ascospores 50% black ; unordered asci 16:1:64:5:14 ( N = 163). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercrosses with T ( I V ; V R ) A R l l r , NM146 and R2366. N M l 4 l break point assigned to IVR because R2366 maps in IVR. FGSC 2025, 1479.

T( I;V)NM143 Reciprocal translocation. IR (near nur) interchanged with VR (near i d ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 22:2:63:3:11 (N = 139). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercroes with T ( I R ;VR)P6166. FGSC 1549, 1550.

T ( I;IV)NM144 Reciprocal translocation. I R (linked 0s-1, distal) interchanged with IVR proximal to trp-4. “Creamy” morphology. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 57:4:31:4:4 ( N = 721, and 22:4:63:0:12 ( N = 8LKowles). Origin:

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Em a, UV. Detected by Perkins, analyzed by Perkins, Kowles. Generates viable duplications from crosses with T ( I R ; I V R )NMlGO, NM164, and NM172. Assignment of NM144 to riglit arm of IV is thus confirmed. Intercrosses infertile with T ( I ; I V ) N M 1 4 0 , NM167, T64M19. FGSC 1336, 1335. References: Kowles, 1972, 1973.

T(IV;V)NM145

Reciprocal translocation. IVR (linked p d z ) interchanged with VR (between at and id).Wild phenotype. Homozygous-fertile. T x N ascospores 60-75% black; unordered asci 18:50:27:3:2 ( N = 277). Origin: Em a, UV. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores from T x N become black. Germination of black ascospores is 6O-75p/o0.No viable duplications are recovered from T x N . Generates viable duplications from intercrosses with T ( I V R ; V R ) A R l l r , N M l 4 1 , and RI355. Breakpoint assigned to IVR on this basis. F W C 2098, 2099.

T(I;III)NM146

Reciprocal translocation. I (loosely linked m t ) interchanged with I11 (loosely linked acr-2). Wild phenotype. Homozygous-fertile. 2' X N ascospores 50% black; unordered asci 15:5:62:5:13 ( N = 61). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T ( I ; I I I ) N M l O 7 , NM109, N M l U , or NM13G. FGSC 2449, 2450.

T(I;IV)NM147 bs

See T ( I ; I V ) N M l 3 9 bs

T(I1 --j V)NM149

Translocation, probably quasiterminal. A IIL segment including ro-3 and distal markers is translocated to the tip of VR near his-G. Wild phenotype. Homozygousfertile. T x N ascospores 757'0 black or less; unordered asci 13:46:3?3:3:0 ( N = 206). Cytology: Chromosome break points identified near the end of the long arm of 2, and in chromosome 6 (Barry). Origin: Em a, W. Detected and analyzed genetically by Perkins. Used as het-c tester for studying vegetative incompatibility. FGSC 1483, 1482 (het-c) ; 2011, 2012 ( h e t - C ) . References: Perkins, 1969a,b, 1975; Barry and Perkins, 1969; Mylyk, 1972, 1975. Duplications: D p ( I I L + VRINM149. One-third of surviving progeny from T x N . Wild phenotype unless heteroaygous for vegetative incompatibility genes. The speed of somatic escape from inhibition in het-clhet-C duplications is increased by mei-3 (Newmeyer and Galeaazi, 1976b). After escape, individual duplications vary from highly barren to relatively fertile, when crossed by nonduplication. Markers shown covered: ro-3, het-c, pyr-4, het-6, cys-5, pi, col-10. Markers shown not covered : thr-2, thr-3, arg-5. There is evidence also for a second class of duplications that are not heterozygous for I I L markers. Possibly these arise by 3 : l segregation, and they may involve VR.

T(II;III)NM150

Reciprocal translocation. I1 (linked arg-6) interchanged with IIIR (linked un.6). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 11:0:63:6:20 ( N = 54). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T ( I I ; I I I R ) N M 1 6 1 . FGSC 2060, 2061.

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T ( I V + I)NM152 Insertional translocation. An extensive segment of IVR, including loci from pyr-3 through pyr-8, is inserted in I, ten units from arg-3 (probably IR). Wild phenotype. Homozygous-fertile. T x N ascospores, 75% black, or less; unordered asci 18: 16:44:9: 13 ( N = 324). Cytology: Preliminary cytological examination. Acentric fragments are produced which become pycnotic (Barry, 1973). However, their frequency is lower than would be expected if such a long insertion were inverted. Origin: Em a, UV. Detected and analyzed by Perkins. Data on IV breakpoint obtained by A. Radford. FGSC 1752, 1753. Duplications: Dp(IVR + I)NM162. One-third of surviving progeny from T x N. Wild phenotype. Duplications scorable as barren with 90% confidence. Markers shown covered: pyr-3, trp-4, met-2, cot-1, pyr-2. Markers shown not covered: pdx-1, COG,$, cys-4.

T ( 1I;V ) ALS154 Reciprocal translocation. IIR (near fl) interchanged with VR (near id).Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 11:40:32:6:11 ( N = 88). Origin: rg cr a, W. Detected and analyzed by Perkins. Poor germination among black ascospores and asymmetrical unordered ascus distribution suggest that some duplication-deficiency ascospores become black. FGSC 2062, 2063.

T(I;VII)NM155 Reciprocal translocation. I R (near our, probably proximal) interchanged with VIIR (linked mo(P11G3) and met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 36:3:22:8:31 ( N = 74). Origin: Em a, UV. Detected and analyzed by Perkins. FGSC 1877, 1878. T ( IV;VII)NM156 Reciprocal translocation. IV (linked pdz) interchanged with VII (probably R ; linked wc and arg-10). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 42:7:33:7:11 ( N = 272). Origin Em a, UV. Detected and analyzed by Perkins. Intercrosses show not identical with T(IV;VII) NMllS or NM168. FGSC 1921, 1922. T ( V;VI)NM157 Reciprocal translocation. VR (between at and al-3) interchanged with VIR (near trp-8, probably distal). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 19:3:46:6:26 ( N = 72). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T(VR ;VI)NM162b. FGSC 2648, 2649. T ( IV;VII)NM158 Reciprocal translocation. IVR (linked cot-1) interchanged with VIIR (near arg-10, probably distal). Wild phenotype. Homozygous-fertile. T X N ascopores > 50% black; unordered asci 37:11:35:9:9 ( N = 151). Origin: Em a, W. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(IVR ;VII)ARlO. Assignment to IVR is based on this observation. Intercross shows not identical with T(IV;VII)NMlBG. FGSC 2026, 2027.

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T(IV + VI)ALS159 Quasiterminal translocation. All IVR markers are translocated to the right end of VI beyond trp-2. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 33:22:32:5:8 ( N = 259). Origin: rg cr a, UV. Detected and analyzed by Perkins. FGSC 2100,2101. Duplications: D p ( I V R + VIR)ALS169. One-third of viable progeny from T x N . Usually recognizable by reduced conidiation on minimal slants at 34°C or patches of hyphae without conidia. Barrenness of duplications is exceptionally stable in crosses to nonduplications. A few individual perithecia become fertile. Markers shown covered: pyr-1, un-8, pdx, pur-3, cot-1, cys-4, uvs-2. T ( V;VII ) NM159 Reciprocal translocation. V (near a l ) interchanged with VII (near wc). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black (defective spores become brown) ; unordered asci 25:2:44:3:25 ( N = 88). Origin: Em a, W. Detected and analyzed by Perkins. FGSC 2411, 2412.

T ( 1;IV ) NM160 Reciprocal translocation. I R (right of nic-2) interchanged with IVR (near COG,$). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 31:6:15:6:42 (N = 209). Origin: Em a, UV. Detected and analyzed by Perkins. Intercrosses indicate identical sequence with T ( I ; I V ) N M l 6 2 and with N M i 6 7 , which is female-sterile. Generates viable duplications from crosses with T ( I R ; IVR)NM140, NMIQQ, and NM172. Strain of origin also contained linked but separable point mutant phe-1 (NM160). FGSC 1338, 1337. T( II;III)C161 aro Reciprocal translocation. IIR (in nro cluster) interchanged with I11 (linked acr-2). Multiple deficiencies in aromatic synthetic enzymes. Homozygous fertility not tested. T x N ascospores 50% black; unordered asci 20:2:48:4:27 (N = 157). Origin: Recognized aberrant by Gross and Fein (1960). I11 linkage found by Perkins. Called arom-2 by Gross and Fein. This differs from the point mutant Y306M81 called arom-2 by Giles et al. (1967). FGSC 2106, 2107. T ( II;III)NMl61 Reciprocal translocation. I1 (linked arg-6) interchanged with IIIR (linked t i p - I ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 18:4:46:7:26 ( N = 179). Origin: Em a, UV. Detected by Perkins. Beske and Phillips (1968) showed I or I1 linked with I11 or VI. Further analysis by Perkins. Generates viable duplications from cross with T ( I I ; I I I R ) A R 6 2 . Intercross shows not identical with T ( I I ; I I I R ) N M 1 6 0 .FGSC 2028, 2029. T ( I;IV)NM162 Structurally identical with T ( 1 ; I V )NM160, q.v. Found in same isolate with T ( V ; V I ) N M 1 6 2 b .FGSC 2589, 2590. Reference: Perkins, 1974. T ( V;VI)NM162b Reciprocal translocation. VR (linked id) interchanged with VI (linked ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered

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asci 15:2:52:6:25 (N = 182). Origin: Em a, UV. Detected and analyzed by Perkins. Found in same isolate with T ( 1 ; I V )NM162. Intercrosses show not identical with T ( V R ; V I R ) N M 1 6 7 or N M 1 7 l . FGSC 2.591, 2592. Reference: Perkins, 1974.

T(I;VI)NM163 Reciprocal translocation. I (linked mt) interchanged with VIL (near chol-2). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 13:0:67:2:18 ( N = 45). Origin: Em a, W. Detected by Perkins. Beske and Phillips (1968) showed I or I1 linked with I11 or VI. Further analysis b y Perkins. FGSC 2030, 2756.

T(1;IV)NMlB.I

Reciprocal translocation. I R (linked al-2, proximal) interchanged with IVR (proximal to trp-4). Wild phenotype. Homozygous-fertile. T X N ascospores 50% black; unordered asci 29:4:27:3:36 ( N 294). Also 32:12:45:1:9 (N = 106Kowles). Cytology: Chromosome 2 is not part of the rearrangement (Barry). Origin: Em a, UV. Detected by Perkins. Genetic analysis by Perkins, Kowles. Generates viable duplications from intercrosses with T ( 1 ; I V )T64M19, NM144, and NM172. FGSC 1341, 1340. References: B'arry and Perkins, 1969; Kowles, 1972,

-

1973.

T(I;VII)ALS167

Reciprocal translocation. I (not separated from rg or cr) interchanged with VII (near w c ) . T x T cross infertile because of markers. T x N ascospores 50% black or more; unordered asci 58:9:6:1:27 ( N = 144). Origin: rg cr a, UV. Detected and analyzed by Perkins. FGSC 2413,2529.

T(1;IV) NM167

Structurally identical with T ( 1 ; l V )NMi60, q.v. Female-sterile. FGSC 1343, 1342.

T ( I;II)NMlB8 Reciprocal translocation. I (probably L-near m t ) interchanged with I I R (near $). Wild phenotype. Homozygous-fertile, T x N ascospores > 50% black; unordered asci 25:24:40:6:5 (N = 185). Origin: Em a, UV. Recognized aberrant by Perkins. Analyzed by Anna Kruszewska. Evidently some inviable duplicationdeficiency ascospores become black. Germination of black ascospores is low. No viable duplications are recovered. FGSC 1923, 1924. T ( I + )NM16Qd Duplication-generating rearrangement in which a distal I R segment containing un-18 is covered in the duplications. Location of the segment is not known in rearranged sequence. Wild phenotype. Homozygous-fertile. T x N ascospores > 75% black; unordered asci 29:54:10:6:1 ( N = 124). Origin: Em a, UV. Detected by Perkins. Analyzed by Perkins and B. C. Turner. Strain of origin also contained T ( I I I R ; V I I ) N M 1 6 9 r .FGSC 2279, 22fUl. Duplications: D p ( I R + )NM169d. Stably barren in crosses by nonduplication. Markers shown covered: un-18. Markers shown not covered: arg-13, so, aro-8, R . Duplications possess the vegetative morphology characteristic of R , and this is known from other rearrangements to be recessive when a duplication is heterozygous R / R + .

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T(II1;VII)NMISSr

Reciprocal translocation. IIIR (linked leu-1 trp-1) interchanged with VIIR (near wc to right). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 34:4:30:1:31 (N = 143). Origin: Em a, W. Detected and analyzed by Perkins. Original isolate also contained T ( I R + )NM169d. FGSC 1816, 1817. Reference : Perkins, 1974.

T(I;IV)NMl7O

Reciprocal translocation. I (linked m l ) interchanged with IV (linked cot-1). “Creamy” morphology. Female-sterile. T x N ascospores 50% black ; unordered asci 11:4:54:9:22 ( N = 46). Origin: Em a, UV. Detected by Perkins. Beske and Phillips (1968) showed I or I1 linked with I V or V. Further analysis by Perkins. FGSC 1489.

T ( V;VI ) NM171

Reciprocal translocation. V (near a t ) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile, T x N ascospores 50% black ; unordered asci 24:3:17:0:55 ( N = 29). Origin: Em a, W. Detected and analyzed by Perkins. Intercross indicates not identical with T ( V R ; V I )NM162b. FGSC 2451, 2452.

T(I;IV)NM172 Reciprocal translocation. I R (linked 0s-1, distal) interchanged with IVR (proximal to t7p-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black or more; unordered asci 10:17:62:5:6 ( N = 314). Also 30:6:46:4:15 (N = 139Kowles). Cytology: Chromosome 2 is not part of the rearrangement (Barry). Origin: Em a, UV. Detected by Perkins. Genetic analysis by Perkins, Kowles. Apparently one class of duplication-deficiency ascospores may become pigmented. Identical structure with T ( I R ;ZVR) NM119 by intercross. Generates viable duplications from intercrosses with T ( l R ; l V R ) N M 1 3 9 , NMl40, NM144, NM160, and NM164. FGSC 1345, 1518. References: Barry and Perkins, 1969; Perkins, 1971a; Kowles, 1972, 1973.

T(I + VII;I;V;VII)AR173

Complex insertional translocation involving I R (near centromere), V (near a t ) and VII (near w c ) . A short proximal I R segment including un-2 and h i s 4 is inserted in VII. Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 41:8:14:7:31 ( N = 212). Origin: 74-OR23-1A,UV. Detected and analyzed by Perkins. FGSC 2468,2469.References: Perkins, 1974. Duplications: D p ( l R + V I I ; I ; V ; V I I ) A R l 7 3 . In one-third of surviving progeny from T x N . Stably barren in crosses. Markers shown covered: un-2 his-2. Markers shown not covered: j r , nit-2, leu-3, m t , arg-1, arg-3, sn, rg, nuc-1, lys-4, met-10, his-3; lys-1, at, al-3, m o ( M 1 8 4 ) , mo(M193-1). (Data on nuc-1-his-3 from R. L. Metzenberg.)

T ( V;VI ) A m 7 4

Reciprocal translocation. VR (linked i d ) interchanged with VI (linked ~ Z O - 1 ) . Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 9:12:53:12:14 ( N = 58). Origin: 74-OR23-1A,W.Detected and analyzed by

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Perkins. Original isolate also contained linked mutant gene resulting in nonblack perithecia and ascospores. FGSC 2678, 2679.

T(VII-IV)ALS175

A duplication-generating translocation involving VII (linked wc) and IV (linked pdz). Either IV is the recipient or pdz is covered by the duplication. Wild phenotype. Homosygous-fertile. T x N ascospores < 50% black; unordered asci 0:3:28:19:51 ( N = 75). Origin: rg cr, W. Detected by Perkins and early analysis by D. A. Smith. FGSC 2931, 2932. Duplications: Dp(Vll-ZV)ALSlT6. Sub-wild vegetative phenotype. Barren in crosses with nonduplication.

T(I;V)AR175

Reciprocal translocation. I (linked mt) interchanged with V (near a t ) . Near-wild phenotype (bleeds high in slant), Homozygous-fertile. T X N ascospores 50% black; unordered asci 39:8:13:5:34 ( N = 165). Origin 74-OR23-1A, W. Detected and analyzed by Perkins. FGSC 2593, 2594.

T(IV;VI)NM175

Reciprocal translocation. IVR (linked pdz) interchanged with VIR (linked ylo-I). Wild phenotype. Homozygous-fertile. T x N ascospores 5oq0 black; unordered asci 49:0:5:5:42 ( N = 41). Origin: Em a, UV. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(IVR;VIR)46602. Arm assignments are on this basis. FGSC 2295, 2293.

T ( I I + V)ALS176 Translocation involving I I R (between bal and arg-6) and V (linked at; probably L) . Probably quasiterminal. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black, or less; unordered asci 24:43:28:3:2 ( N = 126). Origin: rg cr a, W. Detected and analyzed by Perkins. FGSC 2414, 2415. Duplications: Dp(ZIR + VL)ALSlTG. In one-third of viable progeny from T x N . Initially thin, transparent, slow growth. Not stably barren in crosses by nonduplication, mostly behaving like normal sequence. Markers shown covered : arg-6, m o d , cpt, arg-12, fl. (Other markers, in IIL, may be heterozygous in disomics from 3: 1 segregations, which occur with frequencies of several percent.) Markers shown not covered in simple duplications : bnl, pyr-4.

T(1;III;VI;VII)AR176

Complex rearrangement. Linked closely to acr-2 (III), ylo-1 (VI), and wc (VII) and less closely to mt (I). The structural basis is not understood. Wild phenotype. Homozygous-fertile. Ascospores oozed, few shot. T x N ascospores < 25% black; unordered asci 8:6:30:20:36 ( N = 154). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2708, 2709. Duplications: Dp(V1II + VZt)ARlTG. In less than one-third of surviving progeny from T x N . Duplications are recognizable by flat morphology. Very stably barren. Markers shown covered: None. Markers shown not covered: mt, acr-2, rib-1, met-7, nt.

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In( IL + IR)NM176 Pericentric inversion. A distal segment of IL (including ser-3 but not un-3 or m t ) is interchanged with the I R tip. No bona fide euploid recombinant with mating type has been obtained. Wild phenotype. I n x N ascospores 75% black, or more ; unordered asci 43:43: 11: 1 :1 ( N = 1362). Cytology : Inversion id chromosome 1 confirmed cytologically (Barry). Origin: Em a, W. Recognized aberrant by Perkins ; genetic analysis by Turner and Taylor. Generates viable duplications from intercross with In(IL + IRIH4260, and these are A / a heterozygotes which are phenotypically inhibited Dark-Agar types. Break points and genetic behavior resemble In(IL + IR)ARlG. The structure is formally similar to In(lLR)scv’ in Drosophila. FGSC 1613. Reference: Turner et al., 1969. Duplications: Dp(IL + IR)NMlYG. About one-fourth of viable progeny from I n x N . Phenotype nearly wild, but with characteristic growth habit of aerial hyphae. Barren in crosses with nonduplications, eventually becoming fertile by loss of one duplicated I L segment, usually but not always that in the translocated position. Occasionally, duplication may be transmitted to progeny without breakage. Instability increased by mei-3 (Newmeyer and Galeazzi, 1976b). Markers shown covered: fr, un-5, leu-3, cyt-1, cys-6, ser-3. Markers shown not covered: un-3, mt, sue.

T(III;V)AR177

Reciprocal translocation. I11 (linked acr-2) interchanged with V (near at, probably VR). Wild phenotype. Homozygous crosses produce only a few spores. T x N ascospores > 50% black; unordered asci 37:16:34:4:9 ( N = 153). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Apparently one duplicationdeficiency class consists of inviable black ascospores. Germination of black ascospores is 66%. No viable duplications recovered. FGSC 2680, 2681.

T ( I I + I)NM177 Insertional translocation. A segment of I I R between aro-3 and aro-1 is inserted in IL between leu4 and mt. Subtle morphological phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 28:41:29:1:1 ( N = 179). Origin: E m a, UV. Discovered and linkage groups identified by Perkins. Mapping and genetic analysis by A. Kruszewska. Separated with difficulty from closely linked morphological factor originally present. FGSC 1610, 2003. References : Perkins, 1974; Metzenberg et al., 1974; Littlewood et al., 1975. Duplications: Dp(IIR + IL)NM177. I n one-third of surviving progeny from T x N . Duplications are highly stable but sectoring may occur, with loss of material usually from the inserted location (Metzenberg et al., 1974). Vegetative “escapes” are still barren. Markers shown covered: peon (nuc-Z), pe, arg-12. Markers shown not covered: aro-3, aro-I. T(VI1 + IV)ALS179 Translocation, probably quasiterminal. An unmarked distal segment of VIIL is translocated to IVR near uvs-2. Wild phenotype. Homozygous-fertile. 2’ X N ascospores 75% black; unordered asci 18:66:13:2:1 (N = 141). Origin: c r r g a, UV. Detected and analyzed by Perkins. FGSC 2264,2265.

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Duplications: Dp(VIZL + IVR)ALSl79. In one-third of surviving progeny from T x N . Wild phenotype; ecorable as barren. Markers shown covered: None. Markers shown not covered: het-e, nic-3, thid.

T(11+ [IV;V] )AR179

Complex insertional translocation. A IIL segment is inserted, with break points closely linked to pdx (IV) and at (V) in T x N crosses. Near-wild phenotype, slightly less vigorous. Homosygous-barren. Forms perithecia but no ascospores. T x N ascospores < 50% black; unordered asci 0:5:9:34:52 ( N = 65). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2595,2596.

Note added in proof: The short arm of chromosome 2, which carries the nucleolus organizer, is clearly mvolved cytologically. Duplications: Dp(ZIL + [ZV;Vl)AR17Q.In one-third of surviving progeny from T x N . Markers may come uncovered somatically. Barren in crosses. Some are fully stable. Markers shown covered: pyr-4, het-c, rod, thr-2. Markers shown not covered : arg-6.

T(1;IV)ARlSOb

Reciprocal translocation. I (linked m t ) interchanged with IVR (near col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 42:6:29:4:19 ( N = 195). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Separated from T(ZR;IZIR)AR180r, which was present in the same isolate. FGSC 2754, 2755.

T(I;III)AR180r

Reciprocal translocation. I R (linked aur) interchanged with IIIR (linked trp-I). Wild phenotype. T x T crosses show reduced fertility. T x N ascospores 50% black; unordered asci 13:0:55:4:28 ( N = 127). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Separated from T(I;IVR)ARlBOb, which waa present in strain of origin. FGSC 2939, 2940.

T(1I;V)NMlSO

Reciprocal translocation. I I R (near arg-12, to right) interchanged with VR (between lys-1 and inl). Flat vegetative morphology. T x T crosses sterile with no perithecia. T x N ascospores 50% black; unordered asci 25:9:41:4:22 ( N = 69). Origin: Em a, UV. Detected and analyzed by Perkins. FGSC 2031, 1491.

T(1I;VI)ARlSl

Reciprocal translocation. IIR (linked bal and fl) interchanged with VI (linked ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores CO% black; unordered asci 21:7:42:11:20 ( N = 189). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2453, 2454.

T(1;IV)NMlSl

Reciprocal translocation. I (linked m t ) interchanged with IVR (near cot-1 1. Wild phenotype. Homosygous-fertile. T x N ascospores 50% black ; unordered asci 37:2:35:1:24 (N = 241). Origin: Em a, W. Detected and analyzed by Perkins. FGSC 2933, 2934.

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T ( I + )ALS182 Duplication-generating rearrangement, incompletely analyzed but probably with inverted insertion. A breakpoint in I R is between thi-1 and cyh-1. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black; unordered asci 29:37:26: 4:5 ( N = 164). Cytology: Bridges and fragments observed a t meiotic divisions, but abnormal chromosomes not identified. Origin: r g cr a, UV. Detected and analyzed genetically by Perkins. Note added in proof: The IR segment of ALS182, which includes meld, is translocated to the tip of VL, distal to the nucleolus organizer. Viable duplications covering nic-2, cr, and thi-l are produced in crosses of ALSl82 x AR190. Duplications: D p ( I R += )ALS182. In one-third of viable progeny from T x N . Vegetative morphology of duplications is perhaps flat initially. If barren, only fleetingly; quickly become fertile and behave as normal sequence. When gene 1c is heterozygous, both R and R' ascospores result. When a1 is heterozygous, i t sectors vegetatively. Markers shown covered : cyh-1, 01-2, al-1, R, un-18. Markers shown not covered: thi-2, fr, un-6.

T( I;VI)AR182

Reciprocal translocation. I (linked cr) interchanged with VI (linked ylo-I). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 37:8:29:9:17 ( N = 194). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2597. 2598.

T( III;V)NM183 Reciprocal translocation. I11 (linked acr-2) interchanged with V (linked at). Wild phenotype. Homozygous barren. T x N ascospores > 50% black; unordered asci 45:11:29:4:11 ( N = 160). Origin: Em a, UV. Detected and analyzed by Perkins. Intercross shows not identical with T(1ZZ;V)N M l O l . FGSC 2633,2634. T( V;VI)AR184 Reciprocal translocation. V (near a t ) interchanged with VI (near y20-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 35:13:20:8:24 (N = 143). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 2416, 2417. T( III;VI)AR186 Reciprocal translocation. I11 (linked acr-2) interchanged with VI (linked Y~O-1). Wild phenotype. (Slightly flat on synthetic cross medium.) Homozygous-fertile. T x N ascospores < 75% black; unordered asci 44:12:26:6:12 ( N = 173). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Evidently some inviable duplication-deficiency ascospores darken. FGSC 1925, 1926. T( I;IV)NM187 bs See T ( 1 ; I V ) N M I S Qbs. T ( I + V)ARlSO Quasiterminal translocation. Nearly the entire long arm of I, with all known I R markers except un-2, is attached 35 units left of centromere in V. Wild phenotype. Homozygous-fertile. T x N ascospores 75% black or more; unordered asci 32:29:

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31:5:3 (N = 211). Cytology: Break point in chromosome 1 at a or b chromomere, and in 2 in satellite distal to the nucleolus organizer. For pachytene cytology see Fig. 3 in Barry and Perkins (1969). Origin : 74-OR23-1A, W. Detected and analyzed genetically by Perkins. FGSC 1951, 1952. References: Barry and Perkins, 1969; D. E. A. Catcheside, 1969. Duplications: D p ( I R + VL)ARIQO. One-third of viable progeny from T x N . Duplications grow slowly after germination from ascospores. Somatic sectoring is often seen when albino is heterozygous. Duplications break down somatically and premeiotically to give normal sequence by complete loss of the I R segment from the translocation sequence. Barrenness is transient, making duplications difficult to score. The duplication is not transmitted to progeny. D. E. A. Catcheside has used ARl90 to test complementation among albino mutants. Markers shown covered: his-2, nuc-1, me-10, ad-3A, nic-2, cr, cyh-1, aG2, al-1, R . Markers shown not covered: un-2, sn, urg-1, m t . (Information on nuc-1 from R. L. Metzenberg.)

T(I;IV)AR193

Reciprocal translocation. I (linked m t ) interchanged with IV (near p d z ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 41:9:26:5:20 (N = 189). Origin: 74-OR23-1A, W. Detected and analyzed by Perkins. FGSC 2470, 2471.

T(IV;VI)AR207

Reciprocal translocation. IVR (linked pan-1, cot-1) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black (variable); unordered asci 40:9:18:8:24 ( N = 312). Origin: 74-OR23-1A, W. Detected and analyzed by Perkins. FGSC 1927, 1928.

T ( 1;III ) AR208 Reciprocal translocation. I R (right of cr) interchanged with I11 (near acr-2). Wild phenotype. Homozygous-fertile. T x N ascospores 75% black or more; unordered asci 48:21:25:2:4 ( N = 468). Origin: 74-OR23-1A, W. Detected and analyzed by Perkins. Evidently one duplication-deficiency class makes inviable black ascospores. Germination is low among black ascospores. No viable duplications. FGSC 1929. 1930.

T(VI + IV)AR209

Quasiterminal translocation. Right arm of V I with p u n 4 and distal markers translocated tc iight end of IV. Wild phenotype. Homozygous-fertile. T x N ascospores < 75% black; unordered asci 16:33:28:10:13 ( N = 162). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Used as het.9 tester to study vegetative incompatibility. FGSC 1931, 1932 (het-goR).Reference : Mylyk, 1975. Duplications: D p ( V I R + IVR)AR209. In one-third of surviving progeny from T x N . Wild phenotype. Not detectably barren in crosses to non-duplication; duplications behave like fertile normal sequence, apparently by rapid loss of segment in T sequence. Markers shown covered: pun-2, trp-2, het-9, probably rib-1. Markers shown not covered: chol-2, lys-6, ylo-1, ad-1.

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T ( III;IV)AR211 Reciprocal translocation. I11 (near acr-2) interchanged with IV (linked col-4, probably left). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 55:5:33:2:5 ( N = 677). Cytology: Preliminary observations of pachytene chromosomes have indicated abnormalities of 3 and 4. Origin: 74OR23-1A, UV. Detected and analyzed by Perkins. Apparently many duplicationdeficiency ascospores darken. No barren progeny are found, and germination is usually low among black ascospores. FGSC 1933, 1934. T(I;IV)AR212 Reciprocal translocation. IR (near al-1) interchanged with IVR (between pdz, m a t ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 26:2:50:3:19 ( N = 124). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. FGSC 1521, 1522. T ( I;II)AR216 Reciprocal translocation. I R (proximal to a,!+?) interchanged with IIL (between pyr-4 and bal). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black; unordered asci 44:8:16:0:32 (N = 262). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Original isolate also contained a linked but separable albino mutant. FGSC 1950, 1607.

T(I + ;II;VII)AR217

Complex insertional translocation. A central segment of IR, including ad-9 and nic-1, is inserted elsewhere. Other breakpoints are in I1 (near arg-6) and VII (near w c ) . Wild phenotype. Homozygous-barren. T x N ascospores 5070black or more; unordered asci 46:10:13:5:27 ( N = 166). Origin: 74-OR23-1A, W. Detected and analyzed genetically by Perkins. FGSC 2418.

Duplications: D p ( I R + ; I I ; V I I ) A R 2 1 Y . In one-third of surviving progeny from T x N . Slow t o conidiate, pale pigment. Partially barren, with few ascospores shot from perithecia. Markers shown covered. ad-9, cyh-1, al-1, nic-I. Markers shown not covered: his-2, cr, 0s-1.

T ( IV;V)AR221 Reciprocal translocation. IVR (near COZ-4) interchanged with VR (between a t and ilv). Wild phenotype. Homozygous-fertile. T x N ascopores 50% black; unordered asci 37:6:8:7:43 ( N = 183). Origin: 74-OR23-1A, UV. Detected and analyzed by Perkins. Strain of origin also contained linked but separable mutation, ilv (ARBl). FGSC 2034, 2035.

T(I;VI)Y234M419

Reciprocal translocation. I (near m t ) interchanged with VI (between Yl0-1 and trp-2). Wild phenotype. Homozygous-fertile. Translocation ascospores are slow t o blacken. T N ascospores < 50% black because of slow ripening. Unordered asci 19:4:25:8:44 (N = 167). Origin: 74A, UV. Detected and analyzed by Perkins. Strain of origin also contained closely linked point mutant ad-1 (74A-Y234-M419). FGSC 2635, 2636.

x

T ( II;IV)Y256M230 Reciprocal translocation. I1 (near arg-5) interchanged with IV (near Col-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci

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45:5:11:5:33 ( N = 150). Origin: 74A,W. Detected and analyzed by Perkins. Strain of origin also contained unlinked point mutant ylo-2. FGSC 917,1556.

T( I;IV)D304 Reciprocal translocation. I (linked mt, probably right) interchanged with IV (between cys-10 and trp-4). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 46:3:11:3:36 (N = 351). Also 50:4:19:1:26 (N =102Kowles). Intercross with T(ZR;IVR)NMl?Z gives ascus patterns expected if viable duplications are generated, but no such duplications were recovered. Origin : inl (89601), W.Detected by Perkins. Mapped and analyzed by Anna Kruszewska, further data by Kowles. Strain of origin also contained closely linked but separable morphological mutant. FGSC 1443,1444.Reference : Kowles, 1972, 1973. T( III + jIII;VI)D305 A complex translocation involving IIIR distal to trp-1 and VIL near chol-2. Generates viable duplications of a I11 segment. Wild phenotype. Homozygousbarren. T X N ascospores < 50% black; unordered asci 1:8:49:26:17 ( N = 211); T progeny are recovered less frequently than N . Origin: inl (89601), W. Detected and analyzed by Perkins. Used as het-7 tester to study vegetative incompatibility. FGSC 2139,2140. Reference : Mylyk, 1975. Duplications: Dp(ZZZR + )0306. From T x N. Slow or fluffy-like morphology. Variable fertility. Semibarren. Identification difficult, frequency uncertain. 3: 1 segregations are not excluded as the cause of some duplications. Markers shown covered: phe-2, tyr-1, dow, het-7; vet ( 1 ) . Markers shown not covered: acr-2, thi-2, trp-1.

T( IV + I)B362i Duplication-generating translocation. A segment of IV (near p d z ) is translocated to I (linked mt). Wild phenotype. Homozygous fertility not tested. T x N ascospores 80% black; unordered asci 61:20:16:3:0 ( N = 148). Origin: STA, gamma rays. Aneupoidy noted by D. Newmeyer. Extracted, diagnosed, and I linkage shown by D. A. Smith. Originated in same strain with unlinked T(ZZ;VZ)B362r and arg-10 (B362). FGSC 2935. Duplications: Dp(ZV+ Z)B362i. I n one third of viable progeny from I' x N. Stably barren. Markers shown covered: None (only p d z tested). Markers shown not covered: p d z .

T( IIjVI)B362r Reciprocal translocation. I1 (linked bal) interchanged with VI (linked ylo-1). Wild phenotype. Homozygous-fertile. T X N ascospores 50% black ; unordered asci 23:10:31:16:20 (N = 260). Origin: STA, gamma rays. Extracted and analyzed by D. A. Smith. Strain of origin also contained unlinked T(ZV + Z)B362i and arg-10 (B362). T( IVjVII)STL384b Reciprocal translocation. IVR (near col-4 and to right) interchanged with VIIR (near sk). Wild phenotype. T x T sterile with no perithecia. T x N ascospores 75% black; unordered asci 23:42:28:4:3 ( N = 803). Evidently one duplication-

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deficiency class makes inviable black ascospores. Germination among black ascospores is low. No viable duplications. Origin: Detected by P. St. Lawrence in a single aberrant perithecium from a cross mi-1 384a 9 (maternal inheritance) x 74-ORB-1A 8 . Ascus of origin also contained linked but separable T(ZV;VZ) STL384r. Rearrangements resolved and mapped by Perkins. FGSC 2421, 2422.

T( IV;VI)STL384r Reciprocal translocation. IVR (near cot-1) interchanged with VI (linked y b - 1 ) . Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 25:6:53:4:12 (N = 362). Origin: Detected by P. St. Lawrence in a single aberrant perithecium from a cross mi-1 384a 0 (maternal inheritance) x 74-0R231A 8 . Ascus of origin also contained T(lV;VlZ)STLS84b.Rearrangements resolved and mapped by Perkins. FGSC 2419,2420. T ( V;VI ) A420 Reciprocal translocation. V (near a t ) interchanged with VI (near yb-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 55:2:9:0:34 ( N = 170). Origin: Em a, UV. Detected and analyzed by Perkins. Original isolate contained linked but separable mutant trp-6 (Ahmad et al., 1968). FGSC 2334, 2335. T(I;VII)TM429 his3 Reciprocal translocation. I R (at his-!?) interchanged with VII (near met-7). Phenotype his-!?; all three activities are missing. Wild morphology. Homozygous-fertile. T x N ascospores 50% black, or more; unordered asci 30:9:23:7:31 (N = 627). Origin: 429 cot-1 a, W. Translocation identified and his-3 association shown by Angel. VII breakpoint mapped by Perkins. Inviable duplication-deficiency spores darken with aging. FGSC 2530, 2531. References: Angel et al., 1970; Angel, 1971; Catcheside and Angel, 1974. T( I;VI)P649 Reciprocal translocation. I R (near aur) interchanged with VI (near ylo-1). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 33:3:50:5:10 (N = 224). Origin: Spontaneous in backcross of cr thi-1 nit-1 aur nic-1 as-1 a x STA4. Detected and analyzed by Perkins. Generates viable duplications from intercross with T(ZR;VI)ARlS.FGSC 1608, 1609. T( I;VII)S1007 Reciprocal translocation. I (near his-$) interchanged with VII (near met-7). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black ; unordered asci 58:5:2:2:33 ( N = 205). Cytology: Reciprocal translocation confirmed. Break points are in the long arm of chromosome 1 and near the middle of chromosome 7 (Barry). Origin: Detected by Perkins in as71 strain S1007-2(1-4)a (unlinked asn mutant was X-ray induced). Generates viable duplications from intercross with T(I;VII)K79 met-7. FGSC 227, 224. References: Barry, 1967; Perkins el at., 1962.

T(III;V;VII)P1156

Complex interchange, Break points near leu4 (IIIR), al-3 (VR), thi-3 (VIIL). Wild phenotype. Homozygous-fertile. T x N ascospores < 50% black; unordered

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asci 14:1:49:19:18 ( N = 125). Origin: Detected among progeny of a cross n i c J roS, and analyzed by Perkins. FGSC 2599,2800.

x

Duplications: About one-fifth of progeny from T x N are stably barren. Their constitution is not understood. Failure to obtain consistent marker ratios among barrens suggests origin by nondisjunction (3: 1 segregations).

T(IV + VII;I;II;IV)S1229 arg pe

Complex translocation. Interchange points in IL (near arg-3 and m t ) ; in I I R at p e ; in IVR, with markers between pdx and p y r S inserted into VII near thi-3. Arms of groups I, 11, and IV translocated in progressive interchange. Inseparable from pe, which was present in the stock of origin, and from requirement for arginine, citrulline, or ornithine, which arose simultaneously with the aberration. Homozygous-barren. A few ascospores are produced, however. T x N ascospores 40% black; unordered asci 24:2:27:10:36 ( N = 704). Cytology: Initial cytological examination (1960) found chromosomes 1, 2, 6, and 7 aberrant. However, chromosome 2 was later shown not to be involved (1969). Origin: pe fl Y8743-21-(13-7)a, X-rays. Identified as aberrant by Barratt and Garnjobst. Genetic and cytological analysis by Barry. FGSC 2946, 268. References: Barratt et a l , 1954; Barry, 1960a,b; Barry and Perkins, 1969. Duplications: D p ( I V R + VII)S1229. One third of viable progeny from T x N . Wild, but grows at slower rate. Crosses with either duplication or non-duplication strains are almost barren, producing very few spores. Duplications are stable through crosses, and segregate 1:1 in progeny from D p x N . Markers shown covered: p t , met-1, cys(oxD'), col-4, arg-2. Markers shown not covered: pyr-1, pdx, pyr-3, h i s d , trp-4, pan-i, cot-1, m e t d , his-4, pyr-2. (FGSC 264, 265 are Si229 Duplications.)

T(I S V)S1325 nic-2

Insertional translocation. A long central segment of I R is inserted in inverted order into VR between his-1 and inl. No viable duplications are produced; thus a short mutual VR + IR insertion is postulated. The rearrangement is inseparable from the nic-2 phenotype with which it arose. Wild morphology. Homozygousfertile. T x N ascospores 50% black or less; unordered asci 5:0:71:3:21 ( N = 223). Cytology : Singleton observed acentric chromosome fragments and dicentric bridges in the meiotic divisions in the ascus and concluded a paracentric inversion was present, thus confirming St. Lawrence's genetic interpretation of an inversion in linkage group IR. Further genetic and cytological investigation by N. E. Murray, Perkins, and Barry showed the aberration to be actually an inverted insertion of a long segment from I R (chromosome 1) into VR (chromosome 2). The acentric fragments persist in micronuclei and replicate. Origin: pe fl Y874321(13-7)a, X-rays. Detected and I involvement analyzed by St. Lawrence. V involvement shown by Perkins and Murray following evidence of Newmeyer that another chromosome was involved. The inserted segment includes thi-1, met-6, ad-9, a M , and al-1. FGSC 1558, 1557. References: St. Lawrence and Singleton, 1963; Murray, 1968a; Barry and Perkins, 1969; Barry, 1973.

T(1;V)C-1670 pk-1

Reciprocal translocation. IR (near centromere) interchanged with VR (at bis) . Phenotype pk-1 ( p e a k 4 morphology (allele of bis) . Homozygous-fertile. T x

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N ascospores 50% black; unordered asci 33:3:46:3:15 ( N = 270). Cytology: Reciprocal translocation involving chromosome 2 shown by Phillips. Origin : 74A, p propiolactone. Detected and linkage groups identified by J. C. Murray. Generates viable duplications from intercrosses with T ( I R;VR )36703 and ALSIII. FGSC 483, 2761. References: J. C. Murray, 1959; Phillips, 1967; Perkins, 1971a.

T(I;VII)P1676

Reciprocal translocation. I (linked m t ) interchanged with VII (near W C ) . Wild phenotype. Homozygous fertile. T x N ascospores 50% black; unordered asci 61 :8 :10: 1 :20 ( N = 277). Origin : Spontaneous in cross 74-OR8-la x trp-I. Detected and analyzed by Perkins. FGSC 1935, 1936.

T ( V;VI) JH2003

Reciprocal translocation. V (linked at) interchanged with VI (near ylo-I). Wild phenotype. Homozygous-fertile. T x N ascospores > 50% black (probably because some defective white spores disintegrate) ; unordered asci 53: ll :16:3:16 (N = 220). Origin: Detected by Perkins in nit(JH.2003) (Silver and McElroy, 19541, which was obtained by FGSC from J. R. S. Fincham. Freed of nit mutation. FGSC 2423,2424.

T ( 1V;V) R2355

Reciprocal translocation. IVR (near cot-I) interchanged with VR (proximal to his-I). Wild phenotype. Homozygous-fertile. T x N ascospores 50% black; unordered asci 17:0:65: 1:17 ( N = 155). Cytology: Cytological examinations by R. L. Phillips and by J. R. Singleton (unpublished) confirmed the reciprocal translocation, and found chromosome 2 involvement. Origin: inl (896011, UV. Detected and analyzed genetically by Perkins et al. A component of alcoy linkage tester. Generates viable duplications from intercrosses with T(IV;V)ARIIT,NM141, and NM146. FGSC %I, 222. References: Phillips, 1967; Perkins et al., 1962, 1969; Perkins, 1971a.

T(I1 + IV)R2394

Insertional translocation. A I1 segment near bal (probably IIL) is translocated to IV (linked pdx). Wild phenotype. Homozygous-fertile. T x N ascospores > 7570 black; unordered asci 31:49:17:3:0 ( N = 121). Origin: inE (89601), UV. Recognized aberrant by Garnjobst and Tatum (1967). Analyzed by Perkins. FGSC 2757, 2758. Duplications: D p ( I I + I V ) R 2 9 4 , present in one third of viable progeny from T x N . Barren. Markers shown covered: None. Markers shown not covered: bal,

Pyr-4,

11.

T( + I)R2472 arg

Duplication-generating translocation. A segment of unknown constitution is translocated to I (linked m t ) . Flat vegetative growth; not separated from a requirement for arginine or aspartate or asparagine. Not homozygous-fertile. T x N ascospores 50% black; unordered asci 4:30:23:26:17 ( N = 100). Origin: inl (89601), W. Recognized aberrant by Garnjobst and Tatum (1967). Preliminary analysis by Perkins.


di#lycarida choline phonphotnnrferasa

CHiOR

R’O$H

1.2-Diglyceridc

CHiOH

1.7.8.1

CMP

FHiOR R’OfH

7

+

CH@--P--OCH~CH~N(CH,)3

I

OH Phoophrrldylcholine (Lecithin)

FIG.5 (continued)

of the cell wall. Mahadevan and Mahadkar (1970a) have described that, in a particular morphological mutant called spco-I, the earlier and higher frequency of branching was correlated with a greater amount of these autolyzing enzymes as compared to wild-type strains (see Table 3). These authors have described both glucanase and protease activity associated with the cell wall of hleurospora, which can be extracted with p-mercaptoethanol. The activity of these enzymes in the mutant was almost 6 times higher than that found in the wild-type strain (Mahadevan and Mahadkar, 1970a). Of these two enzymes the activity of proteases was found to be much higher than that of the glucanase. The fact that the increased activity of proteases in spco-I lead to morphological changes suggests the role of cell wall peptide in morphogenesis (Wrathal and Tatum, 1974). Abramsky and Tatum have recently described changes in the morphology of a particular mutant strain (smco-9) due t o the inactivation of an enzyme commonly called “branching enzyme” (a-lj4-glucan: a-1,4glucan-6-glycosyltransferase, EC 2.4.1.18), which catalyzes the synthesis of a-1,4 linkages during the biosynthesis of polysaccharides. The authors have characterized a thermolabile inhibitor (MW = 100,000) from the

368

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C. MISHRA

mutant strain which caused a drastic reduction in the activity of the branching enzyme. In the mutant cell extract the activity of the branching enzyme could not be detected unless the inhibitor was removed by DEAE chromatography. The inhibitor, which was present in the mutant strain only, was found to affect the activity of other enzymes of polysaccharide biosynthesis. The branching enzyme characterized from the mutant and wild-type strains had a molecular weight of 140,000 and was similar in other physicochemical properties. These authors have further found that the inability of the mutant to synthesize a-1,6 linkages due to reduced activity as branching enzyme could be overcome, however, by adding sugars (having such ~ 1 , link6 ages) to the growth medium as shown by the wild-type-like growth of the mutant strain on media containing isomaltose or potato starch as the sole carbon source (see Table 4) (Abramsky and Tatum, 1976). I n the morphological mutant called snowflake (sn),the biochemical defect seems to involve cellular accumulation of microfilaments 78 A in diameter and up to several micrometers in length (Allen et al., 1974). The microfilament in this mutant strain (sn) seems to run in all directions TABLE 4 Differences in the Growth Characteristics of Different Neurospora Strains on Different Sugars as Sole Carbon Source Morphology and growth pattern on media with ~~

Strains

Glucose

1. Wild type 2. gal-1

Wild typeWild type

3. gab8

Wild type

4. smco-8

Wild type

5. smco-9

Colonial

6. Osmotic mutants (R2361, R2473)

Wild type

Galactose

~~

Iomaltose

References

Colonial __ Rand (1975) Colonial or no Rand (1975) growth Rand (1975) Colonial or no growth Rand (1975) Colonial or no growth Wild type Abramsky and Tatum (1976) Wild type or Rand (1975) even better growth

The spreading mycelial growth characteristic of the wild-type strain (RL3-8A) ati seen on glucose medium. smco-9 was found to have colonial morphology on glucose, sucrose, and maltose but wild-type-like morphology on potato starch and isomaltose (Abramsky and Tatum, 1976).

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GENETICS OF

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(see Fig. 2B) and sometimes up to the plasma membrane and even inside the nuclei. According to Sussman and his collaborators, the altered morphology of snowflake is related to accumulation of these microfilaments which may affect several physiological processes. Microfilaments are known to control cytoplasmic streaming (O’Brien and Thimann, 1966), and also morphogenesis of animal cells (Wessells et al., 1971). Wood and Luck ( 1971) have reported the occurrence of “paracrystalline inclusions’’ in the mitochondrial mutant (mi-1) of Neurospora or induction of similar microfilaments in the wild-type strain when ethidium bromide or euflavin was added to the growth medium. According to Wood and Luck (1971), these “paracrystalline inclusions” are not products of a mitochondrial gene, but their cellular crystallization is due to a mitochondrial defect. Another interesting mutant is strain 4520% isolated by Houlahan et al. (1949) and described by Dubes (1953) as a temperature-sensitive mutant that required leucine for growth a t low and intermediate temperatures; however, a t high temperature (37°C) its growth was severely restricted regardless of leucine supplementation ; it was designated leu-5. The biochemical lesion of this mutant has been elucidated by Gross and his collaborators a t Duke University (Printz and Gross, 1967; Weeks and Gross, 1971). According to these authors, a number of proteins synthesized during the growth of this mutant (4520%) were structurally altered and enzymically defective (some of the enzymes examined were trytophan synthetase, alcohol dehydrogenase, NADase, and 5-dihydroshikimic reductase, all of which were rendered more thermolabile in the mutant). On the basis of these results authors concluded that the phenotype of strain 4520% resulted from a decrease in the fidelity with which leucine is incorporated into protein due to a defective leucyl-tRNA synthetase with altered binding properties. This conclusion was supported by their finding that the mutant strain 4520% lacked the mitochondrial species of this enzyme whereas the cytoplasmic enzyme was found to have reduced affinity for leucyl tRNA (Weeks and Gross, 1971). Rand (1975) has attempted to identify the primary biochemical lesions in a number of mutants based on their growth characteristics on galactose or glucose as a carbon source. As discussed earlier he has shown that the wild-type strain of Neurospora grew as tightly restricted pellets in galactose medium as opposed to its filamentous growth in glucose medium. He has tested a large number of morphological mutants described earlier by Garnjobst and Tatum for their ability to grow on galactose. Among the mutants tested only semicolonial-8 (snico-8) was found to be unable to grow on galactose (or grow as restricted colonies) whereas all the osmotic mutant strains tested were found to grow better on galactose than on glucose. Rand has also described two new mutations, gal-1 and gal-2, that were

3 70

N. C. MISHRA

unable to grow on galactose (or grew as restricted pellets). Although he could not identify the primary biochemical defect of the semicolonial-8 or the osmotic mutations, he found that gal-1 and gal-2 were defective for the enzymes galactokinase and UDPG pyrophosphorylase, the enzymes of the Leloir pathways (Leloir, 1951). I n the gal-1 mutant galactokinase specific activity and its heat stability were much reduced although there was no change in K , values of the mutant enzyme as compared to the wild type (Rand, 1975). The gab2 enzyme (UDPG pyrophosphorylase) has reduced specific activity, altered Iilr,sand lowered heat stability (see Table 3 ) . On the basis of these data, the author has suggested that gal-1 and gal-2 may represent mutation in the structural genes for the enzymes galactokinase and UDPG pyrophosphorylase, respectively. Furthermore, the g a l 4 mutant seems to respond to arginine supplementation, Thus the behavior of gal-2 seems to be similar to that of the mutant described by Houlahan et al. (1949), which has been elucidated by Gross and his collaborators (Printz and Gross, 1967) ; Weeks and Gross, 1971). Therefore, the arginine response of gal-2 can be explained on a similar basis: i.e., gal-2 mutation may cause a change in the fidelity of the translation mechanism. In case of the mutants listed in Table 4, although the specific activity of the enzymes involved is lowered owing to genetic mutations, it is not yet known whether or not this is due to changes in the structure of the enzymes involved. I n all these cases of enzyme defects the unequivocal proof (see Tables 1-3) for a change in the structure of an enzyme could be established only through amino acid sequence analysis of the polypeptide in question. However, the changes in the physical parameters (heat stability, electrophoretic, and K,,, studies) and the effect of reversion and suppressor mutation do indicate that the enzyme defect a t least in PGM, GGPD, and 6PGD mutant is due to a structural change. However, no such data are available for a large number of morphological mutants (see Table 4). b. Genetics and Biochemistry of the Phenocopy of Genetic Mutants. Chemical induction of morphological changes provides another interesting and useful approach to the understanding of colonial morphology in Neurospora. Tatum, Barratt, and Cutter were the first to induce colonial growth in Neurospora by the addition of L-sorbose to the medium (Tatum et al., 1949). Their discovery was very useful, as it led to the development of a methodology for high-resolution genetics of Neurospora besides its role in the understanding of morphogenesis in Neurospora. Soon it was demonstrated by Stadler (1959) that this sorbose effect was genetically controlled ; Stadler described a particular mutation called patch, which can grow as spreading filaments even in the presence of sorbose. The sorbose-grown culture of the wild-type strain (with colonial morphology)

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was found to have altered cell-wall composition (deTerra and Tatum, 1963; Mahadevan and Tatum, 1965). Mahadevan and Tatum showed that in the sorbose-grown culture, there was a decrease in the level of fraction I11 with a concomitant increase in the level of fraction I. Such an increase in the amount of fraction I in a sorbose-grown culture of the wild-type Neurospora has been visualized by scanning electron microscopy (Mishra, 1975). SEM studies by Mishra have shown the presence of several facetlike structures on the surface of the sorbose-grown culture whereas the normal culture showed a smooth surface. The chemical analysis of the cell wall of the patch mutant by Mahadevan and Tatum revealed no significant changes in its cell wall composition when this mutant (patch) was grown in the presence or in the absence of L-sorbose. Likewise, the surface architecture of the mutant strain (patch), when examined by SEM, was found to remain unchanged in the presence of sorbose (Mishra, 1975). Thus, the fact that sorbose cac cause a dramatic effect in the morphology, cell-wall con~position,and the surface architecture of the wild type but not of the mutant (patch) strain strengthens, the conclusion that the cell-wall composition is the main determinant of the morphology of Neurospora. Mechanism of sorbose effects. Crocken and Tatum (1968) have described that sorbose is taken up by the same transport system as that utilized by glucose. These authors have further provided evidence for the counterflow of sorbose in the presence of high concentrations of glucose in the growth medium. The fact that sorbose and glucose can be transported by the same transport system might explain the role of increasing concentration of glucose on the reversal of the sorbose effect. Scarborough has confirmed the presence of a common transport system for both glucose and sorbose in Neurospora, using spheroplasts or mutant ( s l ) in Neurospora (Scarborough and Schultz, 1974) lacking a cell wall. Later Mishra and Tatum (1972) showed that sorbose causes morphological changes in the wild-type strain by inhibiting the activity of polysaccharide synthetase in Neurospora (1972). Both in vivo and in vitro activities of the wildtype p-1,3-glucan and glycogen synthetases were found to be inhibited by sorbose whereas the activity of these enzymes from the mutant strains (patch) remains unaffected. The idea that a chemical which can interfere with the activity of the enzymes that synthesize the cell wall polymers was further investigated by examining the role of CAMP on morphogenesis. The rationale behind this approach was that excess of CAMP, which is known to regulate the level of carbohydrate synthetase, would lead to morphological changes. Mishra (1976) has observed such morphological changes in Neurosporu when CAMP was added to the growth medium (see Fig. 6). Such a para-

372

N. C. MISHRA

FIQ.6. The effect of cAMP on the morphology of the wild-type strain (RL 3-8A) of Neurospora crassa. The wild-type strain was grown on solid medium containing glucose (0.1%) without or with cAMP a t 25°C in 5-cm petri plates. (A) The wildtype strain showing normal morphology on medium without CAMP. (B) The wildtype strain showing a colonial morphology (with restricted growth) on a medium with cAMP (6 mg/ml).

morphogenetic effect of cAMP can also be elicited by its dibutyryl derivative, but not by 3’-AMP, B’-AMP, or 2’-3’-cyclic AMP (Mishra, 1976). Similarly, theophylline (an inhibitor of phosphodiesterases) should cause morphological changes by increasing the in vivo level of CAMP; such an effect of theophylline has also been observed (N. C. Mishra, unpublished; Scott and Soloman, 1975). The insensitiveness of patch to sorbose can be explained by either of the assumptions that ( 1 ) the uptake of sorbose in patch is decreased as a result of the mutation or (2) the enzymes of cell wall biosynthesis have been so changed as to become insensitive to sorbose. I n sorbose uptake using l*C-labeled sorbose, no significant difference was seen between the wild-type and the mutant strains (Mishra and Tatum, 1972). I n view of this fact, the latter hypothesis seems more plausible (i.e., the nature of the cell wall synthesizing enzymes has changed). Although, the sorbose uptake remains unimpaired in the patch mutant. Klingmuller has described a number of mutations that cause sorbose-resistance in mutant strains of Neurospora which appear to be impaired in the sorbose transport system and have decreased uptake of sorbose (Klingmuller, 1971). In these mutants a lipid component of the cell wall-membrane complex seems to be impaired. Recent work by Scarborough (1971) has led to a similar conclusion

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

373

regarding the involvement of a membrane component in the uptake of sorbose in Neurospora. Scarborough found that, in the inositol-requiring mutants of Neurospora, sorbose uptake was inhibited under limiting concentration of inositol in the growth medium (Scarborough, 1971). Recently, Murayama and Ishikawa (1973) have described a sorboseresistant mutation (T9) in Neurospora crassa with pleiotropic effects ; the pleiotropy of this mutation includes sensitivity to high osmolarity of the growth medium, altered elution profile of the enzyme glucoamylase on gel filtration, and a high activity of extracellular acid phosphatase. These authors have explained the pleiotropy of this sorbose-resistant mutation on the assumption that the mutation (T9)causes a primary alteration of the cell wall or membrane and that secondarily this leads to other effects. The fact that these authors found no difference in the cell wall composition of the wild type and the mutant (T9) excludes the idea of a change in the cell wall (Murayama and Ishikawa, 1973). Recently, Rand (1975) has initiated an exciting new aspect of Neurospora morphology by describing the induction of morphological changes in the wild-type strain by galactose. The wild-type strain was found to grow as tightly restricted colonies (Rand, 1975) when grown in a medium containing galactose as the sole carbon source. It has also been shown that the galactose effect was maximal in the presence of xylose, but glucose reversed the effect of galactose. The biochemistry of the galactose effect can be understood in terms of the inhibitory effect on certain enzymes of cell wall biosynthesis ; however, this remains to be elucidated. Mahadevan and Tatum (1965) have indicated the presence of galactosamine in fraction I of the Neurospora cell wall and a galactosamine polymer has been isolated from Neurospora cell wall (Harold, 1962). Recently, Mishra and Forsthoefel (1976) have described an enzyme in Neurospora that can synthesize a polymer of galactose. Reissig and his collaborators (Reissig and Glasgow, 1971 ; Glasgow and Reissig, 1974) have approached the problem of Neurospora morphogenesis in a novel way by examining the effect of a purified cell wall component on the growth of this mold. These workers have isolated a phasespecific mucopolysaccharide called MP from the wild-type culture of Neurospora. The M P is characteristically present in the growth medium of the wild-type culture at stationary phase. This mucopolysaccharide was found to inhibit growth and agglutination of Neurospora cells and was also to precipitate purified membrane protein. I n certain morphological mutants a phase of slow growth was markcd by the production of this mucopolysaccharide (Reissig and Glasgow, 1971). The M P is a polymer of galactosamine (galactosaminoglycan) and has a molecular wcight of approximately 1 x lo6; its biological activity can be abolished

374

N. C. MISHRA

by its complete acetylation. Polyphosphate was also found to bind MP and found to cause its inactivation in vitro. Harold has earlier described a polymer of galactosamine from Neurospora which binds with phosphates (Harold, 1962). On the basis of these characteristics of MP, Reissig and Glasgow have suggested its role in the regulation of growth and morphogenesis in Neurospora. Additional evidence is provided by the results of an experiment in which these workers have utilized the antagonistic effect of polyphosphate on M P produced by mutant cultures. At lower concentrations (50-400 pg/ml) polyphosphate was found to stimulate the growth of the mutant culture ( c o t ) . After 8 hours of growth the mutant (cot) which produced M P showed signs of agglutination. However, when the growth medium was supplemented with polyphosphate (100 pg/ml) the agglutination was prevented. These authors have also shown that M P can cause inactivation of conidia as evidenced by the inability of the treated conidia to germinate and to take up radioactive metabolites from the growth medium. Studies with radioactive M P showed that a t least lo” molecules of M P bind per cell to cause a lethal effect (Glasgow and Reissig, 1974). Thus the results of their studies clearly suggest a physiological role for M P and polyphosphate in the growth of Neurospora. The role of galactosamine polymer in determining the morphology of Neurospora is further revealed by the fact that certain morphological mutants have deficient levels of UDP-N-acetyl glucosamine-4-epimerase (Edson, 1972) ; this enzyme is necessary for the synthesis of galactosamine, a component of the hyphal wall (Harold, 1962; Mahadevan and Tatum, 1965). However, Schmidt and Brody (1975) have suggested that the galactosamine polymer cannot be a major shape determinant of Neurospora hyphae. This conclusion is based on their finding that the level of galactosamine was always much lower in the growing front than in the older region of the band ( b d ) mutant of Neurospora. This mutant strain ( b d ) possesses a circadian conidiation and shows zonation in its growth as band (conidiating) and interband (nonconidiating) regions (Sargent and Woodward, 1969), which has been found suitable for elucidation of morphogenesis and circadian rhythm as discussed later in this paper. c. Genetics and Biochemistry of the Suppressor of the Morphological Mutants. The study of suppressor mutations has been extremely useful in understanding the genetics and biochemistry of gene structure and function (Yanofsky, 1966; Gorini, 1970). Therefore, the study of such mutations has received much attention in the elucidation of the biochemistry of morphogenesis in Neurospora. The suppressor of morphological mutants was first described by Srb (1957). Srb found that a particular morphological mutant mel-3 usually reverts to the wild-type growth form,

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

375

and this change was found to result from a mutation in a gene unlinked to mel-3. Later Giles (1951) described several suppressors of the inositolrequiring (inos) mutants of Neurospora; inos strains were shown to have colonial morphology when grown on medium containing limiting amounts of inositol. Mishra and Threlkeld (1967) have described an allele specific suppressor of the ragged mutation in Yeurospora. The suppressor mutation su-2 could act only on rg-2, not on the rg-1 mutation. The double mutant rg-2-su-2 has a wild-type-like phosphoglucomutase in Neurospora (Mishra and Tatum, 1970a). Brody has isolated two suppressor mutations, su-B and su-C, which are allele-specific to the morphological mutants balloon and col-2, respectively (Scott and Brody, 1973). Both suppressors are unlinked to the mutations that they suppress. Since the primary defect in the morphological mutants balloon and col-2 was elucidated by Tatum's group a t Rockefeller University, it provided a unique opportunity to investigate the biochemistry of these suppressor mutations. Such biochemical studies were carried out by Scott and Brody. The double mutants su-B, bal and su-C, col-2 have a more spreading morphology and were found to have altered kinetic parameters of the enzyme involved (i.e., G6PD) (see Table 2.). The su-C mutation was found, however, to alter the nature of the enzyme G6PD even when present alone in the wild-type background (see Table 2 ) , although the su-B mutation by itself did not show any effect on the morphology. Both su-C and su-B were found to be dominant over their respective alleles su-C' and su-B' in the balanced heterokaryon. These authors have invoked several possible assumptions in order to explain the unusual behavior of the su-C gene in altering the properties of G6PD in the wildtype background. Some of these explanations are: (1) su-C is a structural gene for one of the subunits to GGPD enzyme. The mutation by itself does not impair the function of the enzyme to an extent that can lead to morphological change; however, this mutation su-C, when in combination with col-2 perhaps can overcome the impairment caused by the col-$2 mutation and thus cause the suppressor effect; (2) the su-C mutation may cause mistranslation of a codon other than the one altered by col-2; (3) su-C may code for an enzyme that can cause some posttranslational modification (DeLange et al., 1968; Kingdon et al., 1967) of G6PD; (4) su-C may be a regulatory gene that can control the appearance of a GGPD subunit. Future work is required to support one or another of these possibilities regarding the su-C function. Modifiers of crisp. Lindegren (1936) first described the crisp (cr)mutation in Neurospora; since then Garnjobst and Tatum (1967) have obtained 14 c ~ i s pstrains of independent origin. On the basis of linkage and complementation analyses, these authors found that crisp mutations were

376

N. C. MISHRA

in either of three closely clustered loci designated cr-1, cr-2, and 0-3 (their gene order being centromere-cr-1-cr-3-cr-2) (Garnjobst and Tatum, 1967). Garnjobst and Tatum have suggested that these three loci may be involved in a sequence of reactions biochemically related to the crisp phenotype; there is some indication that crisp morphology may be associated with defective functioning of the enzymes of the Krebs cycle. Garnjobst and Tatum have described genetic modification of the crisp phenotype controlled by five unlinked genes ; these mutant genes either merely modify the crisp phenotype (m-1, m-2, and m-3 genes) or completely suppress the crisp phenotype (m-4 and m-5 genes). The m-1 mutation shows no locus specificity and acts on all CT-1,cr-2, and m-3 alleles. It is not known whether other modifiers, especially m-4 and m-5, show some nonspecificity. Further work is required to elucidate the biochemical basis of the crisp mutants and their modifiers; it would be of interest to see the effect of these modifiers on the enzyme adenyl cyclase, since its level is reported to be drastically reduced in the cl-isp mutant (Terenzi et al., 1974). 2. Altered Enzyme to Altered Morphology and Pleiotropy

Data presented in the Tables 1-3 clearly establish the enzymic defect of a particular morphological mutant whose normal functioning is required for the maintenance of the wild-type morphology. The pleiotropic effects of altered morphology in the morphological mutants seem to arise from the primary enzyme defect due to a gene mutation. At first glance it does not seem obvious how an altered enzyme leads to an altered morphology. However, after a closer look, a meaningful relation between changes in enzyme and in morphology can be established. It seems that in all these cases the morphology is altered via a defect in cell wall or cell-membrane biosynthesis. a. MorphoEogical Changes via Direct Effects on Cell-Wall and CellMembrane Biosynthesis. This is obvious in the case of the PGM mutants, since PGM catalyzes an enzymatic reaction a t the branch point in carbohydrate biosynthesis as shown below: C-1-P -+ UDPG -+ glucan I

4

C-6-P

1

Glycolysis

Thus it can be conceived that a defective PGM would lead to a reduction in the level of cell-wall polymers. The ragged mutants have indeed been shown to have a reduced level of fraction I11 (p-1,3-glucan) (Mahadevan and Tatum, 1965; N. C. Mishra, unpublished)-a major component of

BIOCHEMICAL GENETICS OF

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MORPHOGENESIS

377

the Neurospora cell wall. Much insight has been obtained about the regulation of PGM in Neurospora (Mishra and Tatum, 1970a) by the studies of the ragged mutants. The study of the behavior of PGMI and PGMII a t different ionic concentrations of Mg2+showthat they exist as two distinct molecular species a t low Mg2+concentration whereas a t high Mg2+ concentration they interact to form a single complex molecular species with 2-fold increase in molecular weight and a change in the structural conformation of the enzyme. The result of gel filtration does provide evidence for such interaction in PGMI and PGMII (Mishra and Tatum, 1970a). Mishra and Tatum have also shown that in vitro mutant PGMI interacts with wild-type PGMII a t high Mg2+concentration, and the resultant enzyme complex has the properties of the mutant enzymes as determined by their increased thermolability and K m values for substrate (Mishra and Tatum, 1970a). No interaction between two isozymes as seen in Neurospora PGMs had been reported previously. Such an interaction between PGMI and P G M I I under physiological conditions would provide a regulatory mechanism for control of the production of the two isozymes in vivo. A common regulation of the two isozymes is evidenced by the fact that in the ragged mutants (although each is defective for only one form of P G M ) , the level of the wild-type-like isozyme is also significantly reduced. The fact that both PGMs are required for the expression of the wild-type morphology supports the idea that a complex PGM molecule is the physiologically active form of the enzyme. The nature of the agent promoting such a complex formation in vivo remains to be determined. The role of Mg2+ in vivo is uncertain since a high concentration is required for the in vitro interaction (Mishra and Tatum, 1970a). However, cations like MgZ+, spermine, spermidine, and Tris-HC1 are known to cause aggregation of yeast UDP-galactose-4-epimerase (Darrow and Rodstrom, 1966). The polyamine cations may be involved in vivo in such interactions of PGM (s) ; spermidine has been found to occur in Neurospora in abundance (Bachrach and Cohen, 1961) and has been found to stimulate the in vitro activity of PGM (N. C . Mishra, unpublished). The decrease in the cell wall polymer (p-1,3-glucan) in rugged mutants seems to be a direct consequence of the defective PGM. However, modification of glucan biosynthesis may also arise in part from the allosteric effects of the different phosphorylated sugars (such as G-6-P) which accumulate in significant excess in the mutants owing to altered P G M (Brody and Tatum, 1967b; N. C. Mishra, unpublished) as reported by Fiengold et al. (1958). Interaction of metabolic pathways has long been known to produce multiple effects (Chaikoff, 1953). Such morphological changes via a direct effect on biosynthesis of cell wall polymers is further

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N. C. MISHRA

evidenced by the study of sorbose; sorbose, which leads to the production of a phenocopy of the morphological mutation in the wild-type strain of Neurospora, also causes changes in the levels of cell wall polymers by inhibition of polysaccharide synthetases. The pleiotropy of the sorbose effect is not seen, however, in the mutant strain patch; none of the enzymes of cell wall biosynthesis in this mutant (patch) are affected by sorbose (Mishra and Tatum, 1972). Changes in cell membrane. Work with the choline- and inositol-requiring mutants of Neurospora has indicated the role of lipid metabolism and membranes in the morphogenesis of Neurospora (Crocken and Nyc, 1964; Pina and Tatum, 1967). The choline- and inositol-requiring mutants behave as conditional morphologicals, and the morphological alterations are caused only under suboptimal conditions of choline and inositol in the growth medium. Both choline and inositol are integral parts of the cell membrane, and a defective membrane formed under suboptimal conditions of these chemicals may disrupt the function of the membranebound enzymes in cell-wall biosynthesis. In both cases the inability of these mutant strains to synthesize a particular lipid has been related to particular enzyme deficiencies (see Table 1) (Crocken and Nyc, 1964; Pina and Tatum, 1967). The nystatin-resistant mutants of Neurospora with altered membrane composition (Grindle, 1974) may cause morphological changes in a similar manner by the disruption of the function of membrane-bound enzymes in cell-wall biosynthesis. However, the Neurospora mutants that require fatty acids for growth were found to have normal morphology although growth was much slower (Lein and Lein, 1949; Henny and Keith, 1971). b. Morphological Changes via Indirect Effect on Cell-Wall and CellMembrane Biosynthesis. The effect of a defective G6PD on the changes in the morphology of the col-2, balloon, and frost mutants is not as direct as that of PGM in the ragged mutants. It has been suggested that a defective G6PD would cause a reduction in the amounts of NADPH that are essential for the synthesis of the components of cell membrane. Therefore, it can be visualized that a defect in cell membrane due to reduced NADPH level in these mutants (col-2, balloon, and frost) might lead to improper functioning of the enzymes of cell wall biosynthesis since some of these enzymes are membrane bound. Much evidence for the above conceptual scheme of the pleiotropic effects of mutations in the pentose pathways has been provided by the work of Brody (1970, 1972). Brody has shown that in the G6PD and 6PGD mutants of Neurospora the levels of NADP and NADPH are reduced. Since NADPH is used primarily for synthesis of fatty acids and their desaturation, a deficiency in the cellular amount of NADPH would cause considerable reduction in the fatty acid biosyn-

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thesis; Brody and Nyc (1970) have found changes in the composition of unsaturated fatty acids in balloon, col-2, and col-3 mutants of Neurospora. However, supplementation with precursors of NADP (such as tryptophan and nicotinic acid) has failed to relieve the morphological abnormalities of col-2 mutants or to increase the level of pyrimidine nucleotide in either wild-type or the col-2 mutant (Brody, 1972). However, this may be explained on the basis of different nucleotide pools and their compartmentalization. The other possible manner in which the cell wall biosynthesis may be impaired in these mutants is by modification of the enzymes by phosphorylated sugars that accumulate in large excess in the mutants (Brody and Tatum, 1966; Scott and Tatum, 1970). Recently, Scott has found that frost, a G6PD mutant has also altered adenyl cyclase; this has created an apparent paradox since i t implies that the single mutation (frost) affects both unrelated enzymes, such as G6PD and adenyl cyclase (Scott and Soloman, 1974). This difficulty has been overcome by the assumption that the altered adenyl cyclase of the frost mutant is a secondary consequence of the G6PD defect mediated by a defective membrane. As discussed earlier in this section, these G6PD mutants have impaired membrane due to a reduced level of available NADPH. The fact that addition of linolenic acid to the growth medium can alleviate the severity of the morphological changes in the frost mutant and leads to reversal toward wild-type-like growth supports this assumption. It can further be argued that the reduced level of adenyl cyclase reported by Terenzi et al. (1974) in the crisp-1 mutant may also have arisen as a secondary effect of membrane impairment. c . Cyclic-AMP, the Ubiquitous Regulatory Molecule. The role of this ubiquitous molecule was conceptualized in view of the following facts: (1) The morphology of Neurospora is determined by the chemical composition of its cell wall, and the latter can be influenced by the regulation of the enzymes of cell-wall biosynthesis, which are controlled by cAMP (Traut and Lipmann, 1963). (2) The addition of cAMP to the growth medium can influence the growth pattern of the wild-type strain of Neurospora (Mishra, 1976) as seen in Fig. 6. Further evidence regarding the role of cAMP in morphogenesis has been reported. (1) Addition of theophylline to the growth medium can cause morphological changes in Neurospora. Theophylline is an inhibitor of phosphodiesterase and thus can lead to excessive accumulation of cAMP in vivo. (2) Various effectors of CAMP (such as histamine, caffeine, quinidine, and serotonin) can cause semicolonial to colonial growth in Neurospora (Scott and Soloman, 1975), although, in general higher concentrations of these chemicals are required to cause morphological changes in Neurospora than in a mammalian system (Robinson et al.,

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1971). (3) In a particular morphological mutant frost, a low level of CAMP and a defective adenyl cyclase have been reported (Scott, in Scott et al., 1974). (4) In the mutant cr-1 with reduced specific activity of adenyl cyclase, addition of CAMPto the growth medium was found to overcome the pleiotropic effect of this mutation (Terenzi et al., 1974). ( 5 ) The presence of a membrane-bound adenyl cyclase and of a particulate phosphodiesterase has been reported in Neurospora (Flawia and Torres 1972a,b; Scott and Soloman, 1974). These studies of the gene-enzyme relationships among morphological mutants, their phenocopies and suppressors clearly suggest that the morphology of Neurospora is determined by cell-wall-membrane complex in this organism. Any direct or indirect disruption in the structure and function of this complex may cause changes in morphology of Neurospora. Recent studies have shown that some hydrolysis of cell-wall structure is an essential part of the normal development of the wild-type strain ; an increased activity of certain cell-wall hydrolyzing enzymes in Neurospora during hyphal development has been demonstrated (Mahadevan and Mahadkar, 1970a; Mahadevan and Rao, 1970; Gratzner and Sheehan, 1969; Gratzner, 1972; Sternberg and Sussman, 1974). Ill. Conidiogenesis

Formation of conidia and their development marks a distinct stage in the differentiation of Neurospora. The conidial development has been extensively studied in order to understand the biochemical basis of differentiation in Neurospora. Some of the earlier studies were directed toward structure and function analysis of conidia (Lowry et al., 1967) or describing the physiological conditions that stimulate conidial development (Weiss and Turian, 1968; Turian and Bianchi, 1971) ; soon genetic mutants were described that can interrupt or disturb conidiation ; peach ( p e ) , fluffy (flu), and aconidial were such mutations (Barrat and Garnjobst, 1949). Recently, some other interesting phase-specific mutants have been described (Selitrennikoff et al., 1974; Matsuyama et al., 1974), and it is believed that a biochemical genetic approach will soon be adopted in the study of Neurospora conidiogenesis. AND SURFACE ARCHITECTURE OF CONIDIA A. ULTRASTRUCTURE

Electron microscopic studies carried out by Sussman’s group at the University of Michigan described the complex nature of the conidial

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structure (Lowry et al., 1967). Their studies also revealed the differences in the origin of the micro- and macroconidia. The outermost layer of the hyphal wall was found to be dissolved during the formation of the microconidia. Thus the microconidial wall was found to be different in constitution from that of the macroconidial wall. Such differences in the surface of the micro- and macroconidia (due to their different origin) has been visualized by scanning electron microscopy (see Fig. 4) (Mishra, 1975). The macroconidial surface, although more sculptured, was found to be very similar in its surface architecture to the vegetative hyphae, whereas the surface architecture of microconidia lacked the sculptured structure and instead showed several fibrillar structures (Mishra, 1975). The macroconidia also showed the presence of a collarlike structure that delimited the chain of macroconidia from the conidiophore (Mishra, 1975). The ragged mutant and some of the osmotic mutants were found to show significant differences in their surface architecture as compared to the wild-type macroconidia (Mishra, 1975). B. BIOCHEMISTRY AND GENETICSOF CONIDIOGENEYIS The study of the conidial formation and its eventual germination into a mycelial structure provides a good model system to probe into molecular processes underlying differentiation in Neurospora. The biochemical processes underlying these morphogenetic developments are distinct and just being elucidated. I n general, the biochemistry of the cellular content of conidia seems to be different from that of the vegetative hyphae, as one would expect. Therefore, one of the approaches to understand conidial differentiation has been to compare the mycelia and conidia in order t o identify the possible biochemical differences. A number of enzymatic activities have been found to be greatly increased in conidia as compared with mycelia. The enzymes whose elevated levels have been established in conidia are: NAD nucleotidase (Zalokar and Cochrane, 1956), p-galactosidase (Zalokar, 1954), p-glucosidase (Eberhardt, 1961), isocitrate lyase (Turian, 1961), trehalase (Hill and Sussman, 1963). Also the level of certain low-molecular-weight substances has been found to increase during conidiation (Fahey et al., 1975). Tuveson et al. (1967) have shown that NADP-dependent glutamic dehydrogenase cannot be detected in Neurospora conidia before germination starts. Zalokar (1954) found that conidial formation can be prevented by addition of Tween-80, a surface-active agent, which inhibits the formation of aerial hyphae. In a related study, Turian (1957) found marked reduction in conidial formation when diphenylamine was added to the growth medium to

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inhibit carotenoid production. Later, Weiss and Turian (1966) found that the relative activities of glycolytic and oxidative pathways in the cell regulate both conidiation and pigment synthesis. When glycolysis was inhibited, conidiation occurred , whereas any disruption of oxidative metabolism in normally conidiating cultures by iodoacetate, sodium fluoride, and PCMB resulted in suppressed conidiation. These authors also suggested that the control is exerted a t the stage of pyruvate, a common branch point of these two pathways. They also found a lower concentration of pyruvate carboxylase, but a higher concentration of NADase activities, in conidia (Weiss and Turian, 1966). The role of NADase in conidiation was suggested earlier by Zalokar and Cochrane (1956). I n a particular mutant called fluffy, which lacks conidiation, the NADase activity was found to be much reduced (Urey, 1971) , indicating the role of NADase in conidiation. However, its role in conidiation has been disputed by recent biochemical genetic studies of Siegel’s group a t UCLA. These workers have isolated some NADase-less mutants of Neurospora that have normal conidiation (Selitrennikoff et al., 1974). Siegel has recently described the series of events that leads to the formation of conidia in Neurospora (Siegel et al., 1974). Siegel has made a distinction among three cell types: vegetative hyphae, aerial hyphae, and conidia. According to Siegel’s group, the vegetative hyphae can be called upon to form the intermediary cell type, the aerial hyphae which then will produce conidiophore and asexual spores known as macroconidia. Matsuyama et al. (1974) have described the stages in the formation of conidiophores and emphasized the differences between aerial hyphae and conidiophores. The role of aerial hyphae in conidiation was also evidenced by Zalokar (1954). He showed that conidiation was inhibited by preventing the formation of aerial hyphae after treatment with Tween-80, a surface-active agent. Siegel and his collaborators (1974) have also developed the methodology for obtaining these three cell types, and thus their biochemistry can be compared. The three cell types differ in their cell-wall composition; Mahadevan and Mahadkar found that the level of fraction I in conidial wall was almost 40% higher than that in the hyphal wall. Also, the amino sugar content of the conidia was much less as compared to the mycelial mat which produced these conidia in the bd mutant of Neurospora; on the basis of this finding, Schmidt and Brody (1975) have suggested the presence of a mechanism for spatial distribution of galactosamine in Neurospora. Siegel et al. (1974) found a difference in the chemical composition of the cell wall of vegetative and aerial hyphae. The development of micro- and macroconidia are controlled by different genes (Barratt and Garnjobst, 1949). Several mutations are known to affect conidiation in Neurospora. Most

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of the conidiation mutants have effects on other phases of the cell cycle. However, recently Siege1 and his collaborators described mutations that are phase specific and affect conidiation only. Matsuyama et al. (1974) have detailed the developmentally linked events during conidiogenesis. According to these authors, formation of conidia includes the following steps: (1) formation of conidiophores from aerial hyphae, (2) migration of nuclei to the top of conidiophore, (3) swelling and budding of the conidiophore tip, and (4) septation. These authors (Matsuyama et al., 1974; Selitrennikoff e t al., 1974) have isolated and characterized several phase-specific mutants that are defective in one or other of the developmental steps. The authors have mainly studied fl ( f l u f f y ) ,acon-2, aeon-3, csp-1, csp-2 mutations, which are located in separate chromosomes of Neurospora. They have tried to map the sequence of developmentally linked events by the phenotypic characteristics of the double mutants. Besides these phase-specific mutants, these authors have studied the effect of other mutations, such as flufly (fl) on conidiation. According to these authors, the fluffy strain is blocked a t step 3 and is also less efficient in earlier steps as compared to the wild type; the mutant acon-3 can successfully complete steps 1 and 2, although i t rarely produces a swollen tip ; acon-2 is expressed earlier and nuclei are seldom seen in the conidiophore tip; and csp-1 and csp-2 are blocked in the last step, i.e., separation of conidia. Thus the sequence of developmental steps for which these mutants are defective can be mapped as acon-2, acon-3, f l u f f y ,and csp-1 or csp-2. Among all these mutants, csp-1 and csp-2 have been studied in greater detail (Selitrennikoff e t al., 1974). These conidial separation mutants (csp-1 and csp-2) complement in heterokaryon to produce conidia like the wild-type strain, and therefore these mutations are recessive. These authors have also found that the cell wall-associated autolytic activity of the aerial hyphae was directly related with the conversion of the preconidial chains into free conidia, and they have measured the autolytic activity of wild-type aerial hyphae and compared it with that of the csp-1 and csp-2 mutants. The csp-1 and csp-2 were found to have only about 18% and 36% of the wild-type autolytic activity (Selitrennikoff et al., 1974). The nature of the autolytic enzymes involved in conidial separation, which are presumably controlled by csp-1 and csp-2 genes, has not yet been characterized. Conidial germination is very poorly understood. Recently Horowitz’s group a t California Institute of Technology has described and identified a substance essential for the germination of conidia (Charlang and Horowitz, 1971). The germination factor (GF) is heat stable and dialyzable; it is present in the growth medium and can be extracted from the mycelium (Charlang and Horowitz, 1971), and has now been identi-

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fied as a polyalcohol (Horowitz, 1974). I n the medium of low water content, conidial germination was inhibited by loss of the germination factor from the conidia. The germination factor is very active, and a very low concentration 1-0.1 pg/ml can stimulate conidial germination (Charlang and Horowitz, 1971). Surface-active agents like tyrocidin and Tween-80 were also found to induce loss of the germination factor from conidia. When tyrocidin (20 pg/ml) was added to a dense conidial suspension ( los conidia/ml) , there was immediate and rapid loss of the germination factor to the medium. However, after 1 hour of incubation this was reversed; the germination factor began to disappear from the medium, and the germination of conidia could occur after 3 hours of incubation. When 20 pg/ml of tyrocidin was added to a conidial suspension containing only lo7 conidia/ml, there was no recovery of the loss of GF, and these conidia failed to germinate ; however, 2 pg/ml of tyrocidin was ineffective when added to a similar conidial suspension (i.e., lo7 conidia/ml). Mack and Slayman (1966) earlier reported that a concentration of tyrocidin of 0.5 pg/ml could inhibit the log-phase culture; therefore, the conidia seem to be less sensitive to tyrocidin than are hyphal cells. The difference in sensitivity toward tyrocidin of these two cell types (hypae and conidia) again points to the differences in the biochemical makeup discussed earlier. This discovery of germination factor by Charlang and Horowitz (1971) seems to lead to a better understanding of the molecular processes involved in conidial differentiation. Bhagwat and Mahadevan (1970) have described the presence of long-term mRNA in the conidia of Neurospora; these authors have also reported differences in the profile of RNA during different stages of germination. These findings of Bhagwat and Mahadevan have been recently confirmed (Mirkes, 1974). It is believed that soon the molecular aspects of conidial differentiation will begin to be revealed. A start in this direction has been made by the work of Loo (1975a,b). She has recently isolated a number of temperature-sensitive mutants ; these have normal germination and hyphal growth a t 25OC, but both are greatly inhibited a t 35OC. The temperature-sensitive phenotype was found to be due to a single gene mutation which behaved as a recessive in the heterokaryon. Of the several mutants studied, two have been biochemically analyzed ; these were found to have altered polysome profiles as compared to the wild-type strain. These mutations seem to affect either the initiation of protein synthesis (as in mutant 34Cts, now designated as p s i - I ) or the ribosomal processing [as in another mutant designated rip-1 (Loo, 1975a,b) 1. Earlier, Inoue and Ishikawa (1970) also described several temperature-sensitive mutants that were unable to germinate a t high temperature, presumably because of disruption in macromolecular synthesis.

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C. CIRCADIAN RHYTHM Conidiation in Neurospora is a manifestation of circadian rhythm. Pittendrigh et al. (1959) and Sussman et al. (1964) independently recognized the significance of the circadian rhythm and the utilization of Neurospora mutants in the elucidation of the biochemical mechanism underlying this biological phenomenon. Circadian rhythm can be expressed as periodic formation of conidia as in certain mutant strains, patch (Stadler, 1959), timex (Sargent et al., 1966; Sargent and Woodward, 1969), or band (Sargent and Woodward, 1969), whereas in other mutant strains e.g., ( c l o c k ) , perodicity can be seen as changes in the hyphal branching frequency (Sussman et al., 1964). It was soon discovered that certain wild-type strains of Neurospora could phenocopy the periodicity in conidiation or branching of these mutants under certain growth conditions, such as aeration of the culture or proper concentration of sorbose in the growth medium (Sargent and Woodward, 1969; Sussman et al., 1964; Feldman and Hoyle, 1974). The conidiation of Neurospora is truly a circadian rhythm because it is not affected by temperature, it can be maintained in the dark and entrained by light and has a periodicity of approximately a day. The fact that a particular strain showing periodicity of conidiation also shows a single gene difference suggests that circadian rhythm is genetically controlled in these strains of Neurospora. Although the periodicity of conidiation is approximately 24 hours, Feldman and Hoyle (1974) have recently reported mutations (frq-I, frq-2, frq-3, and frq-4), which can decrease the length of periodicity in the band mutants of Neurospora. The mutations of chromosome VII (frq-I, frq-2, and frq-3) are closely linked to each other, whereas frq-4 is unlinked to either of them (Feldman and Hoyle, 1974). Since the length of periodicity is intrinsic t o the timing mechanism, these authors (Feldman and Hoyle, 1974) have concluded that these mutations affect the clock mechanism itself. Following a biochemical genetic approach, these mutants have been biochemically analyzed to elucidate the molecular mechanism behind the biological clocks. Among the different mutants showing circadian rhythm, band ( b d ) has been extensively studied because i t shows clear zonation during its growth and the conidiating (band) and the nonconidiating (interband) regions can easily be separated for chemical analyses. Brody, in an attempt to elucidate the biochemical basis of the biological rhythm, has shown that in the bd mutant the level of NADH, and NADPH, and NADP was lower in the conidiating region while the NAD level was higher as compared to the nonconidiating region (Brody and Harris, 1973). The exact nature of the spatial difference in the concentration of different

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pyrimidine nucleotides in the band ( b d ) mutant is not yet known. Hochberg and Sargent (1974) have reported the rhythmic activity of several enzymes during circadian conidiation in the band ( b d ) mutant of Neurospora. I n the band ( b d ) mutant a number of enzymes (such as NADase, isocitrate lyase, glyceraldehydephospate dehydrogenase, 6PGD and G6PD) were found to oscillate with the visible morphological changes. Some wild-type strains and the flufll~mutant, which showed a circadian osciliation of nucleic acid metabolism (Martens and Sargent, 1974), was, however, found to lack the oscillation of these enzymes. The role of NADase activity in circadian conidiation is aiso refuted by the fact that NADaseless mutant of Neurospora are not impaired in conidiation and also that the fluffy strain which shows circadian morphological alterations lacks any detectable NADase activity. These data regarding the fluctuation in enzyme activity during conidiation could therefore be interpreted to suggest that they are not part of the time keeping mechanism of the clock per se. However, Brody has recently observed circadian oscillations in the adenylate energy charge and in NAD:NADH redox ratio. The adenylate energy charge was found to decrease from 0.93 to 0.65 whereas the NAD:NADH redox ratio increased from 1.6 to 3.6 in the growing front of the band mutant strain (Brody, 1974). Since these parameters (i.e., high redox ratio and low energy charge) are associated with partial uncoupling of mitochondrial oxidative phosphorylation, Brody has proposed the latter (uncoupling of mitochondria1 oxidative phosphorylation) to be a critical process for the cellular biological clock. Support for the above scheme (Brody, 1974; Delmar and Brody, 1975) is provided by experiments with dinitrophenol ( D N P ), an uncoupler of oxidative phosphorylation ; in such experiments D N P was found to raise the redox ratio (from 1.8 to 3.1) and to lower the energy charge (0.93 to 0.80) of the growing front (nonconidiating region) only. No such effect of D N P was seen, however, on the conidiating region of the band ( b d ) strain (Delmar and Brody, 1975). The involvement of oxidative phosphorylation in the biological clock implies the role of the cell membrane in this process. Presumably, the cell membrane can act as a receptor or transducer of biological signals or both, as suggested earlier (Sweeney, 1969) ; however, its precise role remains to be elucidated. IV. Morphogenesis during Sexual Development

Neurospora undergoes elaborate differentiation during development of its sexual reproductive apparatus. The studies carried out by Srb’s group

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a t Cornell University have been mainly from the genetic point of view and only recently some biochemistry of this significant aspect of development in Neurospora is being elucidated by their group. Another important biochemical study has been carried out by Horowitz and his collaborators a t Cal Tech. Some of these aspects of morphogenetic development in Neurospora have been recently reviewed by Srb et al. (1974). The suitability of Neurospora for developmental genetic studies of the sexual reproductive apparatus has been discussed by Srb et al. (1974). Some of the attributes of Neurospora in such studies are as follows: (1) the cytology of ascus development is well established (McClintock, 1945; Singleton, 1953) ; (2) the different Neurospora species, although similar in physiology and morphology have significant differences in the genetic implications of their sexual reproductive processes in view of the fact that N . crassa and N . sitophila are heterothallic with 8-spored asci ; N . tetrasperma is pseudohomothallic with 4-spored asci whereas N . terricola is a true homothallic species with 8-spored asci; (3) the cytological differences between different species leading to different ascus patterns have been established by Dodge (1942) ; (4) genetic material can be routinely transferred among these Neurospora species.

A. SEXUAL DEVELOPMENT AND THE MATING-TYPE LOCUS The nature of the mating-type locus in Neurospora has remained elusive. N . crassa is heterothallic and occurs as two distinct mating types called A and a. Ordinarily these mating types have two functions, which control ( 1 ) crossing compatibility during sexual reproduction that leads to the formation of perithecia, asci and ascospores and (2) heterokaryon incompatibility; i.e., strains of the opposite mating types cannot form heterokaryons during vegetative growth. Besides the mating-type locus there are several other genes that control heterokaryon incompatibility in Neurospora crassa (Garnjobst, 1955; Garnjobst and Wilson, 1956). This heterokaryon incompatibility of the mating-type locus is never complete, and in N . crassa, heterokaryons betwecn opposite mating types grow poorly and usually segregate out as homokaryon sectors (Gross, 1952). I n N . sitophila the mating-type locus does not inhibit heterokaryon formation between the opposite mating types (Mishra, 1971) whereas in N . tetrasperma the nuclei of opposite mating types can exist together throughout its life cycle; and this N . tetrasperma is a pseudohomothallic species (Dodge, 1941) as opposed to N . dodgei a truly homothallic species (Nelson et al., 1964). I n an extensive study by Newmeyer et al. (1973) the two above-mentioned functions of the N . crassa mating-type locus (ie., crossing compatibility and heterokaryon incompatibility) could

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never be separated. Recently, Newmeyer (1970) has described a mutation called tol for “tolerance”), which allows the two mating-type loci ( A and a ) to coexist in heterokaryon in N . crassa. This locus (tol) is not linked to the mating-type locus. Similar conclusions have been arrived a t by Metzenberg and Ahlgren (1973) regarding the coexistence of the nuclei of opposite mating locus in N . tetrasperma. When the N . tetraspemna mating type loci were separately introduced into a largely N . crassa background by introgression, the organisms, were unable to form heterokaryons between strains of opposite mating types (Metzenberg and Ahlgren, 1973). Other preliminary studies show the involvement of a single mycelium in ascogonal development (Bhattacharya and Feldman, 1974) and the possible involvement of certain hormones in sexual differentiation (Islam, 1973). B. GENETICSAND BIOCHEMISTRY OF PERITHECIAL DEVELOPMENT The sexual differentiation in Neurospora is always accompanied by the characteristic appearance of certain kinds of proteins ; these are tyrosinase, L-amino acid oxidase, and a phase-specific perithecial protein. Much of the work in this area has been directed toward the understanding of the specific role of these proteins and their characterization. These works, mostly by Horowitz’s group a t Cal Tech and by Srb’s group a t Cornell University have been discussed below.

i. Induction of Tyrosinase Activity Production of melanin is an essential part of sexual differentiation in Neurospora. The enzyme tyrosinase, which converts L-tyrosine into melanin, the main pigment of perithecia and ascospores, has been found to be absent from the vegetative cell cycle (Hirsch, 1954). Earlier, Horowitz and his collaborators showed that sexual differentiation as well as the derepression of tyrosinase and L-amino acid oxidase could be induced by starvation conditions (Horowitz et al., 1960; Horowitz, 1965). These workers have described three genes, T, ty-1, and tu-2, that are responsible for tyrosinase activity in Neurospora. Recently, another strain, T-22 has been described, which cannot be induced for tyrosinase production although the gene is not a structural gene for tyrosinase (Horowitz and Macleod, in Feldman and Thayer, 1974) whereas the wild-type strain, 69-113a has a high level of tyrosinase production. All the tyrosine mutants ty-1, ty-2, and T-22 are female sterile. According to Horowitz et al. (1960) , both ty-1 and ty-2 are regulatory genes whereas the structure of tyrosinase is controlled by the alleles of T gene. Horowitz and his collaborators have also shown that inducibility of sexual differ-

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entiation as well as the derepression of tyrosinase and several L-amino acid oxidase depends on starvation condition. They have also found that the de novo synthesis of tyrosinase or L-amino acid oxidase can be stimulated by inhibition of protein biosynthesis or by D-amino acids {Horowitz, 1966; Horowitz et al., 1970). The apparent paradox that an inhibitor of protein biosynthesis stimulates tyrosinase activity in Neurospora was resolved by these workers on the assumption that the partial inhibition of protein synthesis causes a decrease in the level of an unstable protein which acts as a repressor of the tyrosinase genes (Horowitz et al., 1970). However, soon it was realized that the inducer of tyrosinase activity can act only during the stationary phase, never when added to an actively growing culture. These effects are reminiscent of the catabolite repression in bacteria (Magasanik, 1961) and thus suggest an involvement of CAMPin this process. Feldman and Thayer (1974) have reported the effect of cAMP on derepression of tyrosinase activity in different strains of Neurospora. These authors found that cAMP can increase tyrosinase production by 10 to 12-fold in the wild-type strain. They also found that caffeine or theophylline (at concentrations able to inhibit in vitro phosphodiesterase activity) can also increase tyrosinase production by increasing the in vivo level of CAMP. Only 3‘,5‘-cAMP or its dibutyryl or 8-bromo derivatives were active in inducing tyrosinase production ; 2’,3’-cAMP, 3’-AMP, or 5’-AMP were ineffective in this manner. Also, 3’5’-cAMP was found to increase the production of tyrosinase in the ty-1 mutant but not in ty-2 or T-22 strains. These data suggest functional differences among these three mutations (ty-I, ty-2, and T-22) controlling tyrosinase production. I n this relation, an interesting finding is that the inducer of tyrosinase activity cannot overcome the female sterility of the mutant strains ty-1 and ty-2; this suggests that these mutant loci regulate more than one function during sexual development. This conclusion is further supported by the fact that heterokaryons between ty-I and ty-2 are still female sterile although capable of tyrosinase production (Horowitz et al., 1965). 2. Phase-Specific Perithecial Protein

Srb and his collaborators have discovered the presence of a phasespecific protein in different species of Neurospora, which is characteristically present only in perithecia and is absent from vegetative cells and also from ascospores. The concentration of the perithecial proteins was found to increase to maximal level during 5-6 days after fertilization. These authors have found two electrophoretic variants (slow and fast) of the perithecial protein; in both cases there was a parallel increase in

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their amount after fertilization. The two variants of the perithecial protein found in ATeurospora strains of different origin were shown by tetrad analysis to be controlled by members of an allelic pair (Nasarallah and Srb, 1972). The role of this newly discovered phase-specific protein found only in perithecial extract has yet to be understood. Srb and his collaborators are trying to understand whether a t fertilization the synthesis of this protein is induced or whether it is released from some other form of protein that remains undetected by their present technique. The fact that synchronous production of ascogonia can be induced (Vishwanatha-Reddy and Turian, 1972) could be utilized in the study of different proteins or enzymes associated with sexual differentiation in Nezsrospora. These studies could also be aided by the utilization of different female-sterile mutants or a particular perithecial mutant that lacks melanin and instead has orange pigments (Howe and Benson, 1974). It is known that the ascogonia are produced only at 25OC, not at 37OC (Hirsch, 1954 ; Westergaard and Hirsch, 1954; Barbesgaard and Wagner, 1959). However, Vishwanatha-Reddy and Turian have found that the effect of temperature is reversible; i.e., if a 37OC culture is transferred back to 25OC, ascogonia are produced. Therefore, it appears that the increased temperature only prevents the production of a component necessary for ascogonial development rather than destroying this component. These authors have also found that this component is synthesized between 36 and 48 hours of incubation a t 25OC, when i t is no longer affected by transfer to 37OC (i.e., it suggests that the biosynthesis of this component, not the component itself, is temperature sensitive).

C. GENETICSOF Ascus

AND

ASCOSPORE DEVELOPMENT

The different aspects of ascus development (such as its morphology, cytology, and genetic control) have been described by Dodge (1927, 1939, 1942) and others (McClintock, 1945; Dodge et al., 1950; Singleton, 1953). These authors have also discussed the role of the specific spindle behavior responsible for the heterothallic 8-spored asci of N . crassa and the homothallic 4-spored asci of N . tetrasperma. Dodge and later Srb have shown that the behavior of the spindle, which is of such profound genetic consequence, is itself genetically controlled (Dodge, 1939 ; Pincherra and Srb, 1969). The significance of ascus development in biochemical genetic studies have been emphasized by Srb and his collaborators a t Cornell University, and these authors have carried out a systematic study of the genetic control of development in Neurospora. Srb and his collaborators have described a large number of mutants that are defective for shape and size of ascus or ascospores or for delimitation of ascospores. Srb and

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Base (1969) have also developed a unique technique of zygotic complementation to analyze the number and kind of genetic functions controlled by a particular mutation. I n such a test, a cross between two mutants defective for distinct functions yielded wild-type asci or spores, whereas asci or spores with mutant phenotypes were seen when the mutants involved in the cross were defective for the same function (Srb and Base, 1969). Further, the recessiveness or dominance of a mutation was decided by whether or not a cross between the mutant and the wild type led to wild-type or mutant phenotype. These authors have also determined the sequence of developmentally linked mutations by determining the effect of double mutations. I n this elegant manner Srb and his collaborators have initiated what may be called a formal genetics of development in Neurospora. The biochemical changes underlying these developmental mutants of Neurospora, however, remain to be elucidated. A number of mutants defective for a specific ascus or ascospore feature have been listed in Table 5. 1. Ascus Development and Genetic Control of Spindle

The different wild-type strains of hreurospora produce asci with 8 or 4 ascospores in a linear arrangement, these are either heterothallic or homothallic. The specific orientation of the spindle (longitudinal or transverse) during nuclear division in an ascus is known to determine the different kinds of asci present in hr.crassa and N . tetrasperma (Dodge, 1927, 1939, 1942; Colson, 1934; McClintock 1945; Singleton, 1953). According to these authors, the orientation of the spindle parallel to the transverse axis of the ascus during the second and third nuclear divisions in its development leads to the formation of 4 heterokaryotic but homothallic spores in N . tetrasperma; whereas, in hi. crassa (or N . sitophila) the spindle is parallel to the longitudinal axis of the ascus and thus leads to the formation of an ascus with 8 homokaryotic spores, each of which is capable of producing mycelia of only onc mating type. However, the parallelism in spindle orientation in ill. tetraspernza is never complete, and an occasional disruption (in the spindle orientation mechanism) leads to the formation of small homokaryotic ascospores, which belong to either A or a mating types. The orientation of the spindle is genetically controlled; Dodge (1939) described a dominant lethal mutation ( E ) in N . tetrasperma that causes the formation of %spored asci in N . tetraspernaa. Pincherra and Srb (1969) have also shown that a particular N . crassa mutation called peak ( p k ) when introduced into N . tetrasperma also causes formation of asci with 8 spores. These studies are of profound genetic consequence in the sense that a single gene mutation is shown to wipe out the very primary morphological criterion that makes N . tetra-

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TABLE 5 Characteristics of Some of the Neurospora Mutants That Cause Morphological Changes in Ascus o r Ascospore Development

Mutation 1. peak (pk)"

Origin or genetic background

N . crassa N . crassa in N . tetrasperma N . tetrasperma

2. E

N . tetrasperma

3. scumbo (sc)

N . crassa

4 . semicolonial-9 (smco-9) 5 . Round spores (R or r) 6. triangular ( p k t ) 7 . giant (G) 8. indurated (I) 9. lethal ascospore (le-1) 10. lethal ascospore (le-8)

N . crassa

N . crassa N . crassa N . t etrasp erma

N . tetrasperma N . crassa

N . crassa

Characteristics

References

Balloon-shaped ascus with 8 spores in nonlinear arrangement

Murray and Srb (1962)

Balloon-shaped ascus with only 4 spores in nonlinear arrangement 8 Linear spores in each ascus 8 Linear spores in each ascus 8 Linear spores in each ascus Round spores Triangular spores Giant spores Indurated ascus Ascospore fails to germinate Ascospore fails to germinate

Pincherra and Srb (1969) Novak and Srb (1973) Dodge (1939) Srb et al. (1974) Srb et al. (1974) Mitchell (1966) Srb et al. (1973) Dodge (1927) Dodge (1927) Garnjobst and Tatum (1967) Garnjobst and Tatum (1967)

apeak-8, biscuit, and clock are allelic and zygote recessive and peak-1 through peak-6 are zygote dominant.

sperma a distinct species. Studies by Topper (1972) suggest that the longitudinal orientation of the spindle is dominant over the transverse orientation; also the ascus pattern depends not entirely on the spindle orientation, but also on the movement of the centrosome and the attached nuclei. 2, Developmental Mutants, Phenotypic Interaction, and

Biochemical Lesions

Murray and Srb (1962) have described a number of mutations called peak ( p k ) in N . crassa, which leads to a colonial hyphal morphology and ballon-shaped asci with nonlinear arrangement of the ascospores. Srb and Base (1969) have described a large number of other recessive muta-

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tions that affect the ascus shape and spore pattern; these mutations were found to belong to several groups by zygotic complementation tests and were distributed well over the seven chromosomes of N . crassa. Some of these mutants were found to be allelic to previously known mutations, such as peak; however, a great majority were non-allelic to each other and to peak. Other mutations like scumbo (sc) and semicolonial-9 (srnco-9) also give rise to asci of different shapes with nonlinear spores. These mutations (i.e., sc and smco-9) are complementing, and a cross between them produces a linear ascus. Also the peak mutations of independent origin from N . crassa and N . tetrasperma, were found t o be allelic although these have distinct phenotypes in different genetic backgrounds (Novak and Srb, 1973). The peak mutation of N . tetrasperma origin causes the formation of the balloon-shaped asci with 4 nonlinear spores whereas the N . crassa peak mutation causes production of asci with 8 nonlinear spores in both genetic backgrounds (i.e. N . crassa and N . tetraspemna) . Russell and Srb (1972) have described several mutations that modify the phenotypic expression of the peak mutation. Preliminary studies by Srb and his collaborators also suggest a distinct correlation between the abnormal morphology and the behavior of certain orangelles (like centrosome and spindle). Recently, the biochemical defect of peak-2 mutation has been elucidated by Russell and Srb (1974). These authors found the activity of an enzyme involved in chitin biosynthesis [L-glutamine: ~-fructose-6-phosphateamidotransferase (GFAT)] was increased in the peak-2 mutant strain as compared to the wild type. The pealc-2 enzyme (GFAT) activity was found to be more thermolabile a t 35OC. Recently it has been shown that GFAT is subject to feedback inhibition by UDP-GlUNAC (Endo et al., 1970) ; however, the increased enzyme activity seen in peak-2 mutants was not due to differential feedback inhibition (Russel and Srb, 1974). Other Mutations. Heslot (1958) has described a large number of mutations that can alter the shape of the ascus, its size, and the number of spores in Sordariaceae. Mitchell (1966) first described the dominant mutation ( R ), which produces round spores in N . crassa. Srb et al. (1974) have described additional such mutations in A’. crassa. Mutations that cause production of giant spores or indurated asci in N . tetrasperma have been described by Dodge (1927). I n the case of giant spore mutations, the delimination of spores is defective and in eech ascus a single oversized spore is produced which germinates into a heterokaryotic mycelium. Leary and Srb (1969) have described several such recessive mutations in N . crassa. In the case of indurated asci, mutation causes fusion of the ascus wall with the spores, which fail to be delimited. Garnjobst and Tatum (1967) have described ascospore lethal mutations (le-1 and Ze-2).

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These ascospores fail to germinate; these authors also found that some mutant spores could be germinated after treatment with Clorox. Some of these mutants are listed in Table 5. 3. Phenotypic Interaction

Srb and his collaborators (1973, 1974) determined the sequence of developmentally linked events in ascus morphogenesis by examining the effect of a particular mutation on the expression of another mutation controlling a distinct function. I n such studies these authors found that, in the double mutant R, p k (i.e., round spore and balloon-shaped ascus), the spores were round in a balloon-shaped ascus, suggesting that the delimitation of the ascospore was not affected by the geometry of the ascus. However, the double mutant (giant and peak) produced a balloonshaped giant spore, suggesting that spore shape was determined by the ascus shape when the delimitation of spore was defective. At present, the biochemical basis of these mutants that affect the different aspects of ascus and ascospore development is not known ; however, it is imperative that a t least some of these mutants are defective for cell wall membrane metabolism since several colonial mutants have aberrant ascus formation, while others may be defective for certain organelle structures and functions (Srb et al., 1974). However, a start has been made in this direction by Russell and Srb (1974). These authors have compared the wild type and several mutant strains with altered mycelial and ascus morphology for the specific activity of the enzyme L-glutamine: D-fructose 6-phosphate amidotransferase (GFAT) , which is involved in the synthesis of chitin (Leloir and Cardini, 1953). GFAT activity was found to be significantly increased in the crude extracts of six mutants; of these six mutants, two were allelic to pealc-2 and clock while others mapped a t different loci (Russell and Srb, 1974). According to these authors, the peak locus is not a structural gene for the enzyme GFAT, since only two out of the eleven mutants allelic to the peak locus were found to show elevated GFAT activities as compared to that of the wild type. Further work is required to determine whether or not the other four nonallelic mutants showing higher GFAT activities control the structure of this enzyme (Russell and Srb, 1972).

V. Conclusions The cell wall-membrane complex of Neurospora seems to be the main determinant of its morphology. A genetic mutation or a chemical manipulation that directly or indirectly causes changes in this complex (cell

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wall-membrane) leads to an altered morphology. Such changes can be affected in any one of the several ways as summarized: 1. A defect in the enzyme which is directly or indirectly involved in the biosynthesis of the components of the cell wall-membrane complex (Tatum, 1973; Crochen and Nyc, 1964; Pina and Tatum, 1967). 2. An inhibition in the activity of the enzymes of cell-well biosynthesis (Mishra and Tatum, 1972). 3. An overproduction of certain cell wall autolyzing or other enzymes (Mahadevan and Mahadkar, 1970; Russell and Srb, 1974). 4. A disruption in the membrane function either by a mucopolysaccharide (Reissig and Plasgow, 1971) or by an excessive accumulation of microfilament (Allen et al., 1974). 5. Changes in the in v i m level of cAMP (Mishra, 1976; Scott and Soloman, 1975; Terenzi et al., 1974). It is also implied that the development of the conidia and the asci should involve changes in this complex (i.e., cell wall-membrane) of Neurospora. This idea is supported by the observed changes in the cell-wall composition (Mahadevan and Mahadkar, 1970b) and in the surface architecture of some of these fungal structures (Mishra, 1975). Although the primary enzyme defects in a number of the morphological mutants or their phenocopies and suppressors are now established (see Tables 1-3), this still remains to be elucidated for a large number of mutants. Such elucidation of gene-enzyme defects would not only increase our knowledge of the enzymes involved therein, but would also reveal their role in the morphogenesis of Neurospora. Such studies may lead to the discovery of the kinds of defects already described or to some novel kinds. Some of these may involve defects in protein biosynthesis or ribosome assembly or general changes in the fidelity of the translation which are now being described (Loo, 1975a,b; Printz and Gross, 1967). This information not only would provide a better understanding of the morphogenesis and development of Neurospora, but would add to our much needed knowledge of the molecular biology of differentiation in this organism. The recent studies have been successful in determining the sequence of morphogenetic events during conidiogenesis and ascus development (Siege1 et al., 1974; Srb et al., 1974). The characterization of the germination factor described by Charlang and Horowitz (1971) would be of great aid in understanding the conidial germination processes. Further study of the genetic control and regulation of different inducible or repressible proteins during sexual differentiation would lead to a better understanding of the complex processes involved (Horowitz et aE., 1965) and the role of cAMP therein. Also, further characterization of the perithecial pro-

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tein (Srb et aZ., 1973) from wild-type and mutant strains (female sterile and/or other phase-specific mutants) would reveal its role in the sexual development of Neurospora. A biochemical genetic approach would soon be available to reveal the different aspects of conidiation and sexual differentiation (including mating-type reaction and development of ascus and ascospores) . The study of circadian mutants in Neurospora has provided a better understanding of the biochemical basis of the biological clock (Brody, 1974). The role of cell membrane either as acceptor or transducer of signals for biological rhythms would soon be understood by employing appropriate mutant strains in future studies. A number of mutants that are blocked in different developmental pathways during conidiation (Siege1 et al., 1974), sexual differentiation (Horowita et aZ., 1960), and ascus development (Srb et al., 1974) are already known, and their use in future biochemical genetic study would prove fruitful in providing a better insight into the complex processes involved in the development of Neurospora. ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. E. L. Tatum for his interest and also to thank my colleagues for helpful discussions and Barbara Kay O'Dowd for her patient typing of the manuscript.

REFERENCES Abramsky, T., and Tatum, E. L. 1976. Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium. Biochim Biophys. Acta 421, 106-114. Abramsky, T., Scott, W. A., and Tatum, E. L. 1971. Neurospora morphology and 6phosphogluconic acid dehydrogenase. Fed. Proc., Fed. A m . SOC.Exp. Biol. 30, 1069.

Allen, E. D., Lowry, R. J., and Sussman, A. S. 1974. Accumulation of microtilaments in a colonial mutant of N . crassa. J. Ultrastruct. Res. 48, 455-464. Bachman, B. J., and Bonner, D. M. 1959. Protoplasts from Neurospora crassa. J. Bacteriol. 78, 550-556. Bachrach, V., and Cohen, I. 1961. Spermidine in the bacterial cell. J. Gen. Microbiol. 26, 1-9. Barbesgaard, P. Q., and Wagner, S. 1959. Further studies on the biochemical basis of protoperithecia formation in Neurospora crassa. Hereditas 45, 564-572. Barratt, R. W., and Garnjobst, L. 1919. Genetics of a colonial microconidiating mutant strain of Neurospora crassa. Genetics 34, 351-369. ' Bartnick-Garcia, S. 1968. Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu. R e v . Microbiol. 22, 87-108. Beadle, G. W., and Tatum, E. L. 1941. Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. U.S.A. 27, 499-506.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

397

Benzer, S. 1967. Behavior mutants of Drosophila isolated by countercurrent distribution. Proc. Natl. Acad. Sci. U.S.A. 27, 499-506. Bhagwat, A. S., and Mahadevan, P. R. 1970. Conserved mRNA from the conidia of Neurospora crassa. M o t . Gen. Genet. 109, 142-151. Bhattacharya, L., and Feldman, J. 1974. Sexual differentiation 11. Neurospora Newsl. 21, 6. Brody, S. 1970. Correlation between reduced nicotinamide adenine dinucleotide phosphate levels and morphological changes in Neurospora crassa. J . Bacterial. 101, 802. Brody, S. 1972. Regulation of pyrimidine nucleotide levels and ratios in Neurospora crassa. J . Biol. Chem. 247, 6013-6017. Brody, S. 1973. Metabolism, cell walls, and morphogenesis. I n “Developmental ReguIation: Aspects of Cell Differentiation” ( S . J. Coward, ed.), pp. 107-154. Academic Press, New York. Brody, S. 1974. Mitochondria] uncoupling and circadian biochemical oscillations in Neurospora. Fed. Proc., Fed. A m . Sac. Exp. Biol. 33, 1271 (abstr.). Brody, S., and Harris, S. 1973. Circadian rythms in Neurospora: Spatial differences in pyridine nucleotide levels. Science 180, 498-500. Brody, S., and Nyc, J. F. 1970. Altered fatty distribution in mutants of Neurospora crassa. J . Bacterial. 104, 780-786. Brody, S., and Tatum, E. L. 1966. The primary effect of a morphological mutation in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 56, 1290-1297. Brody, S.,and Tatum, E. L. 1967a. Phosphoglucomutase mutants and morphological changes in Neurospora crassa. Proc. Natl. Acad. Sci. USA. 58, 923-930. Brody, S., and Tatum, E. L. 1967b. On the role of glucose-6-phosphate dehydrogenase in the morphology of Neurospora In “Organizational Biosynthesis” (H. J. Vogel, J. 0. Lampen, and V. Bryson, eds.), pp. 295-301. Academic Press, New York. Chaikoff, I. L. 1953. Metabolic blocks in carbohydrate metabolism in diabetes. Harvey Lect. 47, 99-125. Chang, P. L. V., and Trevithick, J. R. 1972. Release of wall-bound invertase and trehalase in Neurospora crassa by hydrolytic enzymes. J . Gen. Microbial. 70, 13-22. Charlang, G. W., and Horowitz, N. H. 1971. Germination and growth of Neurospora a t low water activities. Proc. Natl. Acad. Sci. U.S.A. 68, 260-262. Colson, B. 1934. The cytology and morphology of Neurospora tetrasperma Dodge. Ann. Bat. (London) 48, 211-224. Crocken, B., and Nyc, J. F. 1964. Phospholipid variations in mutant strains of Neurospora crassa. J . Biol. Chem. 239, 1727-1730. Crocken, B., and Tatum, E. L. 1968. The effect of sorbose on metabolism and morphology of Neurospora. Biochim. Biophys. Acta 156, 1-8. Dagley, S., and Nicholson, D. E. 1970. “An Introduction to Metabolic Pathways,” pp. 178-179. Wiley, New York. Darrow, R. A., and Rodstrom, R . 1966. Subunit association and catalytic activity of uridine diphosphate galactose-4-epimerase from yeast. Proc. Natl. Acad. Sci. U S A . 55, 205-211. DeLange, R. J., Kemp, R. G., Riley, W. D., Cooper, R. A., and Krebs, E. G. 1968. Activation of skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3’,5’ monophosphate. J . Biol. Chem. 243,2200-2205.

398

N. C. MISHRA

Delmar, D. P., and Brody, S. 1975. Circadian rhythms in Neurospora crassa: Oscillation in the level of adenine nucleotide. J. Bacteriol. 121, 548-553. deTerra, N., and Tatum, E. L. 1961. Colonial growth of Neurospora. Science 134, 1066-1068. deTerra, N., and Tatum, E. L. 1963. A relationship between cell wall structure and colonial growth in Neurospora. Am. J. Bot. 50, 669-677. Dodge, B. 0. 1927. Nuclear phenomena associated with heterothalliam in the ascomycete Neurospora, J. Agric Res. 35, 289-305. Dodge, B. 0. 1939. A new dominant lethal in Neurospora. The E locus in N. tetrasperma. J. Hered. 30, 467-474. Dodge, B. 0. 1941. A heritable factor complex for heterokaryotic vigour in Neurospora Genetics 27, 140-141. Dodge, B. 0. 1942. Conjugate nuclear division in the fungi. Mycologia 34, 302307. Dodge, B. O., Singleton, J. R., and Rolnick, A. 1950. Studies on lethal E gene in Neurospora tetrasperma including chromosome counts also in razes of N . sitophila. Proc. Am. Philos. Soc. 94, 38-52, Dubes, G. R. 1953. Investigations of some “unknown” mutants of Nezirospora crassa. Ph.D. Thesis, California Institute of Technology, Pasedena. Eberhardt, B. M. 1961. Exogenous enzymes of Neurospora conidia and mycelia J. Celt. Comp. Physiol. 58, 11-16. Edson, C. M. 1972. The biosynthesis of cell wall structural components in Neurospora. Ph.D. Thesis, University of California, San Diego. Endo, A., and Misato, T. 1969. Polyoxin D: A competitive inhibitory of UDPN-acetylglucosamine chitin N-acetyglucosaminyl-transferase in Neurospora crassa. Biochem. Biophys. Res. Commun. 37, 718-722. Endo, A., Kekiki, K., and Misato, T. 1970. Feedback inhibition of L-glutaminen-fructose-6-phosphate o-amido transferase by uridine diphosphate n-acetylglucosamine in Neurospora crassa. J. Bacteriol. 103, 588-594. Fahey, R. C., Brody, S., and Mikolajcyk, S. D. 1975. Changes in the glutothione thiol-disulfide status of Neurospora crassa conidia during germination and aging. J. Bacteriol. 121, 144-151. Feldman, J. F., and Hoyle, M. N. 1974. A direct comparison between circadian and noncircadian rhythms in Neurospora crassa. Plant Physiol. 53, 928-930. Feldman, J. F., and Thayer, J. P. 1974. Cyclic AMP-induced tyrosinase synthesis in Neurospora crassn. Biochem. Biophys. Res. Commun. 61, No. 3, 977-982. Fiengold, D. S., Neufeld, E. F., and Hassid, W. Z. 1958. Synthesis of a p-1,3linked glucan by extracts of PhaseoEus avieus seedlings. J . Bid. Chem. 233, 783-788. Fincham, J. R. S., an’d Day, P. 1971. “Fungal Genetics,” 3rd ed. Blackwell, Oxford. Flawia, M. M., and Torres, H. N. 1972a. Adenyl cyclase activity in Neurospora crassa. J. Biol. Chem. 247,6873-6879. Flawia, M. M., and Torres, H. N. 1972b. Adenyl cyclase activity in Neurospora crassa 11. J . Biol. Chem. 247, 6880-6883. Fuller, R. C., and Tatum, E. L. 1956. Inositol phospho lipid in Neurospora and its relationship to morphology. Am. J . Bot. 43, 361-365. Garnjobst, L. 1955. Further analysis of genetic control of heterokaryosis in Neurospora crassa. A m . J. Bot. 42, 444-448. Garnjobst, L., and Tatum, E. L. 1967. A survey of new morphological mutants in Neurospora crassa. Genetics 57, 579-604.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

399

Garnjobst, L., and Tatum, E. L. 1970. New crisp genes and crisp-modifiers in NeuTospora crassa. Genetics 66, 281-290. Garnjobst, L., and Wilson, J. F. 1956. Heterokaryosis and protoplasmic incompatability in Neurospora crassa. Proc. Natl. Acad. Sci. U S A . 42, 613-618. Giles, N. H. 1951. Studies on the mechanism of reversion in biochemical mutants of Neurospora crassa. Cold Spring Harbor Symp. Quant. Biol. 16, 283-313. Glnsgow, J. E., and Reissig, J. L. 1974. Interaction of galactosaminoglycan with Newospora conidk. J . Bncteriol. 120, 759-766. Gorini, L. 1970. Informational suppressor. Annu. Reu. Genet. 4, 107-134. Gratzner, H. G. 1972. Cell wall alterations associated with hyper production of extracellular enzymes in Neurospora crassa. J . Bacteriol. 111, 443-446. Gratzner, H., and Sheehan, D. N. 1969. Neurospora mutant exhibition hyper production of amylase and invertas.. J . Bacteriol. 97, 544-549. Grindle, M. 1974. The efficacy of various mutagens and polyene antibodies for the induction and isolation of sterol mutants of Neurospora crassa. M o l . Gen. Genet. 130, 81-90. Gross, S . R. 1952. Heterokaryosis between opposite mating types of Neurospora crassa. A m . J . Bot. 39, 574-577. Harold, F. M. 1962. Binding of inorganic polyphosphate to the cell wall of Neurospora crassa. Biochim. Biophgs. Acta 57,59-66. Henry, S. A., and Keith, A. D. 1971. Saturated fatty acid requirer of Neurospora crassa. J . Bacteriol. 106, 174-182. Heslot, H. 1958. Contribution B l’ktude cytogknktique et gknCtique des Sordariacees. Dev. Cytol. Biol. Veg. Italog Snppl. 2, 1-234. Hill, E. P., and Sussman, A. S. 1963. Purification and properties of trehalase(s) from Neurospora. Arch. Biochem. Biophys. 102, 389-396. Hirsch, H. M. 1954. Melanogenesis and the differentiation of protoperithecia in Neurospora crassa. Bncteriol. Proc. 1054, 88 (abstr.). Hochberg, M. L., and Sargent, M. L. 1974. Rhythms of enzyme activity associated with circadian conidiation in Neurospora crassa. J . Bacteriol. 120, 1164-1175. Horowitz, N. H. 1965. Evidence for common control of tyrosinase and L-amino acid oxidase in Neurospora. Biochem. Biophys. Res. Commun. 18, 68fj-692. Horowitz, N. H. 1966. Evidence for common control of tyrosinase and L-amino acid in Neurospora. Biochem. Biophys. Res. Commun. 18, 686-692. Horowitz, N. H. 1974. Membrane and transport: A conidial factor necessary for germination. Neurospora Newl. 21, 5-6. Horowitz, N., Fling, M., McLeod, H. L., and Sueoka, N. 1960. Genetic determination and enzymatic induction of tyrosinase in Neurospora. J. Mol. Biol. 2 , 9 6 1 0 4 . Horowitz, N. H., Fling, M., McLeod, H., and Watanabe, Y. (1965). Structural and regulatory genes controlling tyrosinase systhesis in Neurospora. Cold Spring Harbor Symp. Quant. Biol. 26, 233-238. Horowitz, N. H., Feldman, H. M., and Pall M. L. 1970. Derepression of tyrosinase synthesis in Neurospora by cycloheximide, actinomycin D, and puromycin. J. Biol. Chem. 245, 27842788. Houlahan M. B., Beadle, G. W., and Calhoun, H. G. 1949. Linkage studies with biochemical mutants of Neurospora. Genetics 34, 493-507. Howe, H. B., and Benson, E. W. 1974. A perithecial color mutant of Neurospora crassa. Mol. Gen. Genet. 131, 79-83. Hunsley, D., and Burnett, J. H. 1970. The ultrastructural architecture of the walls of some hyphal fungi. J. Gen. Microbial. 62, 203-218.

400

N. C. MISHRA

Hunsley, D., and Goodday, G. W. 1974. The structure and development of Septa in Neurospora crassa. Protoplasma 82, 125-146. Inoue, H., and Ishikawa, T. 1970. Macromolecule synthesis and germination of conidia in temperature sensitive mutants of Neurospora crassa. Jpn. J . Genet. 45, 357-369. Islam, M. S. 1973. Sex hormones in Neurospora crassa further studies on its biological properties. Mycopathol. Mycol. Appl. 51, 87-97. Jacob, F., and Monod, J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J . M o l . Biol. 3, 318-356. Kingdon, H. S., Shapiro, B. M., and Stadtman, E. R. 1967. Regulation of glutamine synthetase VIII. Proc. N a t . Acad. Sci. 58, 1703-1710. Klingmuller, W. 1971. Cellular components specifically labeled during sorbose stimulation of sugar transport in Neurospora. 2. Naturforsch. Teil B 26, 409413. Leary, J. V., and Srb, A. M. 1969. Giant spores, a new developmental mutant of N . crassa. Neurospora Newsl. 15, 22-23. Lechner, J. F., Fuscaldo, K. E., and Bazinet, G. 1971. Genetic and biochemical studies of the hexose monophosphate shunt in Neurosporu crassa. 11. Characterization of biochemical defects of the morphological mutants colonial-2 and colonial-3. Can. J. Microbiol. 17, 784-794. Lein, J., and Lein, P. S. 1949. Studies on a Neurospora mutant requiring unsaturated fatty acid for growth. J. Bacterial. 58, 595-599. Leloir, L. F. 1951. The enzymatic transformation of uridine diphosphate glucose into galactose derivative. Arch. Biochem. 33, 186-190. Leloir, L. F., and Cardini, C. E. 1953. The biosynthesis of glucosamine. Biochim. Biophys. Acta 12, 15-22. Lindegren, C. C. 1936. A six point map of the sex chromosomes of N . crassa. J . Hered. 27, 251-259. Lindegren, C. C., and Lindegren, G. 1941. X-ray and ultraviolet induced mutations in Neurospora I. J . Hered. 32, 404-412. LOO, M. 1975a. Neurospora crassa temperature-sensitive mutant apparently defective in protein synthesis. J. Bacteriol. 121, 286-295. LOO, M. 1975b. A temperature-sensitive mutant of Neurospora defective in ribosome processing (rip-1). Neurospora Newsl. 22, 10-11. Lowry, R. P., Durkee, T. L., and Sussman, A. S. 1967. Ultrastructural studies of microconidium formation in Neurospora crassa. Am. J . Bot. 45, 397403. McClintock, B. 1945. Neurospora. I. Preliminary observations of the chromosomes of Neurospora crassa. Am. J. Bat. 32, 671-678. Mack, B., and Slayman, C. W. 1966. Mode of action tyrocidine on Neurospora. Biochim. Biophys. Acta 124, 351-361. Magasanik, B. 1961. Catabolite repression. Cold Spring Harbor S y m p . Quant. Biol. 26, 249-256. Mahadevan, P. R., and Mahadkar, V. R. 1970a. Role of enzymes in growth and morphology of Neurospora crass%. Cell-wall-bound enzymes and their possible role in branching. J . Bacteriol. 101, 941-947. Mahadevan, P. R., and Mahadkar, V. R. 1970b. Major constituents of the conidial wall of Neurospora crassa. Indian J. E x p . Biol. 8, 207-210. Mahadevan, P. R., and Rao, S. R. 1970. Enzyme degradation of conidial wall during germination of Neurospora crassa. Indian J . E x p . Biol. 8, 293-297. Mahadevan, P. R., and Tatum, E. L. 1965. Relationship of the major constituents

BIOCHEMICAL GENETICS OF

Neurospora

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401

of the Neurospora crassa cell wall to wild-type and colonial morphology. J. Bacteriol. 90, 1073-1081. Mahadevan, P. R., and Tatum, E. L. 1967. Localization of structural polymers in the cell wall of Neurospora crassa. J. Cell Biol. 35, 295-302. Manocha, M. S., and Colvin, J. R. 1967. Structure and composition of the cell wall of Neurospora crassa. J. Bacteriol. 94, 202-212. Martens, C. L., and Sargent, M. L. 1974. Circadian rhythms of nucleic acid metabolism in Neurospora crassa. J. Bacteriol. 117, 1210-1215. Matile, P. 1966. Inositol deficiency resulting in death: An explanation of its occurrences in Neurospora crassa. Science 66, 86-88. Matsuyama, S. S., Nelson, R. E., and Siegel, R. W. 1974. Mutations specifically blocking differentiation of microbonidia in Neurospora crassa. J. Dev. Biol. 41, 27S287.

Metzenberg, R. L. 1972. Genetic regulatory systems in Neurospora. Annu. Rev. Genet. 6, 111-132. Metzenberg, R. L., and Ahlgren, S. K. 1973. Behavior of Neurospora tetrasperma mating-type genes introgressed into N . crassa. Can. J. Genet. Cytol. 15, 571576.

Mirkes, P. 1974. Polysomes, ribonucleic acid and protein synthesis during germination of Neurospora crassa conidia. J. Bacteriol. 117, 196-202. Mishra, N. C. 1971. Heterokaryosis in Neurospora. Genetics 67, 55-59. Mishra, N. C. 1975. The surface architecture of different Neurospora strains as revealed by scanning electron microscopy and their relation to morphology. J. Microsc. Biol. Cell. 26, 151-159. Mishra, N. C. 1976. The effect of cyclic adenosine monophosphate on the growth of Neurospora crassa. Naturwissenschaften 63,485-486. Mishra, N. C., and Forsthofel, A. 1976. Galactosyl transferases of Neurospora. In preparation. Mishra, N. C., and Tatum, E. L. 1970a. Phosphoglucomutase mutants of Neurospora sitophila and their relation to morphology. Proc. Natl. Acad. Sci. U.S.A. 60, 638-645.

Mishra, N. C., and Tatum, E. L. 1970b. Pleiotrophy of gene mutations in Neurospora. J. Cell Biol. 470, 141a. Mishra, N. C., and Tatum, E. L. 1972. Effects of L-sorbose in polysaccharide synthetase of Neurospora. Proc. Natl. Acad. Sd.U.S.A. 69, 313-317. Mishra, N. C., and Threlkeld, S. F. H. 1967. Variation in the expression of rg mutants in Neurospora. Genetics 55, 113-121. Mitchell, M. B. 1966. Round spore character in N. crassa. Neurospora Newsl. 10, 6. Morgan, M. F., Garnjobst, L., and Tatum, E. L. 1967. Linkage relations of new morphological mutants in linkage group V of Neurospora crassa. Genetics 57, 605-612.

Murayama, T., and Ishikawa, T. 1973. Mutation in Neurospora crassa affecting some of the extracellular enzymes and several growth characteristics. J. Bacteriol. 115, 796-804. Murayama, T., and Ishikawa, T. 1975. Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase. J. Bacteriol. 122, 54-58. Murray, J. C., and Srb, A. 1962. The morphology and genetics of wildtype and seven morphological mutant strains of Neurosporn crassa. Can. J. Bot. 40, 337-349.

402

N . C. MISHRA

Nasarallah, J., and Srb, A. 1972. Genetically related protein variants specifically associated with fruitlng body maturation in Neurospora. Proc. Natl. Acad. Sci. U 8 . A . 70, 1891-1893. Nelson, A. C., Novak, R. O., and Backus, M. P. 1964. A new species of Neurospora from soil. Mycologia 56, 384-392. Newmeyer, D. 1970. A suppressor of the heterokaryon-incornpatability associated with mating type in Neurospora crassa. Can. J . Genet. Cytol. 12, 914-926. Newmeyer, D., Howe, H. B., and Galeazzi, D. R. 1973. A search for complexity at the mating type locus of Neurosporn crassa. Can. J . Genet. Cytol. 15, 577-585. Novak, D. R., and Srb, A. 1973. A dominant round spore mutant in Neurospora tetrasperma and its implications for ascospore development. Can. J . Genet. Cytol. 15, 685-693. O’Brien, T. P., and Thimann, K. U. 1966. Intracellular fibers in oat coleoptile cells and their possible significance in cytoplasmic streaming. Proc. Natl. Acad. Sci. U.S.A. 56, 888-894. Pina, E., and Tatum, E. L. 1967. Inositol biosynthesis in Neurospora crassa. Biochim. Biophys. Acta 136, 265-271. Pincherra, G., and Srb, A. 1969. Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora. A m . J. Bot. 56, 846-852. Pittendrigh. C. S., Bruce, V. G., Rosenweig, N. S., and Rubin, M. L. 1959. A biological clock in Neurosporn. Nature (London) 184, 169-170. Printz, D. B., and Gross, S. 1967. An apparent relationship between mistranlation and an altered leucyl-tRNA synthetase in a conditional lethal mutant of Neurospora crassa. Genetics 55, 451-467. Rand, J. B. 1975. The regulation of sugar transport and galactose metabolism in Neurospora crassa. Ph.D. Thesis, Rockefeller University, New York. Reissig, J. L., and Glasgow, J. E. 1971. Mucopolysaccharide which regulates growth in Neurosporn. J . Bactejiol. 106, 882-889. Robertson, N. F. 1959. Experimental control of hyphal branching and branch form in hypomycetous fungi. J. Linn. Soc. London 56,207-211. Robertson, N. F. 1968. The growth process in fungi. Annu. R e v . Microbiol. 6, 113136. Robertson, N. F., and Rizvi, 5. R. H. 1965. Apical disintegration of hyphae of N . crassa as a response to L-sorbose. Trans. Br. Mycol. Soc. 48, 467-477. Robinson, G. A., Butcher, R. W., and Sutherland, E. W. 1971. “Cyclic AMP.” Academic Press, New York. Russell, P. J., and Srb, A. M. 1972. Dominance modifiers in Neurospora crassa: Phenocopy selection and influence on certain ascus mutants. Genetics 71, 233245. Russell, P. J., and Srb, A. M. 1974. A study of L-glutamine: D-fructose-6-phosphate amidotransferase in certain developmental mutants of Neurospora crassa. Mol. Gen. G e n e f . 129, 77-86. Sargent, M. L.,and Woodward, D. 0. 1969. Genetic determinants of circadian rhythmicity in Neurospora. J. Bncteriol. 97, 861-866. Sargent, M. L., Briggs, W. R., and Woodward, D. 0. 1966. The circadian nature of a rhythm expressed by an invertaseless strain of Neurosporn crnssa. Plant Physiol. 41, 1343-1349. Scarborough, G. A. 1971. Sugar transport in Neurospora crassn. 111. An inositol requirement for the function of the glucose active transport system. Biochem. Biophys. Res. Commun. 43, 96tL974.

BIOCHEMICAL GENETICS OF

Neurospora

MORPHOGENESIS

403

Scarborough, G. A,, and Schulte, T. H. 1974. An improved method for the production of Neurospora crassa spheroplasts and a new method for measuring transport in fragile cells. Anal. Biochem. 61, 441447. Schmidt, J. C., and Brody, S. 1975. Developmental control of glucosamine and galactosamine levels during conidiation in Neurospora crassa. J . Bacteiiol. 122, 1071-1075. Schulte, T. H., and Scarborough, G. A. 1975. Characterization of the glucose transport systems in Neurospora crassa sl. J . Bacteriol. 122, 10761080. Scott, W. A. 1971. Physical properties of glucose 6-phosphate hydrogenase from Neurospora crassa. J . Biol. Chem. 246, 6353. Scott, W. A., and Abramsky, T. 1973a. Neurospora 6-phosphogluconate dehydrogenase. 11. Properties of two purified mutant enzymes. J . Biol. Chem. 248, 3542-3545. Scott, W. A., and Abramsky, T. 197313. Neurospora 6-phosphogluconate dehydrogenase. I. Purification and characterization of the wild-type enzyme. J . BioE. Chem. 248, 3535-3542. Scott, W. A., and Brody, S. 1973. Effects of suppressor mutations on nonallelic glucose 6-phosphate dehydrogenase mutants of Neurospora crassa. Biochem. Genet. 10, 285-295. Scott, W. A., and Soloman, B. 1974. Cyclic 3’,5’-AMP phosphodiesterase of Neurospora crassa. Biochem. Biophys. Res. Commun. 53, 1024-1030. Scott, W. A., and Soloman, B. 1975. Adenosine 3’,5‘-cyclic monophosphate and morphology in Neurospora crassa: Drug indured alterations. J . Bacteriol. 122, 454-463. Scott, W. A., and Tatum, E. L. 1970. Glucose-6-phosphate dehydrogenase and Neurospora morphology. Proc. Ngtl. Acad. Sci. U S A . 66,515-522. Scott, W. A., and Tatum, E . L. 1971. Purification and partial characterization of glucose 6-phosphate dehydrogenase from Neurospora crassa. J . Biol. Chem. 246, 6347-6352. Scott, W. A., Mishra, N. C., and Tatum, E . L. 1974. Biochemical genetics of morphogenesis in Neurospora. Brookhaven Syrnp. Biol. 25, 1-18. Selitrennikoff, C. P., Nelson, R. E., and Siegel, R. W. 1974. Phase-specific genes for macroconidiation in Neurospora crassa. Genetics 78, 679490. Shatkin, A. J., and Tatum, E . L. 1959. Electron microscopy of Neurospora crassa mycelia. J . Biophys. Biochem. Cytol. 6, 423-426. Shatkin, A., and Tatum, E. L. 1961. The relationship of m-inositol to morphology in Neurospora crassn. Am. J . Bat. 48, 760, 771. Siegel, R. W., Selitrennikoff, C. P., and Nelson, R. 1974. Cell wall changes in Neurospora. I n “Results and Problems of Cell Differentiation.” Spring-Verlag, Berlin and New York (in press). Singleton, J. R. 1953. Chromosome morphology and the chromosome cycle in the ascus of Neurospora crassn. Am. J . Bot. 40, 124-143. Srb, A . M. 1957. Exotic growth forms in Neurospora. J . Heredity 48, 146153. Srh, A. M., and Basl, M. 1969. The isolation of mutants affecting ascus development in Neurospora crassa and their analysis by a zygote complementation test. Genet. Res. 13, 303-311. Srb, A. M., and Horowitz, N. H. 1944. The ornithine cycle in Neurospora and its genetic control. J . Biol. Chem. 154, 129-139. Srh, A. M., Bad, M., Bolist, M., and Leary, J. V. 1973. Mutations in Neurospora crassa affecting ascus and ascospore development. J . Hered. 64, 242-246.

404

N. C. MISHRA

Srb, A. M., Nasarallah, J., and Basl, M. 1974. Genetic control of the development of the sexual reproductive apparatus of Neurospora. Brookhaven Symp. Biol. 25, 40-50.

Stadler, D. R. 1959. Genetic control of a cyclic growth pattern in Neurospora. Nature (London) 184, 170-171. Sternberg, D., and Sussman, A. S. 1974. Hyperproduction of some glycosidases in Neurospora crassa. Arch. Microbial. 101, 303-320. Sussman, A. S., Lowry, R. J., and Durkee, T. 1964. Morphology and genetics of a periodic colonial mutant of Neurospora crassa. Am. J . Bat. 51,243-253. Sweeney, B. M. 1969. “Rhythmic Phenomena in Plants.” Academic Press, New York. Tatum, E. L. 1973. Biochemische Genetik der Morphogenese bei Neurospora. Naturwiss. Rundsch. 86, 140-142. Tatum, E. L., Barratt, R. W., and Cutter, U. M. 1949. Chemical induction of colonial garamorphs in Neurospora and syncephalastrum. Science 109, 509-511. Terenzi, H. F., Flawia, M. M., and Torres, H. N. 1974. A Neurospora crassa morphological mutant showing reduced adenylate cyclase activity. Biochem. Biophys. Res. Commun. 58, 990-996. Topper, V. M. 1972. A cytological study of the sexual cycle of Neurospora tetraspermu. Cornell University. M.S. Thesis. Traut, R. R., and Lipmann, F. 1963. Activation of glycogen synthetase by glucose 6-phosphate. J. Biol. Chem. 238, 1213-1221. Trevithick, J. R., and Metzenberg, R. L. 1966. Molecular sieving by Neurospora cell walls during secretion of invertase isozymes. J. Bucteriol. 92, 1010-1015. Tsoi, A., and Douglas, H. C. 1964. Phosphoglucomutase mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta 92, 513-917. Turian, G. 1957. Recherches sur l’action anticarotknogene de la diphknylamine et ses conskquences sur la morphogknese productive chez Allomyces et Neurospora. Physiol. Plant. 10, 667-680. Turian, 0. 1961. L’acBtate et son double effet d’induction isocitratasique et de diff6renciation conidienne chez les Neurospora. C. R. Hebd. Seances Acad. S C ~ . 252, 1374-1376.

Turian, G., and Bianchi, D. E. 1971. Conidiation in Neurospora crassa. Arch. Microbial. 77, 262474. Tuveson, R. W., West, D. J., and Barrat, R. W. 1967. Glutamic acid dehydrogenase in quiescent and germinating conidia of Neurospora crassa. J . Gen. Microbial. 48, 235-248. Urey, J. C. 1971. Enzyme patterns and protein synthesis during synchronous conidiation in Neurospora crassa. Dev. B i d . 26, 17-27. Vishwanatha-Reddy, M., and Turian, G. 1972. Temperature-induced synchronous differentiation of ascogonia in Neurospora. Experientia 28, 99-100. Weeks, O., and Gross, S. 1971. Mutation and “reversion” a t the l e u 4 locus of Neurospom and its effect on the cytoplasmic and mitochondria1 leucyl-tRNA synthetases. Biochem. Genet. 5, 505-516. Weiss, B., and Turian, G. 1966. A study of conidiation in Neurospora crassa. J. Gen. Microbial. 44, 407-418. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M. 1971. Microfilaments in cellular and developmental processes. Science 171, 136143.

BIOCHEMICAL GENETICS OF NeUTOSpOTU MORPHOGENESIS

405

Westergaard, M., and Hirsch, H. 1954. Environmental and genetic control of differentiation in Neurospora. Proc. S y m p . Colston Res. Sac. 7, 171-183. Wood, D. D., and Luck, D. J. L. 1971. A paracystalline inclusion in Neurospora crassa. Induction by ethedium and acridine, isolation and characterization. J. Cell Biol. 51, 249-264. Wood, W. B., Edgar, R. S., King, J., Lielausis, I., and Henninger, M. 1968. Bacteriophage assembly. Fed. Proc., Fed. Am. SOC.E z p . Biol. 27, 1160-1166. Wrathal, R., and Tatum, E. L. 1913. The peptides of the hyphal wall of Neurospora crassa. J . Gen. Microbial. 78, 139. Wrathal, R., and Tatum, E. L. 1974. Hyphal wall peptides and colonial morphology in Neurospora crassa. Biochem. Genet. 12, 59-68. Yanofsky, C. 1966. Amino acid replacements associated with mutations and relationship to in vitro coding data I n “Papers on Bacterial Genetics” (E. A. Adelberg, ed.), 2nd ed., pp. 163-176. Little, Brown, Boston, Massachusetts. Zalokar, M. 1954. Studies on the biosynthesis of carotenoids in Neurospora crassa during development. J. Gen. Microbial. 39, 85. Zalokar, M., and Cochrane, V. W. 1956. Diphosphopyridine nucleotidase in the life cycle of Neurospora crassa. Am. J . Bat. 43, 107.

GENETIC CONTROL OF THE CONTENT, AMINO ACID COMPOSITION, AND PROCESSING PROPERTIES OF PROTEINS IN WHEAT* Calvin F. Konzak Deportment of Agronomy and Soils, and Program in Genetics, Washington Stote University, Pullmon, Washington

I. Introduction , . , . . . . . . . . . . . . . . . 11. Structure of the Wheat Kernel; Genetic Origin of the Tissue Components A. Structure, . . . . . . . . . . . . . . . . . B. Origin, Development, and Ontogenetic Relationships among Tissues. C. Intracellular Sites of Protein Synthesis and Storage . . . . . D. Genetics of Endosperm Proteins . . . . . . . . . . . 111. Origin and Genetic Relationships among Triticum and Closely Related Species . . . . . . . . . . . . . . . . . . IV. Genetic Relationships and Homoeologies among Chromosomes of Cultivated Wheats . . . . . . . . . . . . . . . . V. Role of Environmental Factors in Protein Synthesis and Accumulation in Wheat Endosperm . . . . . . . . . . . . . . . A. Influence on Amino Acid Composition . . . . . . . . . B. Influence on Protein Solubility Fractions . . . . . . . . C. Genetic Regulation and Control of Protein Content, Composition, and Processing Properties . . . . . . . . , . . . . . VI. Role of the Cytoplasm in the Hereditary Control of Protein Content and Composition . . . . . . . . . . . . . . . . . . A. Cytoplasmic Organelle-Nuclear Gene Interactions . . . . . . B. Joint Chloroplast-Nuclear Gene Control of Ribulose-1,5-Diphosphate Carboxylase . . . . . . . . . . . , . . . . . C. Chloroplast-Nuclear Gene Interactions in Nitrogen Reduction . . D. Cytoplasm Source and Protein Composition in Wheat. . . . . VII. Nuclear Gene Control of Protein Content and Composition . . . . A. Genetic Control of 80 S Ribosomal Proteins. . . . . . . . B. Nitrate Reductase Synthesis and Genetic Control of Nitrogen Reduction . . . . . . . . . . . . . . . . . ~

~

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432 432 434 435 435 436 436 437

* Information paper. College of Agriculture Research Center, Washington State University, Pullman, Project No. 1568. Research conducted in part while on Professional Leave September, 1973 to September, 1974 as Special Service Advisor to the Plant Breeding and Genetics Section of the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, Vienna, and completed at Washington State University. 407

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VIII. Genetic Variability in the Nature and Properties of Wheat Proteins. . A. Wheat Endosperm Protein Composition . . . . . . . . . B. Major Factors Affecting Processing Characteristics and Nutritional Value of Wheat Flour . . . . . . . . . . . . . . IX. Genetic Variation for Protein Content and Lysine Composition in Wheat A. Amino Acid Composition of High-Lysine and High-Protein Wheats . B. Site of the High-Protein and High-Lysine Composition in the Wheat Grain. . . . . . . . . . . . . . . . . . . X. Chromosomal Location of Genes Affecting Protein Content, Composition, and Use Properties . . . . . . . . . . . . . . . . XI. General Discussion . . . . . . . . . . . . . . . . XII. Role for Induced Mutations . . . . . . . . . . . . . A. Principles. . . . . . . . . . . . . . . . . . B. Mutations in Protein Structure and the Genetic Code. . . . . C. Efficient Induction of Improved Protein Mutants . . . . . . XIII. Directions for Future Research . . . . . . . . . . . . XIV. Summary and Conclusions. . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

440 440 463 476 481 481 487 528 535 535 539 540 54 1 544 546

I. Introduction

I n the years ahead, wheat, perhaps more than other cereals, can be expected to assume increasingly greater importance as a source of protein for much of the world’s increasing population. Even today, wheat provides more nourishment for more people than does any other food source (Inglett and Anderson, 1974). As the world’s population continues to increase, stresses on available food supplies will make it desirable, if not imperative, that the nutritional value of foods be improved. The nutritional quality of wheat protein, like that of other cereals (except possibly rice), is primarly limited by the amount and balance of four essential amino acids : lysine, threonine, isoleucine, and methionine (Kasarda et al., 1971; Kies and Fox, 1970a,b; Olson and Sander, 1975; ProteinCalorie Advisory Group (PAG) of the United Nations System, 1976). With a better balance of these essential amino acids, particularly of lysine, wheats would contribute appreciably more to the proper nutrition of those people for whom wheat is a main dietary source of protein. Since no wheat cultivar known today has a protein composition approaching the level desired (Schmidt et UI!.,1974; V. A. Johnson et al., 1975b), the development by breeding of high-yielding varieties with an increased quantity of protein with improved nutritional quality is an important scientific challenge. This challenge has stimulated much new research into the biophysical and chemical nature, functional properties, and inheritance of proteins in wheat, to build upon and expand an already solid foundation of information gained from years of intensive research in many countries by geneticists, biochemists, cereal tech-

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nologists, plant physiologists, and agronomists. A number of recent findings point to further opportunities for gaining insight into the mechanisms and nature of the biochemical genetic control of protein synthesis in wheat, into the genetic basis for improving the nutritional value of wheat protein, and into the physical and chemical nature of those protein components responsible for the unique rheological (functional, dough-forming) properties of wheat flour. Much of the new genetic knowledge of wheat has been achieved via the application of recently developed research technology, e.g., electrophoresis, electrofocusing, to analyses of the several series of genetic stocks (termed aneuploids) generously made available by E. R. Sears and colleagues, and through the concerted efforts of the European Wheat Aneuploids Cooperation. Use of these special genetic stocks markedly reduces the genetic variables, adding greater precision to the necessary comparative experiments. Because these stocks also provide the foundation for classical genetic analyses (Sears, 1953, 1954, 1959a, 1966, 1975a; Unrau, 1958; Law, 1968; Law and Worland, 1972, 1973), the genetic engineering of the various aneuploid stocks in ‘Chinese Spring’ wheat is now widely recognized as one of the most creative achievements in modern crop science. An increasing array of chromosome and gene addition, deletion, and substitution derivatives is greatly extending the research potential of these special genetic stocks (Law, 1968; Sears, 1969, 1975a,b). This paper summarizes current information about the inheritance, properties, and amino acid composition of wheat proteins and provides a compilation of evidence for the control of specific proteins and protein subunits, regulatory mechanisms, and quality characteristics by genetic factors located on particular wheat chromosomes. Much of this genetic evidence has been established through the use of aneuploid and related techniques. The purpose of this review is t o examine the genetic basis and potential for increasing the protein content of wheat and improving the nutritional value of wheat protein, by breeding and genetic manipulations, while retaining those processing properties (product-use qualities) required for making such traditional foods as bread, cakes, pastry, noodles, chapattis, and pasta. Although the emphasis here is focused on evidence for genetic control of proteins in the wheat endosperm, consideration also is given to the genetic aspects of the composition and technological properties of the wheat grain and to the roles of environmental factors and cytoplasmic organelles in the synthesis and accumulation of wheat proteins. Knowledge about those areas is no less important t o an understanding of the problems involved in wheat protein improvement, or to the effective and efficient deployment of resources toward

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that goal. More detailed information about the chemical and physical structure of wheat proteins and their function in breadmaking are found in the recent reviews by Hoseney and Finney (1971), Kasarda et al. (l971,1976a), Pomeranz (1971), Simmonds and Orth (1973), and Bushuk and Wrigley (1974). 11. Structure of the Wheat Kernel; Genetic Origin of the Tissue Components

Knowledge of the basic structure of the wheat kernel, its component parts and genetic origin and ontogenetic relationships provides a better foundation for understanding the nature and location of tissues in which proteins have been identified through the exploitation of genetic stocks and modern methods of analysis.

A. STRUCTURE I n botanical terms, the wheat kernel, like that of other cereal grains, is a caryopsis or single-seeded, indehiscent dry fruit. The true seed consists of the embryo, or germ, and endosperm, which are encased within the nucellar epidermis (hyaline layer), seed coat (testa), and fruit coat (pericarp), to form the kernel or caryopsis. The arrangement and composition of the component tissues in the wheat kernel (Fig. 1) are of importance to the nutritional and processing properties of the grain in commerce, while the genetic and ontogenetic origins of the tissue components are of importance in genetic and biochemical genetic analyses. Notably, the aleurone (a layer of single cells, which is removed with the bran upon milling of white flour) contains the highest proportion of the B vitamins and minerals, as well as a significant portion of the protein present in whole wheat grain (Fig. 2). Further details about the component tissue structure and nutrient composition of the wheat kernel can be found in the excellent review by MacMasters et al. (1971). A bibliography on the industrial utilization of cereals also has been published (Pomeranz, 1973a).

B. ORIGIN,DEVELOPMENT, AND ONTOGENETIC RELATIONSHIPS AMONG TISSUES The wheat kernel develops by a process called “double fertilization,” in which two identical nuclei from a single pollen grain enter the embryo sac within the ovary. One pollen nucleus fuses with the egg cell, the FIG. 1. Gross structure of the wheat kernel. (Courtesy of the Wheat Flour Institute, Chicago, Illinois.)

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CALVIN F. KONZAK

VITAMINS THIAMINE

RIBOFLAVIN

ALEURONE LAYER

ALEURONE LAYER

E

PYRIDOXINE ALEURONE LAYER ALEURONE LAYER

-

EMBRYO

PANTOTHENIC A L D

PROTEI N

MINERALS

ALEURONE LAYER

FIQ.2. Vitamin, protein, and mineral distribution in parts of the wheat kernel. (Reproduced from MacMasters, et a l , 1971.)

second forms a triple-fusion nucleus with the two polar nuclei. The two polar nuclei are genetically identical to the egg nucleus as the result of cell divisions that occur during the development of the embryo sac and its contents within the ovary. The zygote formed from the fusion of the egg cell and one pollen nucleus becomes the embryo, or young plant, while the triple-fusion nucleus develops into the endosperm of the mature seed (Yampolsky, 1957; Mares et al., 1975). The fruit coat, seed coat, vascular bundle, and nucellus remnant tissues are derived entirely from ovary tissues formed by the “mother” plant and thus are genetically identical, but may be different in genetic composition from the embryo and endosperm contained within

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(MacMasters et al., 1971 ; Simmonds, 1974). However, these tissues serve an important role in the nutrition of the seed during early grain development (Rijven and Banbury, 1960). I n a hosnozygous, or true-breeding line, the ovary and embryo tissues are genetically identical. I n a heterozygous plant the embryo and endosperm may be genetically different from the ovary tissues. For example, seed color in wheat shows maternal inheritance, since the red-brown phlobaphene color pigment of red wheats is derived from the integument cells of the ovary and is located in the testa portion of the seed-coat tissue. Consequently, the genetic constitution of the plant on which the seeds grow is reflected in the seed-coat color, and all seeds produced on any wheat plant normally have the same seed-coat color. However, the seed embryos (on the same heterozygous pIant) derived from self-pollination will be of three classes with regard to a single red seed coat color gene: 2:0, O:O, and 1 : l ; but the endosperms of the same seeds would carry, respectively, 3:0, 0:O; 1 :2, o r 2 :1 doses of this gene. Endosperm development following fertilization is extremely rapid. I n the first period of endosperm development the early mitoses are not accompanied by cytokinesis. Cell divisions begin with the single large uninucleate, membrane-limited primary endosperm cell as a syncytium. This central cell divides numerous times, producing a multinucleate cell consisting of a peripheral zone of cytoplasm with a large central vacuole. Cytokinesis then occurs, leading to the development of a cellular structure. Disintegration of the nucellus tissue occurs concurrently with cellularization of the multinucleate endosperm; both processes are completed within 1 or 2 days after fertilization. The cellularization first involves the subdivision of the large central vacuole into a number of small vacuoles surrounded by cytoplasmic network and associated nuclei. Cellularization of the endosperm by cytokinesis occurs a t about 4-5 days after anthesis, initiated a t both the dorsal and ventral surfaces of the dense cytoplasmic mass (Simmonds, 1974; Mares et al., 1975), or first near the proembryo (Shealy and Simmonds, 1973), or near the chalaza1 end of the endosperm (Bennett and Smith, 1973). The ventral segment of the central cell is transformed into a layer of thick-walled cells with dense cytoplasm, giving rise to the modified and differentiated aleurone cells of the crease region, which then are no longer meristematic but develop into the pigment strand (Mares et al., 1975). I n the dorsal segment of the endosperm, cytoplasmic lamellae arising a t the plasma membrane project out and grow toward the center of the endosperm. Cell wall material is deposited along the lines of the lamellar projections and within the cytoplasmic strands. The large central vacuole is thus replaced by a mass of thin-walled, highly vacuolated cells. The cells a t the dorsal surface of the endosperm form a meristematic zone identifiable by 7 to

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8 days after anthesis, the zones of meristematic cells functioning in the

further development of the endosperm. Division of these cells both radially and tangentially leads to the formation of both peripheral and prismatic cells, and eventually the outer cell layer becomes the thickwalled aleurone which is found all around the outer surface of the endosperm of the mature kernel, except in the region of the crease (Simmonds, 1974; Mares et al., 1975). Cell formation in the endosperm of the wheat kernel is completed at about 12-14 days after anthesis (Sandstedt, 1946; Evers, 1970 ; Simmonds, 1974). Subsequent development of the endosperm occurs by cell enlargement, each cell expanding in size and developing starch grains and protein bodies according to a sequence related to its time of formation; i.e., the inner endosperm cells begin forming starch granules before the final cell divisions in the outer regions are completed, while the aleurone cell layer differentiates further to become thick-walled (Sandstedt, 1946; Buttrose, 1963 ; Evers, 1970; Simmonds, 1974; Mares et al., 1975). The subaleurone cells (immediately inside the thick-walled aleurone layer) are smaller, have thinner cell walls, and contain smaller starch grains than aleurone cells or cells located in the more central portion of the endosperm. Differences in endosperm cell size and development time are considered to be the principal factors responsible for the often-observed high prote'in content of cells located near the aleurone layer as compared to that of cells of the inner endosperm (Evers, 1970; Kent, 1966). Wheats with high protein content normally have higher amounts of residual organelle materials in subaleurone cells than do wheats with low-protein content (Barlow et al., 1974; Simmonds, 1974). Major genes for high protein content appear to proportionately increase protein subaleurone and inner endosperm cells, but may differentially increase protein in the subaleurone region (C. F. Konzak and N. V. Mung, 1976, unpublished). The crease of the kernel forms as a result of the development and expansion of the endosperm within the ovary wall tissues, to surround the relatively rigid "pigment strand" segment of thick-walled cells located toward the inner side of the ovary wall and ventral side of the kernel (Evers, 1970). In the crease, the seed coat joins with the pigment strand to form a complete covering around the endosperm and embryo (MacMasters et al., 1971). The vascular tissues are also located in the crease area of the kernel, all along the length of the pigment strand (Zee and O'Brien, 1970a). There are four vascular tracheids in the ovary, branching from a basal vascular bundle. Three of the tracheids, two lateral, and one dorsal to the embryo sac, extend through the pericarp in the ovary, but disintegrate during early grain development, leaving only the fourth tracheid, which extends through the funiculus and supplies the

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developing ovule (Simmonds, 1974). The thick-walled tissues of the pigment strand, which are pigmented only in “red” wheats, may serve as a supporting structure. However, the porous, thick-walled pigment strand is also thought to have a role in terminating starch accumulation and may aid in water loss during grain ripening (Zee and O’Brien, 1970a,b; Zee, 1975). The vascular tissues which serve as the supply system for the kernel in the wheat spikelet are described by Hanif and Langer (1972).

C. INTRACELLULAR SITES OF PROTEIN SYNTHESIS AND STORAGE The principal site of soluble protein synthesis appears to be the cells of the aleurone layer of the endosperm, which contain few insoluble protein storage bodies and only small starch grains (Sandstedt, 1946; Sandstedt and Beckord, 1946; Stevens, 1973). The aleurone cells are the last cells of the endosperm with a meristematic capability. They evidently terminate their development according to a predetermined ontogenetic sequence involving cell-wall thickening and accumulation of greater concentrations of soluble proteins (enzymes, inhibitors, etc.) than other cells of the endosperm. Protein synthesis appears to involve various classes of ribosomes, some of which are concerned with soluble protein synthesis while others are involved with the synthesis of storage proteins that are accumulated in protein bodies, clearly distinguishable by electron microscopy (EM), until near maturity. At maturity the dehydration processes compress the thin-walled inner endosperm cells and their inclusions, causing considerable distortion and rupturing of membranes that appear to surround the protein bodies (see Graham e t al., 1962a,b; Aranyi and Hawylewicz, 1969; Simmonds, 1972a; Simmonds e t al., 1973; Barlow et al., 1974; Adams e t al., 1976). The ribosomes associated with storage protein synthesis are attached to or enclosed within a rough endoplasmic reticulum (RER) distinguishable in EM photographs. At 14 days past flowering, well-developed protein bodies occurring singly or in groups are identifiable in vacuolar structures. The protein bodies (one or several) appear to be surrounded by a membrane (Graham et al., 1962b; Barlow e t al., 1974). Granular-appearing osmiophilic areas associated with the protein bodies are thought to be ribosomes. The RER shows little development, but there is evidence of free ribosomes. The R E R shows greater development by 21 days, and there is an increase in the number of mitochondria, which together with the R E R create a potential for rapid protein synthesis.

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The type of protein body synthesized a t 21 days and beyond differs in appearance from that in younger cells, although the gliadin proteins are identical throughout endosperm development (Barlow e t al., 1974). Bushuk and Wrigley (1971) observed changes in the proportion of glutenin proteins toward endosperm maturity. The synthesis of protein bodies after 21 days past flowering appears to involve secretion of the storage protein into swollen lumina of the RER, and as synthesis proceeds the two parts of a double membrane separate and the ends join to form a single membrane around the protein body, outside of which are ribosomes. The protein bodies remain within the lumina of the RER and are not transported elsewhere in the cell. These observations of Barlow et al. (1974) suggest that the origin of protein bodies in wheat is similar to that observed for Capsella and Arachis (Dieckert and Dieckert, 1972), while confirming the occurrence of a lipoprotein membrane around the protein bodies as observed by Buttrose (1963). On the other hand, scanning EM studies of storage proteins of several cereals isolated using a centrifuge technique showed wheat proteins to be unique in their lack of identity in the mature endosperm, suggesting that a t maturity the protein bodies have been replaced in the endosperm by matrix-type material with a more amorphous characteristic. In all cereals, barley, and sorghum, proteolytic activity appears to be associated with insoluble protein, and isolated protein bodies contain several other enzymes possibly associated with surrounding membrane material (Ory and Henningsen, 1969; Tronier and Ory, 1970; Adams and Novellie, 1975; Adams et al., 1976). The protein bodies of immature wheat endosperms are comprised of protein fibrils deposited in a layered or laminar structure (Bernardin and Kasarda, 1973a). I n the EM photographs of Seckinger and Wolf (1973) , the protein bodies of sorghum endosperm were shown to have a nonprolamine core. The sorghum protein bodies were embedded in a cytoplasmic protein matrix, but only a few seemed to be surrounded completely by matrix protein. The protein bodies of developing wheat endosperm vary in size from a fraction of 1 pm to 15 pm. Soluble proteins also formed in the cytoplasm were separable in a centrifuge supernatant fraction, indicating their association with smaller organelles, or free (unattached) ribosomes, but elements of the RER were also isolated (Graham et al., 1962a). Recent EM and solubility fractionation studies of Ingversen (1975) showed, however, that both prolamine (isopropanol soluble) and glutelin (alkali soluble) components are present in the protein bodies of Bomi barley, but the glutelin component occurs primarily in the high-lysine mutant Bomi 1508. Those observations indicate that glutelins may be integral components of protein bodies, not just RER or matrix material.

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D. GENETICSOF ENDOSPERM PROTEINS Because of the “tri-ploid” nature of the endosperm tissues compared with the “di-ploid” embryo, differences in the expression of endosperm proteins with relation to the genetic constitution of the embryo can be observed for individual seeds formed on plants heterozygous for structural or regulatory genes controlling the content, composition, and/or physical properties of proteins. Those observed differences in expression are primarily due to effects of the different dosages of genes contributed by the polar nuclei ( = twice the same gene content as the egg nucleus) us a single dose of genes contributed by the pollen nucleus. Thus, endosperm cells of the F, generation (in seed formed on F, plants) may contain 0, 1, 2, or 3 doses of proteins unique to one of the parents, whereas the embryos will contain 0, 1, or 2 doses; those with one dose represent heterozygotes corresponding to endosperms with one or two doses of the unique proteins. The seeds produced on some of the F, plants from such a cross will contain either 0 or 3 doses of the proteins, expressing the homozygosity of the embryos and the F, plant, while seeds produced on other plants will be genetically similar to those from the F, plant. When the parents differ by more than one protein, various genetic phenomena including linkage and gene interactions will complicate genetic analyses. Solari and Favret (1968), Favret e t al. (1970), Manghers et al. (1973), and Wrigley (1976a) have illustrated and described the different modes of inheritance of prolamine proteins in wheat and the influence of regulatory mechanisms and gene interactions on the quantitative aspects of protein inheritance. With such analytical procedures as electrophoresis and electrofocusing (Fig. 3) of proteins (Wrigley and Shepherd, 1973; Bietz et al., 1975) as well as less specific single-kernel protein estimation techniques (Brunckhorst e t aE., 1974a,b; Mertz et al., 1974), it is even possible to identify protein phenotypes after growing only the F, plant. However, when protein differences are more complex and genetic linkages as well as gene interactions are important t o the identification of desired recombinants, it may be necessary to conduct analyses on seeds from F2or FSplants or their bulk progeny. 111. Origin and Genetic Relationships among Trificum and Closely Related Species

The modern commercially important wheats are allopolyploids with either two (A and B) or three (A, B, D) genomes or sets of seven chromosomes derived from their diploid (AA, BB, and DD genome) progenitor

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FIG.3. Combined gel isoelectric focusing and electrophoresis of proteins from wheat endosperm, by the method of Wrigley and Shepherd (1973). The pattern obtained by electrofocusing in the first dimension appears across the top. The strip a t left shows the pattern given by gel electrophoresis (pH 3) alone. Starch gel was used for the second stage (electrophoresis) in preference to polyacrylamide, since starch gel has proved to be better for resolving this particular mixture of proteins. In general, however, polyacrylamide is preferred. (Reproduced from Wrigley, 1976a.)

species (Table 1).Two main approaches to taxonomic classification of the Triticinae are in use today. The approach followed in this review recognizes Triticum as an evolved hybrid genus, distinguishing Aegilops as a separate genus-mainly because all Aegilops species are wild forms that have followed separate paths of evolution, even though possibly two Aegilops species have contributed whole or parts of whole genomes to progenitors of the cultivated polyploid wheats (MacKey, 1966, 1968b, personal communication, 1975). The second classification system combines all the Aegilops forms into the single genus Triticum on the basis of evidence that the diploid Aegilops forms have contributed equally or more than the diploid Triticum to the genome composition of the polyploid wheats (Bowden, 1959; Morris and Sears, 1967; Sears, 1975a). All the common bread wheats in cultivation are considered subspecies of a single major hexaploid (AABBDD) species Triticum aestivum (L.) Thell. Among the tetraploid (AABB) wheats, only the convariety durum (Desf.) M K of T. (urgidum (L.) Thell. is commercially important today,

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TABLE 1 Subdivision of the Genus Triticum (L.) Sum. According to Genetic Conceptions (MacKey 1975) Monococca Flaksb. 2n = 14

T. monococcum (L.) MK einkorn wheat ssp. boeolicum (Boiss.) MK wild einkorn wheat var. aegilopoides (Bal. ex Korn.) MK var. thaoudar (Reut.) Perc. ssp. monococcum einkorn wheat T . urartu Tum. zweikorn wheat Dicoccoidea Flaksb. 2n = 28

.'2 timopheevi (Zhuk.) MK zanduri wheat ssp. araraticum (Jakubz.) MK wild zanduri wheat ssp. timopheevi zanduri wheat T. turgidum (L.) Thell. emmer wheat ssp. dicoccoides (Korn.) Thell. wild emmer wheat ssp. dicoccum (Schrank) Thell. (true) emmer wheat ssp. paleocolchicum (Men.) NK kolchis wheat ssp. turgidum conv. turgidum Rivet wheat conv. durum (Desf.) MK macaroni wheat conv. turanicum (Jakubs.) MK khorosan wheat conv. polonicum (L.) MK Polish wheat ssp. carthticum (Nevski) MK Persian wheat

Speltoidea Flaksb. 2n = 42

T . zhukovskyi Men. et Er. timon wheat T . aestivum (L.) Thell. dinkel wheat ssp. spelta (L.) Thell. spelt wheat ssp macha (Dek. et Men.) MK macha wheat ssp. vulgare (Vill.) MK bread wheat ssp. compactum (Host) MK club wheat ssp. sphaerococcum (Perc.) MK Indian dwarf wheat

mainly for it.s use in alimentary paste products-spaghetti, macaroni, etc., although in a few areas (southern Italy and North Africa, especially), the flour is used also for flat or leavened breads. The T.aestivum

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T

E T R A

P L

0 I

D

Common Bread Wheals

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wheats may have unique dough functionality properties for breadmaking. Certain of these unique properties are identified with proteins controlled by genes located in the D genome, which is absent from durum and other tetraploid wheats (La Clerc et al., 1918; Yamashita, 1953; Welsh and Hehn, 1964; Wrigley and Moss, 1968; Kaltsikes et al., 1968a; Kerber and Tipples, 1969; Simmonds and Orth, 1973; Schmidt et al., 1974; Bietz et al., 1975; Joppa et al., 1975; Kasarda et al., 1976a). Analyses of the protein composition and properties of wheats are becoming increasingly important not only in research (to identify new sources of genetic variability for the nutritional and agronomic improvement of wheats by breeding, and to better understand the biochemical genetic basis for the different processing quality properties of wheats), but also to trace the evolutionary history of the cultivated species. Certain proteins soluble in 70% ethanol (EtOH), for example, show little intraspecific variation but show distinctive interspecific variations. These EtOH-soluble proteins serve as markers useful for investigations into the phylogenetic and genetic relationships among wheat species and relatives (Hall et al., 1966; B. L. Johnson and Hall, 1966; B. L. Johnson, 1972a,b; Rodriguez-Loperena et al., 1975). A form of diploid einkorn wheat, T. monococcum (L.) MK, ssp. boeoticum (Boiss.) MK, or more likely T. urartu Tum., has contributed the A genome (Chapman et al., 1976), but not the cytoplasm (Suemoto, 1968, 1973; Kihara, 1974) or chloroplasts (Chen et al., 1975), to both tetraploid and hexaploid common wheats (Fig. 4). The “zanduri” group of wheats has evolved parallel to the common wheats. The hexaploid T. zhukovskyi Jakubz. (Genomes AAAAGG) is of most recent origin (B. L. Johnson, 1968), and is cultivated to only a small extent in Georgia, U.S.S.R. The donor of the D genome, Aegilops squarrosa (L.), which differentiates the common hexaploids from the tetraploid wheats, is the one other diploid progenitor of common wheats that is known with relative certainty (Kihara, 1944, 1968, 1974, 1975; Sears, 1975a). In evolutionary history, the D genome addition to the tetraploid genome (AABB) wheats is the most recent of the polyploidization changes that have occurred. The T . aestivum (L.) Thell., ssp. spelta (L.) Thell. (true spelt) progenitor

FIG.4. Probable origin and evolution of cultivated wheats and closely related wild relatives. Parallel evolution of common and zanduri wheat lines of descent. Taxonomy according to MacKey (1975). Schematic incorporates information from B. L. Johnson (1968, 1975, 1976), Maan and Lucken (1972), Shands and Kimber (1973), Xihara (1974), Chen et al. (1975), Chapman et al. (1976), and E. R. Sears (personal communication, 1976). 2’. = Triticum; Ae. = Aegilops; conv. = convariety ; RuDPCase = ribuIose-1,5-diphosphate carboxylase.

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of all common wheats arose cataclysmically through the union of unreduced gametes from a natural hybrid (McFadden and Sears, 1944, 1946; Kihara and Lilienfeld, 1949; Stebbins, 1951) between plants of cultivated tetraploid emmer species, T . turgidum (L.) Thell. ssp. dicoccum (Schrank) Thell. in the Caucasus-Eastern Turkey region, where it is believed that the natural hybridization and polyploidization took place. The putative primitive hexaploid T . spelta, which was created under natural conditions, has been artificially synthesized (McFadden and Sears, 1944,1946; Kihara and Lilienfeld, 1949). The main area of uncertainty today in wheat evolution studies concerns the identity of the B genome donor for the emmer progenitor of modern cultivated wheats. The B genome donor species is still unknown, although many of its characteristics have been determined through genetic and cytogenetic analyses. The most widely accepted hypothesis suggests that the diploid B genome donor is taxonomically identified as belonging to the Sitopsis section of Aegilops (Kihara, 1954, 1959, 1974; Sarkar and Stebbins, 1956). Prime candidates considered are Ae. speltoides Tausch. or subspecies Auscheri, Ae. longissima Schweinf. et Munsch, or Ae. bicornis (Forsk.) Jaub et Sp., or an as yet unidentified species or subspecies form. It is now well established that the cytoplasm of cultivated tetraploid and hexaploid wheats is similar to and derived from their B genome progenitor (Kihara, 1966; Maan and Lucken, 1972; Maan, 1973a,b,c). Certain experiments indicate that there is considerable similarity between the cytoplasms of the common wheats and that of Ae. speltoides (Suemoto, 1968, 1973) while other experiments indicate that their cytoplasms are distinctly different (Maan and Lucken, 1972; Maan, 1973a,b,c; Shands and Kimber, 1973). Recently, T. Mello-Sampayo (personal communication, 1976) has obtained evidence of a high degree of chromosome pairing between B genome chromosomes in tetraploid wheat and a variant of Ae. longissima which shows an intermediate amount of chromosome pairing with T . aestivum chromosomes (possibly due t o suppression of the 5B Ph locus (Larsen and Kimber, 1973 ; E. R. Sears, personal communication, 1976). Aegilops longissima appears to have cytoplasm similar to that of Ae. speltoides as well as a high degree of cytogenetic identity with individuals of that species (Maan and Lucken, 1972; Kimber, 1973) and is in fact considered a variety of Ae. speltoides by Morris and Sears (1967). However, in nature the two forms appear to have different mating systems and population structures (Hillel and Simchen, 1973). Aegilops speltoides is an outbreeder (cross-pollinating) while Ae. longissima is self-pollinated, like the wheats. Since the large subunit proteins of chloroplast fraction I protein ribulose-1,5-diphosphate carboxylase (RuDPCase) located in

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the chloroplasts of a t least one Ae. speltoides collection (and presumably, Ae. longissima and Ae. bicornis) are similar to those of T . turgidum ssp. dicoccum and T . aestivum (L.) Thell. (Chen et al., 1975), it seems quite plausible that a form of Ae. longissima may be the B genome donor to the progenitors of cultivated wheats. The evidence that the Ae. speltoides cytoplasm is the same as that of T . timopheevi (Zhuk.) M K (zanduri wheat) and that both cytoplasms bring about cytoplasmic male sterility in common wheats (Maan and Lucken, 1972) is contradicted by the evidence of Suemoto (1968,1973) indicating that the cytoplasm of another strain of Ae. speltoides does not induce cytosterility t o a tetraploid wheat when the genome transfer is made via backcrossing. The basis for this contradiction merits investigation, since i t suggests that cytoplasm variation exists within Ae. speltoides. This is not unexpected, however, since considerable variation appears to occur also among wild tetraploid wheats (Tanaka and Ishii, 1973). Aegilops speltoides was ruled out as the B genome donor for common wheats by both cytogenetic and protein electrophoresis studies (Kimber and Athwal, 1972; Shands and Kimber, 1973). However, the cytogenetic and biochemical genetic studies and cytoplasm-genome substitution experiments indicate that Ae. speltoides is the source of the second or G genome, recently reclassified as S, and of the cytoplasm for the T . timopheevi (Zhuk.) M K group of zanduri wheats, including cultivated form ssp. timopheevi as well as the wild form, ssp. araraticum (Jakubz.) MK = T. dicoccoides nudiglumis Nebalek (Maan and Lucken, 1972; Shands and Kimber, 1973; Kimber, 1973 ; MacKey, 1966, personal communication, 1975). An alternative hypothesis put forth by B. L. Johnson (1975) is that T. urartu Turn. is the source of the B genome while a form of T. boeoticum is the source of the A genome of both the T. araraticum and T. dicoccoides-T. diwccum lines of descent in wheat. Cytogenetic and morphological evidence shows that even though bivalent chromosome pairing is complete, the F, hybrid of T. boeoticum x T . urartu is sterile (B. L. Johnson, 1975; B. L. Johnson and Dhaliwal, 1976). However, fertility in the F, hybrid is restored in the amphiploid, in accord with widely accepted genetic principles behind the creation of successful polyploid species (Stebbins, 1947, 1951). Except for the presence of two quadrivalents (considered by B. L. Johnson, 1975; B. L. Johnson and Dhaliwal, 1976 as two chromosomal interchanges), normal closed bivalent pairing occurs in the amphiploid. Morphological features of the amphiploid closely resemble those of T. dicoccoides types (B. L. Johnson, personal communication, 1976). The cross of T . boeoticum X T. urartu is only successful with T . boeoticum as the female parent, based on over 700 attempts including crosses with a derivative with a largely

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T. urartu genome in T . boeoticum cytoplasm. The failure of T . urartu x T . boeoticum crosses to yield viable F,, except in culture, suggests that T. urartu cytoplasm may carry a factor that serves as an evolved genetic barrier causing failure of endosperm development, hence preventing the reciprocal combination (B. L. Johnson and Dhaliwal, 1976; Dhaliwal and Johnson, 1976a,b,c ; Dhaliwal, 1976). Thus, T . boeoticum is considered to be the donor of both the A genome and the cytoplasm to the tetraploid wheats. Electrophoresis studies indicate the presence in T. urartu of proteins found in polyploid wheats but not in any of a large number of T . monococcunz (L.) MK. forms examined. The cultured T. urartu X T. boeoticum hybrid is viable and similar to the reciprocal cross. Crosses of the synthetic tetraploid with T. boeoticuwa or T. urartu generally show six or more bivalents, rarely trivalent or other multivalent chromosomal associations (Dhaliwal and Johnson, 1976a,b,c; B. L. Johnson and Dhaliwal, 1976; Dhaliwal, 1976). However, according to new results of Chen et al. (1975) the chloroplast fraction I (RuDPCase) proteins of T. monococcum, ssp. boeoticum and ssp. monococcum are different from those of polyploid wheats, but surprisingly they are similar to those of Ae. squarrosa. Also, when both tetraploid and hexaploid wheat genomes are transferred to T . monococcum ssp. cytoplasms, the plants produced are both male sterile and weak in vigor (Maan, 1973b). Vigor and male fertility are restorable by crossing with R (male fertility restorer) lines carrying genes for fertility restoration from T. monococcum ssp. boeoticum or from T . zhukovskyi (Maan and Lucken, 1970, 1971a). The results demonstrate that these strong cytoplasmically controlled genetic isolation mechanisms are in fact manifestations of nuclear gene-cytoplasm interaction systems. Separate (possibly linked) genes may be concerned with the restoration of vigor and with male fertility. On the basis of the evidence reviewed above and on the facts that (1) cytoplasm variation (Kihara and Tsunewaki, 1968; Maan and Lucken, 1972; Suemoto, 1973; Mukai and Tsunewaki, 1975; Tsunewaki and Endo, 1973) and (2) chloroplast RuDPCase protein variation (Chen et al., 1975) occurs among related Aegilops species, (3) T . boeoticum and T . uartu show the same RuDPCase large subunit proteins as Ae. squarrosa, and (4) T . boeoticum is an old and widely distributed species showing considerable genetic variability, as does T . urartu, it seems possible that variants of T . boeoticum with chloroplast RuDPCase proteins like the bread wheats yet may be found (B. L. Johnson, 1976, and personal communication). Still other evidence, however, tends to contradict the hypothesis that a form of T . urartu provided the B genome, since a high degree of pairing homology was obtained for T. urartu

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chromosomes with the ditelocentric chromosomes of the A genome, but little association with the B genome ditelocentric chromosomes of hexaploid wheat (Chapman et d , 1976) . Electrophoretic analyses indicate that the esterase and acid phosphatase isozymes of T. urartu are different from those in any other diploid wheat species (Jaaska, 1974). The value of isozyme variations for distinguishing genome homoeologies is questionable (Nakai, 1973), although Tang and Hart (1975) and Hart et al. (1976) have demonstrated homoeology of genes on different genomes using isozymes. The reduction of SS bonds by mercaptoethanol prior to sodium dodecyl sulfate (SDS) electrophoresis has revealed the several expected albumin protein bands of T. boeoticum, which complement those of T . urartu to produce the typical isozyme pattern of the tetraploid wheats (B. L. Johnson and Dhaliwal, 1976, and personal communication). The certainty with which chromosome pairing is viewed as being an indication of homoeologies, is also questionable since chromosome pairing itself is controlled by specific genes (Kimber, 1966; Mello-Sampayo, 1971a,b ; B. L. Johnson, personal communication, 1976). A third concept for the origin of the B genome which is currently being investigated involves the possibility that the B genome may be a complex involving whole chromosomes or parts from one or more donors, brought about following the creation of a primary tetraploid form. The primary tetraploid form would produce triploid hybrids with one or several diploid species growing in close association. Fixation of similar tetraploid forms which also hybridize freely to exchange genes would eventually occur, giving rise t o one or a few related forms able to compete successfully with diploids. These new tetraploids could then carry similar A and B genomes and cytoplasm, but their B genome chromosomes might be a mixture or a complex unlike any one of the diploid parental forms contributing chromosomes or parts of chromosomes to that complex. The occurrence of sufficient geographic separation and/or evolution of genetic isolation mechanisms (via mutation or hybridization) would prevent the breakdown of new tetraploid forms by free hybridization with diploid species. The increased adaptability and larger size of the polyploid forms may have made them successful in a wider range of environments, and eventually they became attractive to man as food sources. This concept, essentially of a pivotal form, is embodied in works by Kimber (1973), Kimber and Athwal (1972), Harlan and Zohary (1966), Zohary (1966), Tanaka and Ishii (1973), Vardi (1973), and Harlan (1975). The various cultivated forms of the tetraploid and hexaploid wheats have evolved naturally along separate paths, in part through conscious (though unplanned) selection by man for adaptability to cultivation and

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for specific food and feed uses, as well as for their interesting and attractive differences in plant, spike, and grain morphology (Harlan, 1970, 1975; Harlan and Zohary, 1966; Harris, 1967; Zohary, 1973). With regard to protein content of the various species, Dunstone and Evans (1974) compared cell size, grain size, plant and leaf structure, and percentage nitrogen us grain weight of a number of lines of diploid, tetraploid, and hexaploid wheat species. They noted increases in cell size with polyploidy, but cell size did not increase proportionately with genome content. They suggested that protein content decreased with increasing ploidy. However, recalculation of their data shows that N content per seed is considerably greater for the polyploids and for the cultivated forms. All polyploid cultivated forms have a similar N (protein) content per seed. Recent analyses of a large number of varieties in the USDA World Wheat Collection showed that T. monococcum accessions did not possess a superior percentage of protein as compared with that available in tetraploid and hexaploid wheats (V. A. Johnson and Mattern, 1975; V. A. Johnson et al., 1975b). IV. Genetic Relationships and Homoeologies among Chromosomes of Cultivated Wheats

The cytogenetic, genetic, and biochemical genetic relationships established among and between the 21 pairs of chromosomes in hexaploid wheats show that sufficient homology and genetic compensating ability still exists between the chromosomes of the A, B, and D genomes to consider the three sets of seven chromosomes as nearly homologous or homoeologous series, making possible the construction of unique sets of aneuploid and related stocks useful for genetic analyses. Thus, chromosomes lA, lB, and 1D; 2A, 2B, and 2D, etc., form different homoeologous series, in which any one pair will nearly compensate for the loss or absence of any pair of the other two homoeologous chromosomes in the genome. Evolutionary changes occurring prior to and after and natural synthesis of the tetraploid or hexaploid wheats have differentiated the genetic content of the chromosomes in each homoeologous series such that the compensation is incomplete, and the degree of compensation differs for different homoeologous series (Sears, 1954, 1969). Possibly because of their longer evolutionary history, the chromosomes of tetraploid wheats have become so differentiated that the monosomic series (lacking one chromosome of a pair) were difficult to construct, and the fertility of such plants is comparatively poor (Longwell and Sears, 1963; NoronhaWagner and Mello-Sampayo, 1966; Sears, 1975a). All 14 monosomics of

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durum wheat now have been produced; all are viable, grow well, and set some seeds (Mochizuki, 1968; Joppa, 1973; Sears, 1969, 1975a). Even in hexaploid wheat, many nullisomics and ditelosomics are subnormal, nearly or completely sterile, while nearly all the 42 nullitetrasomics (i.e., lacking a pair of homoeologous chromosomes from one genome but carrying two pairs of a homoeologous chromosome from one of the other two genomes) are reasonably fertile and have essentially normal growth. Likewise, several members of ditelocentric series in hexaploid wheats are viable, but are nearly or completely sterile. These are maintained as heterozygous individuals carrying one or two doses of the other telocentric chromosome arm (Sears, 1954, 1974, 1975a, and personal communication, 1976). Such results demonstrate that, although genetic compensation exists, evolution has produced differentiation in the form of male or female sterility controlled by genes on a particular arm of a chromosome of the series. An extensive array of special genetic stocks has been developed by E. R. Sears and colleagues, not only a t the University of Missouri, but also a t Cambridge, England, and more recently a t many other research laboratories over the world. Lists of the available stocks and progress reports on the construction of new genetic stocks can be found in the European Wheat Aneuploid Cooperation Newsletter published a t the Plant Breeding Institute, Cambridge, e.g., E. W. A. C. Newsletter 4: 1974. Included among the interesting recent developments are the addition and substitution of specific chromosomes from rye (Secale), Agropyron, and Aegilops umbellulata, into wheat, and the progress toward determining their homoeologies (Sears, 1975a). More recently, chromosomes from barley (Hordeum) have been introduced into wheat (Islam et d., 1976). The many diverse genetic stocks constructed already have made wheats unique for genetic, cytogenetic, biochemical genetic, and allied research. Ways in which these stocks may be used in genetics investigations have been described by Kuspira and Unrau (1957, 1958), Law (1968), Law and Worland (1972, 1973), Sears (1954, 1975a), Schmidt et al. (1966), Unrau (1958), Tang and Hart (1975), Hart et al. (1976), and H a r t (1976). V. Role of Environmental Factors in Protein Synthesis and Accumulation in Wheat Endosperm

Some of the nutritional, and most processing characteristics of wheat grain are determined by the quantity and composition of proteins. Although all wheat quality characteristics are genetically controlled, it has

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long been known that the cultivation environment is an important modifier. Numerous studies show N availability and water supply to be the most important environmental factors, but soil and atmospheric conditions, especially temperature, light intensity, and photoperiod, as well as prevailing diseases, may also exert strong effects (Finney and Fryer, 1958; Hehn and Barmore, 1965; Kolderup, 1975; Reitz, 1964). Nitrogen availability, temperature, and water supply influence mainly the quantity of proteins accumulated by a genotype, but those factors also may affect the relative proportions of the different amino acids and protein types (Bushuk and Wrigley, 1974; Jahn-Deesbach and Jurgens, 1973; J. A. Johnson et al., 1972; V. A. Johnson et al., 1973; Kasarda et al., 1971; Kolderup, 1975; Mitra and Bhatia, 1973; Orth and Bushuk, 1973b, Pomeranz et al., 1966b, 1974, 1975a; Rhodes and Jenkins, 1975). A. INFLUENCE ON AMINOACID COMPOSITION Lawrence et al. (1958) observed that the lysine composition of wheats varied inversely with protein content and noted differences in the protein composition among wheat varieties and species in response to the cultural environment. A negative curvilinear relation of lysine to protein content was observed in analyses of over 12,600 common wheats in the United States Department of Agriculture (USDA) World Wheat Collection by V. A. Johnson, Mattern, and colleagues of the University of Nebraska, USDA-ARS: United States Agency for International Development (AID) Program (V. A. Johnson et al., 1968a,b, 1975a,b; Vogel et al., 1973; V. A. Johnson and Mattern, 1972, 1975). The negative curvilinear relation for lysine was most pronounced in the range between 7 and 15% protein. I n the range above 16% protein there was little influence of protein and lysine content on lysine per unit protein. Juliano (1972) showed a similar relationship of lysine per unit protein for protein variability above 10% in rice. The variability in protein and lysine content among 6000 durum wheats in the USDA collection was much like that found for common wheats (V. A. Johnson et al., 1973, 1975b). In a later study, less than 1% of 3399 durums analyzed showed significantly higher lysine/protein than the average, while about 4% showed higher than average protein (V. A. Johnson and Mattern, 1975). The nonlinear relations of lysine to protein content complicate the identification of the genetic vs environmental components of lysine variation. Over 50% of the variation in lysine content per unit protein in wheats from the USDA World Wheat Collection is attributable to variation in protein content, largely due to environmental or

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nongenetic causes (V. A. Johnson et al., 1973; Vogel et al., 1973). Since nitrate availability of the cultural environment (Chmeleva and Medredev, 1973; Gunthardt and McGinnis, 1957; Jahn-Deesbach and Jiirgens, 1973; Mitra and Bhatia, 1973) or chemical treatments using Terbacil and Simazine (Strbac et al., 1974) may disproportionately increase the amounts of the lower lysine gluten proteins, it is not surprising that a nonlinear relation of lysine to total protein was observed for a wide range of genotypes (V. A. Johnson and Mattern, 1975; V. A. Johnson et al., 1975b). Heritability estimates for dibasic amino acid (DBAA) content of wheat were only about 27% for F,/F, and F,/F, generation comparisons for 51 wheat lines grown in 6 environments, but they were 96% for thousand-kernel weight (TKW) and 77% for flour recovery (Sharma et al., 1973). A negative relation of lysine to protein (and differences in genotype-environment interactions), also was observed for a group of winter wheats grown in an international array of environments (Stroike and Johnson, 1972). Analyses of essentially single-background genotypes grown in a single environment (location/year) in some cases show a nearly linear relationship of Udy protein or DBC values (based on pH 2.0 buffered acilane orange dye-binding capacity, a measure of dibasic amino acids, including lysine; Udy, 1971) relative to total protein (Siddiqui and Doll, 1973; C. F. Konzak, unpublished data on induced mutations, 1974). Siddiqui (1972) also observed different regression values, but essentially linear relations of DBC value us protein for substitution lines of ‘Hope’ and ‘Timstein’ us S-615 in the same (Chinese Spring) background. Thus, it seems that the variability of genotypeenvironment interactions for protein and lysine may be appreciably less for materials of similar genetic background (as induced mutants) if the cultural environment is also relatively uniform. The increased content of gluten proteins induced by higher nitrate availability may improve the quality of the protein for breadmaking (Kasarda et al., 1971; P. J. Mattern, personal communication, 1974). However, it is not yet clear whether culture under high nitrate availability will increase or reduce the chances for identifying high lysine-producing genotypes. Presumably the total protein production and the amino acid composition of the proteins are controlled by different genetic systems. Results of a study with barley varieties ‘Hiproly’, ‘Risg mutant 1508’, and ‘Maris Mink’, suggest that the greatest differences in grain protein content will be found under conditions of relatively low N supply, while lysine content differences may be relatively stable and unaffected by N supply. However, such results should not be taken as representative of all potential genetic sources for protein and lysine improvement, as they concern only one high-protein and two high-lysine “drastic” mutant gene sources in one crop, barley.

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B. INFLUENCE ON PROTEIN SOLUBILITY FRACTIONS Solubility fractionation studies indicate that the protein components of wheat flours may be somewhat disproportionately affected by culture under different environmental regimes (see Kasarda et al., 1971; Mitra and Bhatia, 1973; Orth and Bushuk, 1972, 1973b). This may to a certain extent result also from a hastened or extended maturity, due to weather conditions in particular locations and years. However, Tanaka and Bushuk (1972) observed little influence of protein content on the proportion of proteins in five modified “Osborne” solubility groups (albumins = water soluble, globulins = salt water soluble, gliadins = alcohol soluble, glutenins = acetic acid soluble, insoluble residue). Endosperm solubility fractionation analyses of 70 wheat samples from the Northern Regional Performance Nursery showed that, as protein content increased, saltsoluble proteins decreased according to similar but opposite trends. However, the percentage of acid-soluble proteins decreased slightly and alkaline-soluble proteins decreased moderately with increasing protein content (Ulmer, 1973; V. A. Johnson and Mattern, 1975). These results suggest definite trends toward an increased proportion of prolamine proteins as protein content increases over different environments and are consistent with the observation of lower 1ysine:protein ratios a t higher protein contents. C. GENETICREGULATION AND CONTROL OF PROTEIN CONTENT, COMPOSITION, AND PROCESSING PROPERTIES The functional or processing properties of wheat and wheat flour also may show responses to environmental conditions, and these may in part be a reflection of the influence of environment on the quantity and composition of wheat proteins (Kasarda et al., 1971, 1976a; Pomeranz, 1971). Some of the environmental interactions involved are variety dependent, and thus genotype dependent (G. Rubenthaler, personal communication, 1974; J . A. Johnson et al., 1972; V. A. Johnson et al., 1972a,b; Orth and Bushuk, 197313; V. A. Johnson and Mattern, 1975). Sharma et al. (1973) compared thousand-kernel weight (TKW) , DBC value, sedimentation value (SV) and flour recovery (FR) for 16 varieties and 51 line selections grown under 6 environments over 2 crop seasons in India. They found that the varieties and selections differed significantly from one another with regard to the reEponse of each trait to environments favoring increased response. Of the four traits studied, TKW and FR were most stable and had high heritability as calculated from the regression of F, upon F, values. Least stable was DBC value, which also had the lowest

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heritability. Biuret protein values for the 16 varieties indicated that protein content was consistently higher for some varieties than for others. Jain et al. (1975, 1976) determined that high TKW has great selective value for advancing protein yield in wheat. I n a similar, but more detailed study, McGuire and McNeal (1974) showed that bread wheat varieties diflered with respect to their loaf volume, mixing-time requirements, and flour yields in response to Montana State environments favoring increases in those quality parameters. The comparably high us low flour yields characteristic of ‘Fortuna’ and ‘Centana’, for example, were relatively stable to culture under environments in which other varieties showed either increased or decreased flour yields. Likewise, the variety ‘Manitou’ showed a low response to environments favoring increases in mixing time, while the variety ‘Sheridan’ showed a relatively high response to the same environments. Such similar results from studies conducted continents apart strongly indicate that the responsiveness us stability of kernel characteristics and protein composition and functional (breadmaking) properties to environmental influences is genetically controlled. Environmental factors may assert considerable influence on the lysine composition, but the genotype-environment interactions also may be subject to genetic regulation. The environmental responsiveness of protein and lysine/protein composition of several wheat accessions selected for lysine content from the USDA World Collection was found by V. A. Johnson et al. (1972) and V. A. Johnson and Mattern (1975) to vary rather widely over different years and environments, but certain varieties had high protein or high lysine and high protein more consistently than others. Genotype-related differences in grain yield, protein, and lysine also were observed for wheat and triticale varieties grown a t several test locations (Ruckman et al., 1973). Likewise, durum wheat selections from local Sicilian populations showed marked differences in protein content stability when grown a t 3 different locations in Italy (Porceddu et al., 1973). High molecular weight (MW) glutenin is already present in the endosperm at 14 days past flowering, and the accumulation rates of different solubility classes of proteins vary for different wheats grown under glasshouse conditions. Ratios between gliadin and glutenin proteins change during endosperm development, suggesting that genetic factors also control the rates a t which the various storage proteins are accumulated in the endosperm (Bushuk and Wrigley, 1971). High-protein wheats synthesize greater amounts of protein in their subaleurone tissues (Barlow et al., 1974). The results indicate that there is ample opportunity for environment to modify the final protein composition, depending on the degree of genetic control (regulation) asserted over individual proteins

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by varieties (see also Koenig et al., 1964; Mitra and Bhatia, 1973; Manghers et al., 1973). However, a large number of other studies, mainly involving electrophoresis techniques, show that the structure, production (presence or absence), and dosage of specific proteins are under very strong, if not absolute, control of genes (Barber et al., 1968a,b; Bietz and Wall, 1972, 1973a; Bietz et al., 1975; Doekes, 1968, 1973; Doekes and Hack, 1971; Elton and Ewart, 1962; Ewart, 1966a,b; Feillet and Bourdet, 1967; Kasarda et al., 1976a; B. L. Johnson et al., 1967; B. L. Johnson, 1972a,b, 1975; J. A. Johnson et al., 1972; V. A. Johnson et al., 1972; May et al., 1973; Orth and Bushuk, 1973b; Shepherd, 1968; Silano et al., 1969; Wrigley and Shepherd, 1973; Tang and Hart, 1975; Hart et al., 1976). Even in a synthetic polyploid like triticale, it has been shown that the genetic systems for protein synthesis derived from the two parents, durum and rye, both function throughout plant development, essentially affirming the independence and additivity of the genetic systems combined in one nucleus and working in one cytoplasm (Riley and Ewart, 1970; Dexter and Dronzek, 1975a,b; Tang and Hart, 1975). VI. Role of the Cytoplasm in the Hereditary Control of Protein Content and Composition

An understanding of the important cellular components and mechanisms involved in the synthesis of amino acids and proteins may prove to be useful in devising new approaches to the nutritional improvement of proteins (see also Woodbury, 1972). The cytoplasmic proteins are generally among the water- and salt-soluble classes of proteins, some of which contain a high proportion of lysine. Recognized control mechanisms and interrelationships between nuclear genes and cytoplasmic organelles suggest some interesting possibilities and novel approaches toward the goal of improved protein nutritional value. A. CYTOPLASMIC ORGANELLE-NUCLEAR GDNBINTERACTIONS Nuclear-cytoplasmic interactions involved in the synthesis of plant proteins are indicated in a wide variety of investigations, but only in a few cases is there strong documentary evidence. Beck et al. (1971) have reported that 63 nuclear gene loci control the biogenesis, structure, and function of yeast mitochondria, while von Wettstein et al. (1971) have described 198 chloroplast mutations controlled by 86 genetic loci in barley. Nuclear genes in barley maintain a very strong control over both

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pigment synthesis and chloroplast biogenesis (Henningsen et al., 1973 ; von Wettstein et al., 1974). On the other hand, several maternally inherited chlorophyll mutants in barley are well known (Nilan, 1964; Robertson, 1971). In wheat, the genome of the cultivar ‘Salmon’ was found to perform better in Aegilops caudata cytoplasm than in its own (Kihara and Tsunewaki, 1964). Similarly, field trials of several alloplasmic lines derived by substituting the genomes of two hard red spring T . aestivum cultivars ‘Chris’ and ‘Selkirk’ into cytoplasms from T . macha, T . dicoccoides, or Ae. squarrosa, showed some alloplasmic lines t o be significantly earlier heading and higher yielding than the original cultivars (Busch and Maan, 1974). Defective chloroplasts in a variegated mutant of Nicotiana tabacum L. carry a mutation in chloroplast DNA (Wong-Stahl and Wildman, 1973). The DNA alteration was identified by a difference in DNA buoyant density in CsC1, and by EM analyses which showed a mismatched DNA region corresponding to about 500-1000 base pairs. At least 40% of the genetic information in chloroplast DNA is expressed in mature chloroplasts ; but this genetic information accounts for only four proteins-about 10% of the identifiable products ; i.e., the results suggest that chloroplast DNA codes for relatively few structural and enzymic proteins and that its major function is concerned with regulation. Close associations between the informational contents of chloroplast DNA and nuclear DNA have been demonstrated in several other systems (see Kawashima and Wildman, 1971; Wildman et al., 1973, 1975). I n Nicotiana, nuclear DNA is known to code for (1) two of the numerous ribosomal proteins composing the 50 S subunit of the chloroplast ribosome (Bourque and Wildman, 1972), (2) a thylakoid membrane protein to which chlorophyll pigments of photosystem I1 are attached for ferredoxin (about 12,000 MW) which is involved in electron transport and is required for the photosynthetic function of chloroplasts, and (3) the large subunit of fraction I (RuDPCase) protein (Wildman et al., 1975). It is known, however, that RuDPCase is rapidly converted in the dark to membrane proteins (Wildman et al., 1973; Kannangara, 1969; Huffaker and Peterson, 1974). RuDPCase catalyzes the critical step of CO, fixation and also serves as an important temporary protein storage system in the plant (Kannangara, 1969; Huffaker and Peterson, 1974). About 40% of the soluble protein in the green plant leaves appears to be in the form of RuDPCase (Chan et al., 1972; Wildman et al., 1973). The mechanism for breakdown of this protein, the nature of the proteases responsible, the size of the peptide units released and their method of transport to the seed have not been determined. The chloroplast DNA also has been shown to carry two genes that

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code for its ribosomal RNA as determined from analyses of the buoyant density of chloroplast DNA and analyses of 23 S and 16 S rRNAs (Thomas and Tewari, 1974; see, also, Whitfeld et al., 1973). There also may be a physical interaction between chloroplasts and the cytoplasm via the vehicle of mitochondria. The movement of mitochondria into and out of the chloroplasts and their apparent involvement in starch granule accumulation in living cells has been photographed (Wildman et al.. 1974). CHLOROPLAST-NUCLEAR GENECONTROL OF B. JOINT RIBULOSE-1,5-DIPHOSPHATE CARBOXYLASE In tobacco species hybrids, chloroplast and nuclear DNA each code for different subunits of the dimeric fraction I protein, RuDPCase (Singh and Wildman, 1973; McFadden, 1973; McFadden and Tabita, 1974). A similar system exists in peas (Boulter et al., 1972). The large subunit of RuDPCase is made on chloroplast ribosomes in peas and in wheat (Roy and Jagendorf, 1974). RuDPCase is the enzyme responsible for photorespiration, and the oxygenase function is located in the large subunit (Andrews et al., 1973; Bowes et al., 1971; Bowes and Ogren, 1972; Lorimer et al., 1973; Ogren and Bowes, 1970). The large subunit of RuDPCase of N . tabacum has a molecular weight (MW) of 56 kilodaltons (Kung et al., 1974a). The small subunit of this enzyme is comprised of two polypeptides of 125 kilodaltons and is made in the cytoplasm on 80 S ribosomes. The large subunit of the RuDPCase of different tobacco species hybrids is comprised of three polypeptides, and the small subunit is comprised of one to four polypetides (Kung et al., 1974b). Each of 15 species of Nicotiana has a different combination of polypeptides, as indicated by the isoelectric points of the proteins on electrophoresis. I n the hybrid N . glutinosa P x N . tabacum 8 , the three large subunit polypeptides were the same as in the female parent (two from each in the doubled hybrid). Changes in the “Osborne” solubility properties of the fraction I proteins are correlated with changes in the composition of the small subunit (Sakano et al., 1974a,b). Fraction I protein thus appears to be an unique genetic marker (Kung, 1976). The large RuDPCase subunit proteins coded for by chloroplast DNA are similar or identical for an Ae. speltoides accession, the tetraploid wheats, T . dicoccum, T . turgidum, T . timopheevi, and the hexaploid wheat T. aestivum, but are distinctly different in electrophoretic mobility from those of T . boeoticum, T. monococcum, T. urartu, and Ae. squarrosa (Chen et al., 1975). Their results (albeit based on single accessions of each species) suggest that the chloroplast genome is similar for Ae.

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speltoides and cultivated polyploid wheats, supporting other evidence that a n Aegilops species similar to Ae. speltoides may be the B genome progenitor of our modern wheats. The results also demonstrate th a t the products of ribosomes under the genetic control of chloroplast DNA (several genes) may interact with the products of ribosomes under the control of nuclear genes to synthesize the important plant enzyme RuDPCase (Wildman et al., 1975). C. CHLOROPLAST-NUCLEAR GENEINTERACTIONS IN NITROGEN REDUCTION Activity of the enzyme nitrate reductase, which reduces nitrate (NO,) to nitrite (NO,), may be associated with grain protein content of wheat. Nitrate reductase is the first, and possibly the main, limiting enzyme involved in the synthesis of protein from nitrate, the primary form of N utilized by plants. Cereals can also utilize some ammonium N, but the ammonium form of N is rarely available in soils. The nitrite reductase function (NO,+NH,) seems to be located within the chloroplasts (Polya and Jagendorf, 1971a,b ; Jaworski and Key, 1972; Dalling et al., 1972; Boulter et al., 1972; Magalhaes et al., 1974; Neyra and Hageman, 1974). The control of nitrite reductase by chloroplasts may be one of regulation via chloroplast DNA, although the requirement for energy and great efficiency of nitrite reductase suggests that this enzyme could be localized in chloroplast ribosomes. Ferredoxin, also located in the chloroplasts, is required for nitrite reductase activity and for RuDPCase activity (Paneque et al., 1964; Miflin, 1974; Warner and Kleinhofs, 1974; Neyra and Hageman, 1974; Wildman et al., 1975; Kwanyuen and Wildman, 1975 ; Klepper, 1975).

D. CYTOPLASM SOURCE AND PROTEIN COMPOSITION IN WHEAT Relatively little specific information is available about the effects of nuclear-cytoplasmic interactions on wheat protein content and amino acid composition. Apparently the T. timopheevi cytoplasm, extensively used in hybrid wheat development, causes no appreciable modification in use quality properties of the grain from tetraploid or hexaploid wheats. Electrophoretic studies are needed to prove that protein differences do not exist. It is known that several restorer lines in T. timopheevi cytoplasm have high protein (G. Rubenthaler, personal communication, 1974; V. A. Johnson and Mattern, 1975). Differences between the electrophoretic patterns of acid phosphatase proteins produced by triticales with cytoplasm from wheat us cytoplasm from rye, have been observed

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recently, providing further evidence that the control of certain proteins in the Gramineae involves nuclear-cytoplasmic “gene” interactions (J. Rimpau and G. Robbelen, personal communication, 1974). Marked differences in the effects of different cytoplasm sources on gene expression in hexaploid and tetraploid wheats, including effects on spike fertility and characteristics as well as on plant development and vigor, have been reported (Maan and Lucken, 1971a,b, 1972; Maan, 1973a,b, 1975; Suemoto, 1973; Tsunewaki and Endo, 1973; Endo and Tsunewaki, 1975; Mukai and Tsunewaki, 1975). Two malate dehydrogenase (MDH) isozymes of rye were absent when single chromosomes of rye were added to wheat, suggesting that rye cytoplasm plus two rye chromosomes may be required for their expression, or that the rye M D H genes were epistatic to those of wheat (Bergman and Maan, 1973). VII. Nuclear Gene Control of Protein Content and Composition

Nuclear genes appear to carry the codons specific for nitrate reductase and for many other enzymes, as well as for enzyme inhibitors and storage proteins, although there are indications that some structural proteins are coded for by mitochondrial DNA. A. GENETICCONTROL OF 80 S RIBOSOMAL PROTEINS The number of 80 S ribosomal proteins identifiable by two-dimensional gel electrophoresis is correlated strongly with chromosome numbers when the protein extractions were prepared from hexaploid and tetraploid wheat, diploid barley, diploid rye, common bean, garden peas, or spinach (Nagabhushan et al., 1974). The correlations between chromosome number and 80 S ribosomal proteins were highest for the hexaploid and tetraploid wheats and diploid barley, as compared to the other crop plants, and strongest for the number of basic us acidic or total proteins. The lower correlation of acidic proteins with chromosome number suggests a stronger contribution of cytoplasmic organelles to their control. Evidence for nuclear gene control over mitochondria has been reported recently ; the mitochondrial mass and mitochondrial DNA in diploid yeast is nearly double that in haploids (Grimes et al., 1974), the nucleolus organizer appears to be an important site for RNA synthesis (Jain et al., 1968a,b). Group 1 chromosomes are involved in the control of RNA genes in wheat (Flavell and Smith, 1974a,b). Further details about the cytoplasmic systems and organelles involved in protein synthesis are described by Boulter e t al. (1972) and Hertel (1974).

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Current information about ribosome structure has been reviewed recently (Maugh, 1975). A summary of the proteins now known to be controlled by genes in wheat is presented later.

B. NITRATEREDUCTASE SYNTHESIS AND GENETICCONTROL OF NITROGEN REDUCTION Nitrate reductase is synthesized on the 80 S ribosomes and appears to be localized in the cytoplasm of plant cells. Nitrite reductase which further reduces the nitrite to ammonia is localized in chloroplasts. Nitrate reductase activity (NRA) is NADH (reduced nicotinamide adenine dinucleotide) dependent with requirements both for photosynthesis and respiration ; whereas increased CO, concentration causes an increase in malate but a reduction both in nitrate accumulation and NRA (Wells and Hageman, 1974; Aslam et al., 1973; Purvis et al., 1974), illustrating the competition for photosynthetic energy by carbohydrate production and nitrogen metabolism. The pivotal compound involved in the energy (electron) supply is ferredoxin. Ferredoxin donates electrons directly to nitrate reductase, but to fix C02 in photosynthesis, ferredoxin first reduces NADP to NADPH. Nitrate reduction utilizes 3-phosphoglyceraldehyde, the first sugar formed by photosynthesis, which passes from the chloroplast to the cytoplasm, where it is oxidized by NAD-dependent triose phosphate dehydrogenase, resulting in the reduction of NAD to NADH, which also is used for nitrate reduction. Nitrite formed in the first reduction enters the chloroplast for further reduction by direct photosynthetic energy as indicated above. I n the reduction of a single nitrate molecule to NH,, 8 electrons may be used in reducing 4 molecules of CO, to sugar (Klepper, 1975). NRA in wheat increases with N fertilization (Hernandez et al., 1974), indicating that the synthesis of nitrate reductase is induced by nitrate. Both nitrate and nitrite reductase from wheat are quite unstable in vitro but have been electrophoresed (Upcroft and Done, 1974). 1. Genetic Regulators or Inhibitors of N R A

A low-MW protein isolated from several plant species apparently serves a regulatory function in stimulating in vivo NRA (Ku, 1973). NRA inhibitors occur in leaves of several plant species-corn, beans, and spinach (Ku et al., 1973). The low-MW type inhibitors are heat stable. The lowMW inhibitors appear to be organic acids. One high-MW inhibitor is heat labile, another is heat stable. The heat-labile, high-MW inhibitor appears to be a protein, the heat-stable high-MW inhibitor has not yet been characterized (Purvis et al., 1974; Ku et al., 1973). Malate con-

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centration in eight maize genotypes was negatively correlated with nitrate concentration. An enzyme isolated from maize roots causes the inactivation of NRA, and the in vitro inactivation of NRA by the enzyme was prevented by including phenylmethyl sulfonyl fluoride (but not by other trypsin inhibitors) in the extraction medium, suggesting the involvement of a serine residue a t the active site of the inhibitor enzyme. The enzyme involved may be a protease (estimated MW 44,000) which, like NRA, is located in the cell cytoplasm and shows optimum activity a t neutral pH. An extract containing the NRA-inactivating enzyme was found to degrade casein but had no peptidase activity (Wallace, 1974). The increased stability of NRA by the use of 3% (w/v) bovine serum albumin or casein in the NRA extraction medium (Schrader e t al., 1974) suggests involvement of the same enzyme studied by Wallace (1974). The biochemical evidence for regulators and inhibitors of NRA also supports wheat genetics data. Nitrate reduction in wheat is under the control of genes on several chromosomes from each of the three genomes; chromosomes 2A, 7A, and 7B of Chinese Spring were found to carry inhibitors or regulators of NRA (Edwards, 1973,1974).Some of the results reported by Edwards (1974) were not confirmed by E. L. Deckard (personal communication, 1975), although those for chromosome 2A have been confirmed by Warner and Konzak (1975). The substitution of Chinese Spring chromosomes 2A, 2B, and 2D by the homoeologous chromosome from rye significantly increased grain protein content (Jagannathan and Bhatia, 1972; N. Darvey, personal communication, 1974), suggesting that a homoeologous series of NRA inhibitors may be located in the group 2 chromosomes of common wheat. The evidence for inhibitors of NRA, and their identification with particular chromosomes, provides a basis for the genetic removal of these factors as a means to increase the genetic potential for protein production in wheat. Analyses of the group 2 and 7 nullitetrasomie and ditelosomic lines for the inhibitor enzyme may permit greater resolution of the cellular function of the enzyme and its role as an inhibitor of NRA. A number of mechanisms for enzyme regulation have been suggested (Hammes and Wu, 1971). Wheat varieties differ in their response to additional nitrate, in their ability to utilize available soil nitrate, and in their ability to translocate the N into grain protein (V. A. Johnson e t al., 1967, 1973, 197513; V. A. Johnson and Mattern, 1975; Edwards, 1973, 1974; Hernandez et al., 19741, as do oat varieties (Cataldo et al., 1975). Genetic variation for NRA apparently also occurs between individuals within a cultivar (Klepper, 1975), indicating that selection to improve protein productivity may be effective in cultivars derived from F2 to F, populations or from bulks of lines.

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2. Genetic Variation in Response to N and in Protein Content

Seasonal levels of NRA among several wheat genotypes may vary 2-fold or more, and there may be marked differences in the amounts of N translocated to the grain (Edwards, 1973, 1974; Dalling et al., 1975). Nitrogen translocation efficiency also differed among wheat varieties and was correlated with grain protein yield, but not with percent grain protein. Genetic studies demonstrated that NRA was highly heritable but polygenically controlled. Predicted responses to selection for high NRA were 2- to 3-fold greater than the predicted response for yield on protein. Sampling for NRA a t the onset of tillering appeared to be highly effective for selecting high-yielding lines, but was less effective for protein content. Similarly, NRA was associated with vegetative N in wheat (Eilrich and Hageman, 1973; Hernandez et al., 1974; Dalling et al., 1975) and in oats (Cataldo et al., 1975), but not necessarily to grain N. Wheat varieties differ not only with relation to the level of nitrate necessary to induce NRA but also with relation to the rate of response of NRA to increasing nitrate and with relation to the maximum level of nitrate able to induce an increase of NRA (A. Brunetti, personal communication from A. Bozzini, 1974). Wheat varieties may show different seasonal patterns for total NRA per plant and total N content per plant. Dalling et al. (1975) found the cumulative seasonal NRA to be highest for the cultivar ‘Olympic,’ followed by ‘Petit Rojo’ and lowest for ‘Gatcher.’ The selection ‘Argentine IX’ showed the steepest regression slope for total N/per plant with total NRA, with Olympic and Petit Rojo next and about equal. However, the varieties also differed considerably in their ability to translocate N to the grain. Olympic had the lowest translocation efficiency, and Petit Rojo and Gatcher had about equal efficiency. Two groups of varieties showing parallel variation for total seasonal NRA, were distinguishable on the basis of above and below 30% N translocation efficiency (Dalling et al., 1975). The results suggest the independence of NRA and N translocation to the grain, with perhaps fewer genetic factors controlling N translocation. High grain protein thus requires high NRA, plus ability for high N translocation from vegetative to storage tissues (Klepper, 1975). Nittler and Jensen (1974) also observed differences in the ability of barley varieties to respond to available N after being deprived of nitrate in culture solutions for 2 weeks, suggesting that the varieties may differ in their ability to grow under or tolerate conditions of low nitrate availability. The high-protein, high-lysine barley variety ‘Hiproly,’ was found to transfer more N to the grain (per grain basis) and do so longer during grain development than ‘Maris Mink,’ a standard low-protein cultivar (Rhodcs and Mathers, 1974). Hiproly was

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found to take up more nitrate from sand culture media than ‘Ymer’ barley, and to translocate more nitrate to the seedling tops in pots over a range of N nutrition levels (Edgar and Draper, 1975). When grown to maturity under gravel culture conditions in a glasshouse, the grain of Hiproly was found to have double the protein content of Risg 1508 and Maris Mink a t the lowest N level, whereas all had similar protein (gm/100 gm dry matter) a t the high N level. The protein of RisZ 1508 contained significantly more lysine than either Hiproly or Maris Mink a t all N levels. Wheat varieties also may differ markedly in their relative yield response to low vs high inputs of N fertilizer (S. A. Qureshi and A. Hafiz, personal communication, 1974). The results suggest that varieties able to produce well a t low N levels may lack NRA inhibitors, hence the genetic mechanisms involved in the response of varieties to low nitrate levels may be general in nature, and can be exploited by breeding to develop cultivars with greater N use efficiency.

VIII. Genetic Variability in the Nature and Properties of Wheat Proteins

A. WHEATENDOSPERM PROTEIN COMPOSITION Among the cereal grains, polyploid wheats have unique properties for making leavened bakery products. These unique properties are largely due to their protein composition. Hexaploid wheats are usually superior for breadmaking (Kerber, 1964; Kaltsikes et al., 1968a,b; Dronzek et al., 1970; Kasarda et al., 1971, 1976a; Bietz et al., 1975; Irvine, 1971). An extracted tetraploid that retained part of the D genome ( l A / l D substitution) had greatly improved baking quality (Kaltsikes et al., 1968a ; Konarev et al., 1972). A chromosome 1D addition line of tetraploid wheat has higher protein content and similar characteristics (Joppa et al., 1975). The uniqueness of the wheat endosperm proteins is reflected in their functional properties and in compositional differences as determined by protein fractionation, amino acid analyses, electrophoresis, electrofocusing, and electron microscope studies. Such differences are also reflected in the range of genetic variability for the species (B. L. Johnson et al., 1967; Kasarda et al., 1971, 1976a,b; Huebner and Wall, 1975; Bietz et al., 1975; Simmonds and Orth, 1973; Rodriguez-Loperena et al., 1975).

1. Protein Composition a. Solubility Fractionation of Wheat Proteins. Various modifications have been made of the procedure of Osborne (1907, 1924) for fraction-

GENETIC CONTROL OF PROTEINS IN WHEAT

441

ating proteins by their extraction in specific solvents. The most widely used solvents are water for albumins, saline for globulins, alcohol for gliadins or prolamines, and acetic acid for glutelins (wheat = glutenins), or NaOH for residue. Numerous protein extraction and fractionation methods have been reported in the literature (see Finney, 1943; Lee and Wrigley, 1963; Jones et al., 1963; Maes, 1966; Mattern et al., 1968a; Huebner, 1970; Landry and Moureaux, 1970; Chen and Bushuk, 1970a,b; Orth and Bushuk, 1972; Gavrilyuk et al., 1973; Bietz and Wall, 1975; Bietz et al., 1975). Most extraction methods show differences between varieties and species of wheats and other crops, but still more standardization is required ; consequently, the results from many experiments are difficult to compare. The greatest difficulties and variations in results have been encountered in the extraction and characterization of the "insoluble" fraction of the glutelin (glutenin) protein as well as in separating from among the glutelins those protein components soluble in alcohol or salt solutions (Finney, 1943; Inamine et nl., 1967; Hoseney et al., 1969c, 1970a; Mecham et al., 1972; Bieta e t al., 1973, 1975; Bietz and Wall, 1975; Huebner and Wall, 1974, 1975). But it should be recognized a t the outset that the solubility group method of fractionation is a completely artificial, very crude and preliminary means for proteh classification. Many proteins have multiple solubilities. Some proteins are complexes associated with lipids or carbohydrates ; others are polypeptides with different amounts of polar us nonpolar components. Some protein subcomponents differ from others only in electrical charge and often may be temporarily complexed until separated by solvent sequences, or by chemical treatment, e.g. reduction, alkylation, or electrofocusing (Kanazawa and Yonezawa, 1973, 1974a,b; Bietz and Wall, 1975). Even the albumins do not all conform to the classical solubility concepts. Some albumin proteins remain in flour after repeated water extraction, but can be extracted by salt solutions, with 0.26 M (NH,) .SO, most suitable (Minetti et nl., 1975). Those results indicated presence of two closely related albumin pools, one bound by salt linkages to some water-insoluble components. Both alcohol and water extracts of flour include such carbohydrate-protein complexes as arabinogalactan-peptides which are present in amounts up to 0.1% (w/w) in wheat endosperm (Fincher et al., 1974; Kasarda et al., 1976a). a-Amylase enzyme appears concentrated in aleurone cells and apparently is contained in sedimentable (lysosomal) fractions, sometimes difficult to isolate (Gibson and Paleg, 1975). However, there is abundant a-amylase in the inner endosperm cells. Protein fractionation studies generally indicate that there are wide differences in the relative proportions of albumins, globulins, prolamines, and glutelins comprising the proteins of the different major cereals

442

CALVIN F. KONZAK

TABLE 2 Protein Content and Solubility Fractions of Cereal Grainsa Protein fractions ( % of total protein) Cereal grain

Protein ( % dwb)*

Wheat Rice Maize Sorghum Rye Barley Oats

10-15 8-10 7-13 9-13 9-14 10-16 8-14

a

Albumins, Globulins, water salt soluble soluble 3-5

Trace Trace Trace 5-10 3-4 1

610 2-(8)? 5-6

Trace 5-10 10-20 -80

Prolamines, alcohol soluble

Glutelins, alkali soluble

40-50 (gliadin) (1-5) (oryein) 50-55 (zein) >60 (kafirin) 30-50 (secalin) 35-45 (hordein) 10-1.5 (avenin)

30-40 85-90 30-45

Considerable 30-50 35-45 5

Source: Brohult and Sandgren (1954); ? and orysin added by author.

* Percent, dry weight basis.

(Table 2 ) . The higher proportions of globulin or glutelin us prolamine proteins in oats and rice are in marked contrast to those of wheat, rye, barley, sorghum, and maize. The amino acid composition of the different cereal proteins shows considerable variation (Table 3) , but analyses in greater detail are necessary to reveal the genetic basis for the differences. Ewart (1967a,b, 1968) compared electrophoretic patterns of several different cereal proteins and amino acid compositions of the various proTABLE 3 Protein Content and Amino Acid ComDosition of Selected Cerealsa

Food Oats Barley Wheat Rye Sorghum Maize Rice References Whole egg Beef (round) Pork (ham, fresh)

Amino acid composition ( % of total protein)

Protein (%)

Lys Met Thr Trp

14.2 12.8 12.3 12.1 11.0 10.0 7.5

3.7 1 . 5 3.4 1.4 2.8 1.5 4 . 1 1.6 2.7 1.7 2.9 1.9 4.0 1.8

12.8 19.5 15.2

6.4 3 . 1 5.0 8.7 2.5 4.4 8.2 2.5 4.6

3.3 3.4 2.9 3.7 3.6 4.0 3.9

1.3 1.3 1.2 1.1 1.1 0.6 1.1

Ile

Leu

Tyr Phe Val Arg

5.2 4.3 4.3 4.3 5.4 4.6 4.7

7.5 6.9 6.7 6.7 16.1 13.0 8.6

3.7 5 . 3 3.6 5.2 3.7 4 . 9 3.2 4.7 2.8 5.0 6 . 1 4.5 4.6 5 . 0

1.7 6 . 6 1 . 2 5.2 1.3 5 . 1

6.0 5.0 4.6 5.2 5.7

6.6 5.1 4.8 4.9 3.8 lj.1 3.5 7.0 5.8

8.8 4.3 5.8 7.4 6.6 8 . 2 3.4 4.1 5.6 5.4 7.4 3.6 3.9 5.2 6 . 1

,. Source: Orr and Watt (1957): ? and oryein added by author.

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443

tein solubility fractions. His observations indicated that the proteins of oats and maize were markedly different from those of wheat, with only wheat proteins having the composition and properties necessary for bread dough. Some rye proteins were similar to those of wheat. The close relation of rye to wheat is indicated also by the ease with which rye chromosomes appear to compensate genetically for individual wheat chromosomes (Shepherd, 1968, 1973; Sears, 1969). The solubility properties of proteins from the various species appear to be related to a number of chemical, physical and physical-chemical factors. Differences in molecular weight, conformation, electrochemical charges, number and exposure of hydrophophilic vs hydrophobic groups, intra- and intermolecular disulfide bonding, and associations with carbohydrates and lipids, all affect the ease with which proteins can be dispersed in various solvents. However, solubility characteristics also reflect genetically controlled alterations in protein structure, as has been shown for changes in the gene-controlled small subunit of crystalline fraction I (RuDPCase) protein (Sakano et al., 1974a). Until recently, a large portion of the glutenin proteins from wheat has remained unextracted and reported as an insoluble residue, or the total glutenin fraction has been dissolved in urea, which disrupts hydrogen bonding (Simmonds, 1962; Ewart, 196713; Orth and Bushuk, 1972; Bietz and Wall, 1975; Mecham et al., 1972; Cole et al., 1973; Bietz et al., 1975; Huebner e t al., 1974). The conversion of disulfide bonds from intra- to interchain positions occurs by SH-SS exchange reactions (Kanazawa and Yonezawa, 1974a,b). The removal of lipids by petroleum ether improves the proportion of the protein components extractable from wheat flour (Simmonds and Wrigley, 1972). However, petroleum ether also removes certain lipoproteins, and decreases the solubility of the high-MW fraction extractable from flour by 55% ethanol (Garcia-Olmedo et al., 1968; Charbonnier, 1973). The use of a detergent in the extracting solution also helps to release molecules of lower MW, e.g., albumins, globulins, and gliadins, which may be bound among the non-water-soluble and insoluble material (Bietz and Wall, 1973a,b, 1975; Huebner and Wall, 1975; Cole et al., 1973). Wheat amylases are sometimes tightly bound to glutenin (possibly membrane) proteins, but the catalytic sites of these enzymes are evidently located some distance away from the binding site (Rothfus and Kennel, 1970). Reduction and alkylation of the disulfide groups and the use of HgC1, also has allowed separation and electrophoresis of many relatively insoluble high M W glutenin components (Rothfus and Crow, 1968; Mecham et al., 1972; Cole et al., 1973; Huebner et al., 1974; Bietz and Wall, 1975).

444

CALVIN F. KONZAK

It is now clear that the solubility characteristics of proteins also may be changed in some way by the solubilization process, since proteins insoluble in alcohol, for example, become soluble in alcohol after removal from their native tissues or after extraction in another solvent and drying (Bieta and Wall, 1975; Kasarda et al., 1976a). Minetti et al. (1975) likewise found that a large portion of albumins could not be extracted by water, but after extraction by salt solutions they proved to be soluble in distilled water. Bieta and Wall (1975) have defined as glutenins only those proteins that prove to be insoluble in alcohol even after reduction and alkylation, and are soluble in dilute acid. Alcoholsoluble proteins are considered to be gliadins, although some gliadin components prove to be water soluble (Preston and Woodbury, 1975, 1976; Kasarda et al., 1976a). b. Amino Acid Composition of Solubility Fractions. Amino acid analyses of several solubility group fractions from wheat flours have been made by Simmonds (1962) and by Ewart (1968). Simmonds (1962) analyzed the fractions of flours from only two varieties (one low and one higher in protein), but observed only minor variations in amino acid composition. In both varieties the soluble (albumin globulin) proteins were highest in lysine, while protein of the gluten group (gliadin, glutenin, and insoluble residue) was higher in glutamic acid and lower in lysine. Low-protein flour had a higher proportion of soluble proteins. More detailed fractionations of wheat flour by Bushuk and Wrigley (1974) showed that of the soluble proteins, the globulins were highest in lysine. Of the gluten proteins, the “insoluble” residue protein (which remained after extraction of flour by aIcohol and acetic acid) contained more lysine than the lower MW glutenin and gliadin fractions. More recent work of Mecham et al. (1972), Cole et al. (1973), Bietz and Wall (1975), and Bietz et al. (1975) suggest that the “insoluble” proteins are high-MW glutenins. Numerous results reviewed here exemplify the complexities of problems faced by cereal chemists even in the first phase of protein identification: extraction of the proteins from within the storage tissues of the endosperm. A more extensive analysis of the problems involved and progress made in the extraction and identification of individual wheat proteins can be found in a recent review on wheat proteins by Kasarda et al. (1976a). c. Molecular Weight of Protein Components. The water-soluble albumins are of MW under 20,000; salt-soluble globulins of MW 29,000 to 200,000; alcohol soluble gliadins of MW about 25,000-100,000 with most in the range of 36,000; and the relatively less-soluble glutenins of MW to lo6 (Meredith and Wren, 1966). Jones et al. (1961) showed the MW

+

GENETIC CONTROL OF PROTEINS I N WHEAT

445

of purified y - and p-gliadins to be 31,000-37,000 and glutenin M W to average 1.5 t o 2 X loG. The gliadin components of wheat have been generally classified into four main groups (a,alpha; p, beta; y , gamma; and O , omega) based on their characteristics on gel electrophoresis in aluminum lactate buffer. There appear to be several different protein components in each gliadin fraction and with more recent electrofocusing-electrophoresis methods their genetic relationships should soon be clarified (Wrigley, 1968a,b, 1969, 1970a,b, 1971a,b, 1972a,b, 1976a,b; Wrigley and Shepherd, 1973; Leaback and Wrigley, 1976). The size of gliadin molecules appears to be variable. After reduction of the disulfide bonds in gliadin, using sodium dodecyl sulfate (SDS) , and electrophoresis on SDS polyacrylamide gels, Bietz and Wall (1972) obtained components of MW 11,400, 44,200, 69,300, and 78,100 in moderate amounts, while components of M W 25,600, 48,800, and 57,300 were present in trace amounts. SDS-polyacrylamide electrophoresis is widely used for molecular weight determinations and appears to be reasonably reliable (Weber and Osborn, 1969). Ewart (1973) obtained M W values of 32,000-36,200 for a-gliadins, M W 37,800-38,000 for Pgliadins, and MW 38,20044,100 for 7-gliadins. Charbonnier (1974) reported the "-gliadins to have M W values of 64,500-73,000 by gel filtration methods, while Booth and Ewart (1969) found M W 76,000 and 79,000 for these proteins by sedimentation equilibrium measurements. Hamauzu et al. (1974) suggested that the high protein content of the "-gliadins interfered with the complexing by SDS, and the absence of disulfide might allow these proteins to assume conformations that are more expanded than other gliadins in the SDS solvent. Hence, the gel filtration method would show those proteins to have apparently high MW in comparison to other gliadins. Bietz and Wall (1972) determined M W of 36,500 for yl-gliadin and 44,000 for 7,-gliadin by SDS-gel electrophoresis whereas Sexson and Wu (1972) obtained values of 30,300 and 34,700 for these same components by the sedimentation equilibrium method. Platt et al. (19741, Kasarda (1976), and Kasarda et al. (1976b) found that the aggregatable fraction of a-gliadin, which included 4 or 5 subcomponents, appears as a single protein of MW 36,000 on SDS electrophoresis, whereas Platt and Kasarda (1971) obtained a value of 32,000 by amino acid analyses. Stevens (1973) obtained results suggesting that gliadins are comprised of single polypeptide chains containing a n average of four SS bonds each. Other gliadin components have not been described in detail, nor have the more recently developed methods of electrofocusing been widely applied to their study. It is likely that several homoeologous gliadins of the subcomponents observed by Wrigley and Shepherd (1973) will have different M W values and/or differences in molecular charge.

446

CALVIN F. KONZAK

Interestingly, a low-MW component (MW 18,000),considered to be gliadin on the basis of solubility and amino acid composition, also had unusually low amounts of glutamic acid and unusually high amounts of methionine and cystine residues. At acid pH the mobility was less than that of a-gliadin, and a t pH 8.9 the molecule was still positively charged (Ewart, 1975). Because of its small size, this gliadin may represent a protein form resulting from an intramolecular chain termination mutation, or may be the 18,000 MW component found by Bietz and Wall (1972) in reduced glutenin. More recently, a protein of 10,000 MW classified as gliadin because of EtOH solubility was shown to have an amino acid composition similar to albumins (Preston and Woodbury, 1976). Wheat varieties also may have different numbers and relative quantities of gliadin components, although no clear relation with processing properties has been observed (Doekes, 1973; Elton and Ewart 1962; Khrabrova et al., 1973; Tanaka and Bushuk, 1973a; Wrigley and Shepherd, 1974), and may have homoeologous or homologous proteins differing in structure and amino acid composition. The definition of glutenin proteins is an even greater problem, because preparations isolated by many different workers and by many different methods often show considerably less similarity than gliadin or albumin preparations. However, this research area is currently receiving considerable attention by cereal chemists. The most widely applicable definition of glutenin is that this fraction includes less-soluble components, primarily proteins of high MW. Bietz and Wall (1975) indicate that, by the use of various extractants, glutenins from flours sequentially extracted in 0.04 M NaCI, 70% ethanol, 0.1 N acetic acid (HOAC), 0.2 M HgC1, and 0.1% 2-mercaptoethanol ( ME ) differed significantly in their genotype-dependent subunit composition. Upon further fractionation of the HOAC and HgCl, extracts with 70% ethanol, 3&50% gliadinlike proteins were obtained from the different wheats. Huebner and Wall (1975) identified an unreduced glutenin with MW over 20,000,000. Gluten also contains a “high MW gliadin” or “low MW glutenin” fraction which has a MW of 104,000-125,000 (Nielsen et al., 1968; Meredith, 1965a,b; Beckwith et al., 1966; Kanazawa and Yonezawa, 1973). However, the degree of purification, reduction, and chemical modification, as well as the methods of analysis, plays a role in the MW estimates obtained. Bieta and Wall (1973b) found that, after reduction and alkylation, 62% of the glutenin that was first extracted by acetic acid was soluble in 70% ethanol. The ethanol fraction was composed of subunits of MW 44,000 and 36,000 estimated by SDS electrophoresis. The fraction insoluble in 70% ethanol had a higher MW. Bietz and Wall (1972) identified

GENETIC CONTROL OF PROTEINS IN WHEAT

447

15 subunit fractions in reduced glutenin with MW 133,000, 124,000, 102,000, 87,200, 79,100, 71,000, 64,300, 49,400, 44,600, 42,200, 36,000, 32,600, 27,500, 18,000, and 11,600. Other investigators have found the highest MW subunits of glutenin to be 152,000 (Orth and Bushuk, 197313) and 104,000 (Hamauzu et al., 1974), or 98,000 (Kasarda et al., 1976a). These variations in highest M W values for glutenin subunits obtained by SDS-gel electrophoresis apparently are dependent somewhat upon the experimental procedures used, i.e., run time, techniques of gel and buffer preparations, etc. (Cole et al., 1973, 1976). The major portion of glutenin is made up of subunits near M W 44,000 and 36,000. The smaller subunits appear to have MW near 12,000, similar to albumins, and may in fact be albumins that wcre bound in some way with the glutenin complex (see Rothfus and Kennel, 1970). Most important, the proportion of glutenin subcomponents in the MW range 35,000-50,000 is sufficient for them to have a considerable influence on the amino acid composition and properties of native gluten. Bietz and Wall (1973b, 1975) noted the gliadinlike character of the 44,000 MW subunits found in the ethanol-soluble proteins of reduced and alkylated glutenin, and in acetic acid or mercuric chloride extracts of flour. They considered these subunits to be distinct from the high M W “purified” glutenin. Wheat flours contain many proteins differing widely in MW and other properties. Results from studies on peptic enzyme hydrolyzates provide evidence that components of gliadin and glutenins of wheat also may differ in primary structure, i.e., amino acid sequence (Bietz and Rothfus, 1970, 1971; Bieta and Wall, 1975; Huebner et al., 1974). The amino acid composition and sequence in “purified” glutenin preparations is currently under investigation (J. A. Bietz, personal communication, 1975). Bietz and Wall (1975) consider that it is no longer adequate to define glutenin by solubility in the classical manner, since it is a complex of proteins with MW ranging into the millions, and with characteristic spectra of disulfide-bonded subunits, some similar t.0 gliadins and others over MW 100,000. They noted that after extraction with acetic acid much of the glutenin remains undissolved either as gel or residue protein, which they found could be solubilized by HgCl,, surfactants, hydrogen bond-breaking agents, or reducing agents. The classical extracts of glutenin were found to contain other proteins separable by reprecipitation upon adjustment of pH, gel filtration chromatography, or SE Sephadex C-50 procedures. Such impurities were considered responsible for inconsistencies in reported properties and composition of glutenin. The observations by Bietz and Wall (1975) appear to support those of Bernardin and Kasarda

448

CALVIN F. KONZAK

(1973a) and Bernardin (1975), which suggest that proteins of the gliadin proteins may exist in unusual conformations or aggregations, which affect their solubilities in water, ethanol, or acetic acid. Thus, Bieta and Wall (1975) found that gel proteins ( = glutenins) first extracted in acetic acid (HOAC) or HOAC-HgCl, could then be solubilized in 70% ethanol, proving to be gliadinlike. Bernardin (1975) considers that most of the gluten proteins may be gliadins, but artifact changes caused by the common protein fractionation procedures temporarily alter their water and ethanol solubilities, which may be reversed once they are separated out by other procedures. Isoelectric focusing also has been applied recently to the separation of component polypeptides of glutenin, and should be useful in biochemical genetics studies (Mita and Yonezawa, 1971). The functional (breadmaking) properties of wheat flour are due largely to the properties and interrelations among the gliadin and glutenin components of gluten (Hoseney and Finney, 1971; Pomeranz, 1971, 1973b,c; Kasarda et al., 1971, 1976a; Bushuk and Wrigley, 1974). Similarly, the glutenin: gliadin ratio is important to pasta-making quality (Wasik and Bushuk, 1975a,b; Matsuo and Irvine, 1975). Other wheat flour proteins and lipids, including glycolipids may exert important modifying effects (Pomeranz et al., 1966a, 1970; Pomeranz, 1971, 1973b,c; Pomeranz and Finney, 1973a,b). Much of the recent research on solubility fractionation indicates that characterization of protein components in terms of the Osborne method does not adequately describe the chemical and physical nature of proteins or their interrelationships (Silano et al., 1969; Silano and Pocchiari, 1971; Mecham et al. 1972; Mecham, 1973; Minetti et al., 1973; Wasik and Bushuk, 1974; Huebner et al., 1974; Huebner and Wall, 1975; Bietz et al., 1975; Bietz and Wall, 1975; Kasarda et al., 1976a). Rather, a number of criteria may be needed, and even then it may not be possible to classify all protein components in simple terms. Moreover, Bernardin and Kasarda (1973a,b), Mecham (1973), and Bernardin (1975) suggested that many features, especially the relative insolubility and molecular sizes of many gluten proteins, may be a consequence and, therefore, are artifacts of chemical and physical changes that occur upon rewetting and during the solubility fractionation processes. In this regard, Charbonnier (1974) found that several protein bands revealed a t p H 8.9 by electrophoresis of a fraction which gave only one band a t pH 3.2 correspond to different amidation levels of the same protein. Likewise, Mosse (1973) showed that partial hydrolysis of amide groups occurs during storage of gliadin in buffer. Torres and Hart (1976) recently found that several homoeologous acid phosphatase isozymes distinguishable by elec-

GENETIC CONTROL O F PROTEINS I N WHEAT

449

trophoresis had essentially the same MW of 58,000 but were distinguishable because of differences in molecular charge. Bernardin and Kasarda (1973b) and Bernardin (1975) observed that marked changes in the physical and chemical characteristics of gliadin take place when high concentrations of those proteins reabsorb water. Rehydration of gluten proteins in flour or in sections of wheat endosperm was observed to be extremely rapid, producing a two-phase system of protein fibrils in highly concentrated form. Also, Shogren et uZ. (1969) showed that pH of the solvent for separating proteins has a major influence on the proportion of gliadins us glutenins extracted from flours, and that increasing amounts of proteins could be extracted with additional water extractions of gluten. Recent work by Bietz and Wall (1973a,b) indicates that as much as 757'6 of wheat protein may be gliadin or gliadinlike components. Bernardin (1975) considers the solubility properties of the protein fibrils to be largely those of gliadin, as expressed at high protein concentrations. When wet with aqueous solvents, gliadin proteins form a hydrated mass, which dissolves to varying degrees depending on the ability of the solvent to dissociate the aggregated proteins in the form of microfibrils lying parallel to one another. The liquid mass is considered to be a liquid crystalline form of the protein where the aggregate exposes a minimal surface to the solvent, while the degree of dissolution reflects the equilibrium concentration of the two phases. Variations in solubility and other properties of proteins may be affected also by small differences in amino acid composition as well as by variations in component subunits comprising the molecules (Kasarda et al., 1971 ; Bernardin, 1975). Binding with lipids, carbohydrates, and other components can also modify solubility. Garcia-Olmedo et uZ. (1975 ; personal communication) have found that the lipoprotein purothionins are soluble either in petroleum ether or in saline, whereas dilute sulfuric acid preferentially extracts the protein moiety, and ether the lipid moiety. The components of glycoproteins, and other carbohydrate protein complexes found in flour, can likewise be expected to be affected by solubility fractionation and chemical modification procedures. p-Amylase has been found adsorbed on glutenin, possibly representing its natural association in a n organelle membrane, although i t is abundant in cell walls and in bran (Kruger, 1973). The complexing of protein subunits can occur in vitro as well as in vivo. Mitra and Bhatia (1971) and Irani and Bhatia (1972) have shown that mixtures of wheat and rye proteins form the same dimer isozymes of alcohol dehydrogenase as appear in protein extracts of triticale or of wheat with a disomic rye chromosome addition. Some of the isozymes formed are combinations of the distinct

450

CALVIN F. RONZAK

monomeric components from wheat with component from rye. H a rt (1971) has obtained similar results for homoeologous isosyme monomers of wheat. 2. Physical Structure of Wheat Gluten Proteins

A number of wheat proteins have been isolated and studied extensively (see Wu and Cluskey, 1965; Wu et al., 1967; Kasarda, 1970; Kasarda et al., 1967, 1971, 1974a,b, 1975, 1976a,b; Mecham, 1973; Bernardin and Kasarda, 1973a,b; Silano et at., 1973; Nimmo et al., 1974; Bedetti et al., 1974). Using transmission EM, Seckinger and Wolf (1970) observed that dispersions of gliadins produced filmlike structures, while glutenins formed strands. Likewise, Crozet et al. (1974) observed that gliadins and four protein fractions high.in gliadin had a smooth, compact, and nearly electron-translucent structure, whereas glutenin and fractions high in glutenin had a granular and fibrillar structure. The fibrils of glutenins had a diameter of 100-700 A and formed a dense network. The structures of albumins and globulins appeared to be much more like glutenins than gliadins. Kasarda et al. (1967) observed reversible aggregation of a-gliadin to fibrils, depending on p H and ionic strength. The fibrils are about 80 A thick and several thousand angstroms long; they dissociate to globular protein subunits at very low ionic strength and pH 3 (0.001 M HCl), but are re-formed by increasing the pH to 5 and the ionic strength to 0.005 M . Bernardin and Kasarda (1973a,b) showed that wheat endosperm protein is present in sheet form, and they suggested that the sheets result from a laminar deposition of the storage protein in the protein bodies of the developing wheat grain. The sheets of endosperm protein in the protein bodies rupture under stress and, when wetted, form webs of protein composed of different-sized fibrils. The larger fibrils proved to be aggregates of the smaller fibrils interacting laterally. The observations suggest that the fibrillar wheat gluten proteins are comprised of subunits bonded together and stabilized by intramolecular disulfide bonds, and that there are no intermolecular bonds or cross-links joining the subunits of the fibrils to one another (Mecham, 1973). Complexing and conformational changes affecting the solubility of proteins may be introduced by disulfide interchange reactions involving glutathione or similar SH-bearing amino acids or SH-peptides. The SH-molecules are brought in contact with the proteins of protein bodies upon rehydration of the flour during dough formation or protein solubility fractionation.

GENETIC CONTROL OF PROTEINS IN WHEAT

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Rather extensive recent work by Orth e t al. (1973, 1974b), using scanning EM, clearly shows differences in the ultrastructure of glutenin proteins that may provide a basis for the different rheological properties of flours from rye, triticale, durum, and common wheats. I n their studies, the glutenin from a hard red spring wheat cultivar ‘Manitou’ had a characteristic fibrous structure, including many thick strands (10 pm in diameter) intertwined with thin strands (1 pm). I n addition there was a general alignment in which cross sections of the fibers appeared cylindrical, suggesting a major unidirectional organization of the fibers ; and there was filmlike material, especially at the point of contact between the strands. On reduction by p-mercaptoethanol and 6 M urea, all the fibrous structure was lost, leaving only an amorphous mass. Four fractions of unreduced glutenin separated by gel filtration had different heterogeneous structures (Orth et al., 1974b). The fraction with highest M W contained disk-like particles connected with fibrous strands. The next highest MW component had large, platelike protein particles of different sizes. The third of the four fractions contained many small particles associated with few fibrous protein strands, and the fourth fraction had small aggregates of rather amorphous particles. The highest MW fraction was much more heterogeneous than the unfractionated, purified glutenin preparation obtained by pH precipitation (Orth and Bushuk, 1973a). I n the same study, the purified glutenin from ‘Canthatch’ appeared similar to that from Manitou, but had more intertwined, stringy fibers containing also some filmy materials. The tetraploid wheats, including a derived tetraploid with Canthatch A and B genomes (‘Tetra-Canthatch’) , contained a large amount of filmy material and large, flat ribbonlike structures quite unlike those of the bread wheats. The structure of the durum and Tetra-Canthakh glutenins was consistent with their lower elasticity and poorer breadmaking properties. Studies also of purified glutenin from Ae. squarrosa, the D genome source for hexaploid wheat, showed this protein to have the fibrous structure typical of the bread wheats; whereas removal of the D genome from the hexaploid wheat, as in Tetra-Canthatch, caused the purified glutenin to lose the more fibrous structure and take on a more amorphous appearance. Purified glutenin from rye had characteristic rodlike structures, but there were very few of the thin fibers typical of bread wheats. Rye glutenin differed from that of durum wheats and Ae. squarrosa. Glutenin from triticale (eelection 6A-190) had structures expected from the combined contributions of its parents (Orth e t al., 1974a), consistent with the relatively lower elasticity and relatively poor rheological properties of its flour. These results are strikingly similar to those reported by Bernardin and Kasarda

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(1973a,b) and Bernardin (1975) in studies on seed and flour proteins and on purified fractions of a-gliadin. A current concept of glutenin molecules is that it is comprised of a mixture of subunit proteins linked together by intrasubunit SS bonds in the form of concatenations (Greenwood and Ewart, 1975). End-to-end linkage of different numbers and sizes of monomer subunits into rather high polymers with MW into the millions may occur, depending probably on the genetic characteristics of the variety. The variety ‘Red River 68’, for example, appeared to contain extremely high MW glutenins compared with other varieties (Huebner and Wall, 1975). Any disulfide cross-linking between subunits is restricted to that permissible in linear polypeptides, which probably are subject to bonding together by secondary forces, such as may involve hydrogen ionic and hydrophobic bonds. The shape of the molecules and their intermolecular associations of the concatenations also depend on p H and other factors, such as hydration and association with free thiols (Kasarda et al., 1976a). Sulfhydryl bonding between glutenin molecules was thought by many to be necessary t o account for the alterations achieved by various chemical treatments and the actions of oxidizers and thiols in dough. However, the appearance of purified glutenin as fibrillar components rather than a more complex or amorphous mass in scanning EMS is not consistent with the concept of intermolecular disulfide bonding, and it is now thought that oxidizers and dough improvers mainly inactivate the free thiols in flour and dough (Greenwood and Ewart, 1975; Kasarda et al., 1976a). 3. Relative Increase of Endosperm Proteins during

Grain Development and Maturation Proteins of the different Osborne solubility classes are laid down in the cereal endosperm a t different rates during the various stages of grain development and maturation (Fig. 5 ) . I n the early stages of endosperm development, the highest proportion of the N is in the form of albumins, globulins, and nonprotein nitrogenous substances (Mitra and Bhatia, 1973; Manghers et al., 1973). As development proceeds, prolamine synthesis increases a t a very rapid rate, while glutenin synthesis increases a t a somewhat slower rate. I n the later stages of development, the larger proportion of N is incorporated into prolamines, while N in the form of soluble and insoluble proteins increases a t slower rates. The relative proportions of different protein components in different wheats appears to be similar during all development stages, with the maximum amount of low-MW materials occurring a t 14 days past flowering, and the minimum a t maturity (Bushuk and Wrigley, 1971). They found the proportion of albumins to be somewhat higher a t 14 days past flowering in the hard red

GENETIC CONTROL O F PROTEINS I N WHEAT

0.9

K

A

453

L NITROGEN

RESIDUAL PROTEINS

LUBLE NITROGEN DAYS OF DEVELOPMENT

FIG.5. Accumulation of different solubility classes of wheat proteins with relation to endosperm development. (Redrawn from Mitra and Bhatia, 1973.)

spring (HRS) wheat cv. RIanitou than in the soft white winter (SWW) cv. ‘Talbot’ or in the durum cv. ‘Stewart 63’, but a t maturity the proportions were the same in all three wheats. Acetic acid-soluble glutenin was found to comprise 2070 of the total protein as early as 14 days past flowering. During the final stages of endosperm development, the glutenin fraction increased markedly in the HRS wheat, less in the SWW wheat, and little, if a t all, in the durum. In maize, mutants opaque-:! ( o p ) and brittle-2 (bt,) and in sorghum,. mutant hily ( h l ) greatly affect the proportions of proteins synthesized during endosperm development (Singh and Axtell, 1973; Misra and Mertz, 1975a,b; Jambunathan et al., 1975). Both single mutants (0202 and bt,bt,) and the double mutants ( orozbtrbtp)had higher levels of albumins and globulins throughout development. Synthesis of the low lysine zein proteins was evident a t 14 days in normal endosperm, but only a t 18 and 21 days for bt,bt, and 0 2 0 y .The net zein synthesis rate was also slower, resulting in 50% less zein a t maturity in each single mutant, while no zein a t all was synthesized by the double mutant o,o,btzbt, (Misra and Mertz, 1975a,b; Misra et al., 1975a,b,c). The rates of growth and dry matter accumulation in developing wheat grains have recently been found t o show periodic fluctuations, with

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frequency maxima of about 3 4 , 6 7 , and 10 days (Jenkins et al., 1974). Another concept beginning to emerge from several studies is that the different isozymes of a given enzyme change markedly in intensity and presence in various tissues of the developing plant and kernel during development. The extent of genetic control over formation us inactivation of the enzymes is still not clear (Glasziou, 1969). Up to 12 peroxidase isozymes occur in different anatomical parts (pericarp, green layer, ovary wall, aleurone, embryo, scutellum) of the kernel, changing in intensity during kernel development with different distributions of total enzyme activity over time for a durum as compared with a bread wheat variety. Similar peroxidase isozymes were present in both species of wheat, although one variant appeared to be absent in the hexaploid (Kruger and La Berge, 1974). Four types of amylase activity including that of P-amylase are distinguishable in developing wheat grain (Meredith and Jenkins, 1973). High levels of amylase activity are associated with outer tissues during early grain development. These appear to be P-amylases (Kato et al., 1974). Thermal stability also varied for two other activities associated with breakdown of soluble starch, with patterns associated with specific cultivars. Proteolytic enzyme levels also change during growth and maturation (Kruger, 1973). Likewise, mono- and o-diphenolase activities varied in hexaploid wheats during kernel development, although the relations of activity to different tissues were not determined. Monophenolase activity was low until about 33-36 days after anthesis after which it rose sharply to a peak a t 42 days to fall again thereafter. Monophenolase was considered to have a role in the formation of seed-coat pigment. Twelve o-diphenolase isozymes were identified in the developing kernel, with the relative intensities (strong to none) varying throughout the development period. No one isozyme was found at all sampling times, and nearly all showed increases and/or decreases in intensity (Taneja et al., 1974). Presumably, the same isozymes described as polyphenolases (PPO) also were observed in different parts of the developing grain, with a large part of the activity in the endosperm (Kruger, 1976). The activities of seven acid phosphatase isozymes also showed tissue and developmental variation in relative intensity as measured in 7-day shoots, leaves, leaf sheaths, stems, peduncles, and in the endosperm and scutellum of kernels. Five other enzymes (anthranilate synthetase, anthranilate-5 phosphorylalase-1 pyrophosphate phosphoribosyl transferase = PR transferase, N-5’-phosphoribosylanthranilate isomerase = PRA isomerase, indole-3-glycerol phosphate synthetase = InGP synthetase, and tryptophan synthetase) in wheat differ in relative intensity in different plant tissues and a t different plant developmental stages (Singh and Widholm, 1974). Isozymes homoeologous

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to each of the major isozymes in hexaploid wheat which are present in one or another of the progenitor species also showed similar developmental and/or tissue specificity (Torres and Hart, 1976). The further identification of isozymes with regard to substrate specificities and responses to inhibitors, should eventually clarify the role of these enzymes in plant biochemistry. Some progress in this regard has been reported for maize esterases (MacDonald and Brewbakcr, 1975), for wheat &-amylases (Tkachuk and Kruger, 1974; Tkachuk, 1975), and for wheat esterases (Cubadda and Quattrucci, 1974). The various molecular forms of enzymes appear to have a role in differentiation, but no clear understanding of their role has yet emerged (Scandelios, 1969, 1974; Hart, 1976, personal communication).

4. Specific High-Lysine Proteins The lipoprotein purothionins from wheat and barley (so named because of their high sulfur content, i.e., cysteine 16%) also have an exceptionally high lysine content and contain about 18% N (Balls et al., 1942; Nimmo et al., 1968, 1974). a. Occurrence and Concentration. Purothionin from barley is similar to, but distinct in composition from that of wheat (Nimmo et al., 1974). Purothionins were not found in rye, maize, or oats (Redman and Fisher, 1968, 1969; Fisher et al., 1968). I n polyploid wheats these lipoproteins occur as two electrophoretically distinct, but analogous forms. Both forms (a,p ) are present in the hexaploid and tetraploid wheats, whereas only the /3 form occurs in the diploid T . monococcum, and the form occurs in several Aegilops species, including Ae. squarrosa and Ae. speltoides (Carbonero and Garcia-Olmedo, 1969; Mecham, 1971). The concentration of purothionins was reported to be appreciably higher in certain varieties of hexaploid wheat than in durum wheat. All of 26 varieties of durum had less than 20 mg of purothionin/100 gm of flour, while all but four out of 40 T. aestivum varieties had over 20 mg/100 gm of flour, and some had as high as 78 mg/100 gm of flour (Carbonero and Garcia-Olmedo, 1969). However, as will be noted later, these differences may be artifacts of the extraction technique, since only about one-tenth as much purothionin could be extracted by petroleum ether as by dilute sulfuric acid (F. GarciaOlmcdo, personal communication, 1975). However, the purothionins may account for more than 0.07 to 0.09 gin of lysine per 16 gm of N, or about 0.1 gm of lysine per 100 gm total protein, in 11.4% protein flour from hexaploid wheat (based on values from Garcia-Olmedo et al., 1968; F. Garcia-Olmedo, personal communication, 1975). This suggests that compared with other seed proteins, purothionins contribute disproportionately to the total lysine composition and, as individual proteins, are a major (Y

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source of the lysine present in wheat. New evidence, however, indicates that the purothionins themselves may possibly be extraction artifacts (Garcia-Olmedo, personal communication, 1976). b. Physical Properties. Recently, Nimmo et al. (1974) presented further data on the physical characterization of wheat purothionins. They found that the LY and p forms, which differ slightly in amino acid composition and have different mobilities in gel electrophoresis, do not differ in other physical properties. Molecular weights for the LY and p forms as determined by sedimentation equilibrium were 5100 and 5300, respectively, whereas MW values for both forms determined were 6100 (by osmotic pressure) and 7500 by gel permeation chromatography. Studies of the circular dichroism spectra of the purothionins suggested a 40% a-helical structure, while intrinsic viscosity data suggested a compactly folded spherical globular structure. The amino acid sequencing studies (60% completed) indicate that LY purothionin has a MW of 5500, with a high cysteine content (four SS bonds/mole) and lysine located in the N-terminal position (Mak and Jones, 1974). Recent evidence of Mak and Jones (cited by Kasarda et al., 1976a) suggests that the remaining residues of apurothionin may correspond to the components identified by HernandezLucas et al. (1974)-one related to the B genome and containing isoleucine, the other related to the D genome and containing no isoleucine. G. Differential Afjinity for Protein Dyes. Purothionins have a strong affinity for acidic protein dyes (Redman and Fisher, 1968; Nimmo et al., 1968; Silano et al., 1969; Lawrence et nl., 1970). Lysine-containing proteins stain green, albumins stain black, and gliadins stain red with aniline blue-black dye (Silano et nl., 1969; Silano and Pocchiari, 1971). This differential staining results from differences in coprecipitation tendencies of the proteins with impurities vs normal components in the dyes (Minetti et al., 1973). The differential staining of electrophoretically separated proteins may be related to their content of free basic amino groups and thus to the lysine and other dibasic amino acid (DBAA) composition (Lawrence et al., 1970). Varieties of durum and bread wheat differ in their content of water-soluble gliadin and albumin proteins detected using aniline blue-black stain on electrophoretic gels (Minetti et al., 1971). Nigrosine dye was found to be useful for identifying proteolipid CM (chloroform-methanol extract) proteins in wheat (Aragoncillo et al., 1975b). Studies using flourescamine (Fluram) and carbocyanine dyes for staining and identification of proteins also are pertinent here: (1) A fluorescent antibody technique using fluorescamine showed differences in the local concentrations of soluble proteins in sections of wheat endosperm (Barlow et al., 1973a). (2) Staining of proteins with fluorescamine prior to separation on polyacrylamide-gel electrophoresis avoided fixation and

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destaining procedures (Zak and Keency, 1974). (3) Fluorescamine labeling did not affect the mobility of proteins on polyacrylamide gels (Pace et al., 1974). (4) Amino acid analyses could successfully be made of Fluram-stained protein bonds cut from polyacrylamide gels (Stein et al., 1974). (5) The cationic carbocyanine dye, “Stains All,” 1 ethyl-2-[3(ethyl -naphtho [ 1,2d]thiazolin - 2 - ylidene) - 2 - methylpropenyl] -naphtha[ 1,2d]thiazolium bromide, differentially stained phosphoproteins, carbohydrate and sulfate carbohydrate-containing proteins of histological sections (Green and Pastewka, 1974a,b). “Stains All” is a sensitive stain for nucleic acids separated electrophoretically on polyacrylamide-agarose composite gels. Ribonucleic acid (RNA) stains bluish-purple, deoxyribonucleic acid (DNA) stains blue, and protein stains red (Dahlberg et al., 1969). The blue-stained proteins of Dahlberg et al., (i.e.) proved to be phosphoproteins (Singer, 1952; Haag et al., 1971 ; Green et al., 1973). The development of differential staining techniques for laboratory identification of higher lysine-containing endosperm proteins may be feasible. Such techniques may prove useful in genetics and breeding experiments and for the identification of specific proteins upon electrophoresis. d . Role in Processing Quality and Biological Properties. Purothionins apparently have no function in breadmaking (Hoseney et al., 1970b), but their high cysteine (SS) composition, relatively small molecular size, and relative abundance suggest that they could play a role in the SS-SH exchange reactions during dough development. Stuart and Harris (1942) , Fernandez de Caleya et al. (1972), and Hernandez-Lucas et al. (1974) showed that the purothionins have in vitro antibiotic or antifungal activity, but their biological function in wheat remains a mystery (Nimmo et al., 1974). Evidently their influence on human and animal nutrition is also unknown, although Stuart and Harris (1942) found purothionin to be toxic when intraperitoneally injected into mice. e. Genetic Control of Synthesis. The synthesis of the (Y and p holoprotein components of the purothionins is controlled by chromosomes of group 1 in wheat (Garcia-Olmedo et al., 1975). The different electrophoretic mobilities characteristic of the and p analogs are evidently due to evolutionary changes that occurred in one of the diploid ancestors (probably the A genome donor) prior to the natural origin of the tetraploid wheats. Synthesis of the lipid moiety of these purothionins is controlled by group 5 chromosomes. Using dilute sulfuric acid instead of petroleum ether to extract purothionins of Chinese Spring wheat, Garcia-Olmedo et al. (1975) observed that the same relative proportion of the protein component was produced by the group 1 chromosomes in each of the three genomes, whereas the quantity of lipid moiety produced by chro(Y

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mosome 5A was less than for 5B, the most being produced by 5D. These results suggest that the differences in purothionin content of wheats reported earlier (Carbonero and Garcia-Olmedo, 1969) may have been due to differences in the degree of extraction of the lipid-protein complex. The lipid components appear to have been identified as phosphatidylcholine (Redman and Fisher, 1968), mono- and digalactosyl diglycerides, and phosphatidyl ethanolamine (Hoseney et al., 1970b). 5. Lysine Content Variation in Other Proteins

Different gliadin components in wheat also appear to vary in lysine content, with 2-fold or greater differences (Booth and Ewart, 1970). HOWever, the entire prolamine fraction (gliadin) contains only about 0.1 gm of lysine per 100 gm of protein (about equal to the contribution of the purothionins) . Because of its greater abundance (25-75%), the prolamine fraction has a marked influence on the average lysine/protein composition of the endosperm. I n several high-lysine maize mutants and in the barley mutant ‘Bomi 1508’ (Ingversen et al., 1973), the prolamine fraction is reduced in proportion to the total with an increase of the salt-soluble (globulin) fraction, which is higher in lysine (Misra et al., 1972). The high prolamine composition of barley, rye, sorghum, maize, and wheat indicates that these crops have a similar potential for genetic alterations to improve the nutritional balance of lysine (Tables 1 and 2). The possibilities of reducing the proportion of low-lysine proteins are greater for maize and barley than for oats or rice, which contain much smaller amounts of prolamines. I n oats, strains with up to 5% lysine in the protein already have been identified (Robbins et al., 1971). Data on the amino acid composition of proteins indicate that variations in content of the four important amino acids occur among specific wheat gluten proteins (Kasarda et al., 1976a). Gliadins (a, p, y, 0 ) generally have low-lysine, high-glutamic acid, and high-proline composition (Ewart, 1973). Tryptophan composition is similar t o that of lysine, but methionine content varies and threonine composition is about three times that of lysine. High MW gliadin (variety not specified) contained about twice as much lysine and methionine, slightly more threonine, and less prolamine than the lower MW types. Several glutenin fractions contained two to four times as much lysine, about 1.5 times as much threonine, about twice as much methionine, and half as much proline as low MW gliadins. However, some glutenin fractions contained only as much lysine as the low MW gliadin, but were generally similar in composition to other glutenins. Recent studies by Huebner et al. (1974) indicate that two higher-lysine components (A, and A,) are present in the glutenin fraction of one hexa-

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ploid wheat. The fraction A, also had a relatively lower leucine content; while A? had a much higher leucine content than the whole glutenin or other glutenin fractions. However, the differences also may represent variations in the relative purity of the two fractions which should be resolved by the amino acid sequencing of purified glutenin protein subunits currently in progress (J. A. Bietz, personal communication, 1975). The prospects of finding or inducing higher lysine variants in wheat may be indicated from evidence about the composition of mutants already identified in maize and barley. Misra et al. (1972) compared the amino acid composition of protein solubility fractions from normal maize with those of several mutants isogenic for genes s u, sh2, shr, bt, and bt, alone or genetic combinations also carrying 0, in inbreds ‘OH 43’, and ‘W22’. The range in Iysine (gm/100 gm of protein) was 3.7 to 6.3 for albumins globulins, 0.1 to 0.5 for zein (prolamine), 0.4 to 0.8 for glutelin 1, 1.4 to 2.8 for glutelin 2, and 6.4 to 7.1 for glutelin 3. The higher lysine composition in f12, bt,, o,, 02bt2,and o1 was due to proportionate increases in the albumin-globulin and glutelin-3 fractions. The 0, gene invariably raised the free amino acid content in the endosperms above the levels achieved by other genes present (Misra et al., 1975). However, the combination of o2 and fi2 or o7 mutant genes did not produce additive effects on lysine (Misra and Mertz, 1975a). I n some maize mutants the lysine increases were associated with the soluble protein fractions, others with the glutelin fraction (Jimenez, 1968; M a and Nelson, 1975). Ingversen and Kgie (1973) showed that the high lysine composition of ‘Hiproly’ barley is due to disproportionate increases in the globulin and glutelin fractions, a t least one or more proteins in each having a higher than normal lysine composition. One of the high-lysine globulin protein subfractions makes up about 46-50% of the salt-soluble protein of Hiproly. Different proportions of four globulin proteins appeared to account for the different higher lysine compositions of Hiproly, CI7115, and ‘Carlsberg’ mutants 20 and 86, but in all mutants the same protein subfraction is proportionately increased. Analyses of Bomi Mutant 1508 indicate that the nutritional composition of proteins can be increased also via another genetic route, since Ingversen et al. (1973) found that there was 300% more lysine in the prolamine fraction of this mutant than in Carlsberg without any change in lysine composition of either the albumin-globulin or insoluble fractions. This seemingly large increase, nevertheless, only raised the lysine in the prolamine (hordein) fraction from 1.0 to 2.9 gin per 100 gm of protcin. Bomi Mutant 1508 also has a significantly increased amount of lysine in its glutelin fraction. The Bomi Mutant 1508 has a genetically stable 44% increased lysine composition controlled by a recessive gene, which is inherited independently from

+

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the hily gene in Hiproly and has an additive effect on lysine content (Muench et al., 1976). Recent studies of Ingversen (1976) indicate that the proportion of electron-dense protein bodies (as estimated by E M after sucrose density sedimentation) differs for Bomi and Bomi 1508. Bomi 1508 has far fewer electron-dense protein bodies, and on electrophoresis several protein bands not found in Bomi were identified in the high-lysine mutant Bomi 1508. Bomi 1508 has a substantially increased level of a-amylase (Munck, 1976). Oram et al. (1975) found that two co-dominant alleles at one locus on chromosome 5 controlling the electophoretic pattern of a group of hordeins in the Bomi and Sultan barleys were not linked with and hypostatic to the recessive locus of Bomi 1508, which suppresses these and certain other hordeins. E M studies of Ingversen (1975) showed further that the protein bodies of Bomi contained both prolamine and glutelin proteins while those of Bomi 1508 contained mostly glutelin. The prolamine component appeared homogeneous in E M while the glutelin component was granular. Similar compositional changes in wheat might improve the nutritional quality of gluten proteins. However, it must also be pointed out that the mutant gene caused the prolamine fraction to decrease from the 29% in Bomi barley to 9% in Mutant 1508, while the albumin-globulin fraction increased from 27% to 4670, respectively, of the total protein. Such changed proportions of prolamine and soluble proteins would probably destroy the breadmaking potential of wheat. Nevertheless, the inexactness of the solubility fractionation procedures leaves the lysine-content data open to question until the isolation and analysis of the specific genecontrolled protein subunits with high lysine can be demonstrated. Although the high-lysine sources investigated so far in barley, maize, and sorghum have shrunken endosperms, one of six recently induced high lysine barley mutants has a nonshrunken endosperm, indicating that the defective shrunken endosperm, which reduces grain yield potential, is not an essential feature of all high-lysine sources (Doll, 1976). The shrunken endosperm of a t least some of the high-lysine sources appears to be due to the effects of a gene closely associated with the high-lysine character (Eslick and Hockett, 1976). 6. Enzyme Inhibitors and Toxic Components of Wheat Endosperm Wheat, like other cereals, appears to contain a number of components shown to act as antimetabolites or toxins in various organisms tested (see also, Kakade, 1974). a. a-Amylase Inhibitors. Among the components in wheat endosperm are albumins shown to have specificities toward a-amylases of different animal species (Kneen and Sandstedt, 1943), but not of cereal a-amylases

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(Sandstedt and Beckord, 1946; Silano et nl., 1975). One, AmI, is active against insect a-amylases (Shainkin and Birk, 1970). Similar a-amylase inhibitors were isolated and studied by Ewart (1969), Fish and Abbott (1969), Feillet and Nimmo (1970), Sodini e t al. (1970), Silano e t d. (1973, 1975) , Saunders and Lang (1973), Petrucci et al. (1974, 1975, cited by Kasarda et al., 1976a). The a-amylase inhibitors of wheats are classified in two families, designated 0.19 and 0.28 on the basis of the mobility of the major component relative to bromophenol blue dye upon gel electrophoresis a t pH 8.5 (Silano e t al., 1973). The 0.28 albumin was the same as an albumin described by Ewart (1969) as well as the 13B albumin of Feillet and Nimmo (1970) and probably the same as AmI, of Shainkin and Birk (1970). The 0.19 inhibitor was the same as the albumin of Fish and Abbott (1969), the 13A albumin of Feillet and Nimmo (1970) and the AmI, inhibitor of Shainkin and Birk (1970). Recent analyses by Redman and Ewart (1973) suggest a close identity between ALB13A and CM2 and ALB13B and CM3 of Garcia-Olmedo and Carbonero (1970). Proteins CMI and CM2 were found to be similar even to amino acid sequence. The 0.19 family of albumins was found to have an M W of approximately 24,000 whereas those of the 0.28 family had an M W of approximately 12,500 (Petrucci et al., 1974). A 60,000-MW component with a-amylase inhibitory activity was also identified. When placed in dissociating solvents, the proteins of 24,000 MW were reduced to about 10,000 MW, accompanied by loss in activity which was regained after removal of the dissociating solvent. The reassociation active a-amylase inhibitors also regained their original electrophoretic characteristics. Inhibitors of the 12,500-MW group were found to inhibit a-amylases from several other insects, particularly those that attack grain or grain products (Silano e t al., 1975). The 60,000-MW inhibitors were active also against mammalian and avian species and all three aamylase inhibitors were active against marine species. The inhibitors were completely inactive against a-amylases from immature or germinating wheat grains of all three ploidy levels, suggesting that these aamylase inhibitors either evolved in nature or were selected by man as protective mechanisms to restrict losses caused by grain insects as well as losses due to birds, etc., since both nature and man would likely retain for further propagation those genotypes not previously eaten by other species, and man would likely have available to reproduce only those grains that survived insect attacks during storage. Petrucci et al. (cited by Kasarda e t al., 1976a) have found that 0.19 a-amylase inhibitor was sensitive to pepsin degradation but was not inactivated by trypsin. Puls and Keup (1973) reported that &-amylase inhibitors from wheat retarded the digestion of raw starch in animals and man, but were relatively in-

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effective on cooked starch. No symptoms were produced by feeding concentrated a-amylase inhibitors to celiac paticnts (Auricchio et al., 1974). However, the influence of the a-amylase inhibitors on starch utilization in animals and man would depend upon their relative abundance in the feed or food. I n any case, it is unlikely that active amylase inhibitors would be beneficial in animal or human nutrition. b. Protease Inhibitors. Trypsin inhibitor albumin proteins are present in wheat endosperm and embryo (Hochstrasser et al., 1969; Mikola and Kirsi, 1972; Petrucci et al., 1974; Minetti et al., 1975). I n one study the trypsin inhibitors made up about 9% of the water-soluble proteins of the endosperm (Mikola and Kirsi, 1972), 0.3% of the seed weight or about 1% of the kernel weight, were about one-third of the total albumins (Petrucci e t al., 1974). The trypsin inhibitors of wheat endosperm are quantitatively important components with MWs similar to the 0.19 and 0.28 a-amylase inhibitors, and both separate out in the water-soluble protein fraction. The trypsin inhibitors are more basic proteins separable from the a-amylase inhibitors by gel electrophoresis a t pH 8.5 (Petrucci et al., 1974). Madl and Tsen (1974a,b) identified trypsin and chymotrypsin inhibitor activities in extracts of rye, wheat, and triticale flours. The trypsin inhibitor content that was high in rye appeared to be intermediate in triticale and comparatively low in wheat. The chymotrypsin inhibitor activity of the extracts was equally high in all three cereals. The trypsin inhibitors from triticale were highly heat stable, retaining considerable activity even after cooking for 1 hour in a boiling water bath. The chymotrypsin inhibitor activity of the triticale extract was destroyed by heating to 5OOC. Trypsin inhibitors may be important components affecting the feeding value of triticales, and selection for low inhibitor activity may prove effective for increasing feed or food nutritional value (Madl and Tsen, 1974b). It is also possible that a similar basis may be found for nutritional differences in wheats. Protease inhibitors may function as regulators of protease activity a t certain stages of plant development, but also appear to have evolved as protective mechanisms against insect damage, etc. (Ryan, 1973). c. Gluten. Certain components of wheat gluten, now tentatively identified as fractions of a-gliadin, appear to be the agents responsible for a human ailment called celiac disease, which affects about one in 2000 people in the United States and about one in 400 in Ireland (Kendall et al., 1972; Mylotte et al., 1973). Celiac disease is due to a genetically controlled specific immune response derived by digestion of a gliadin fraction to a peptide (or peptides) . The immune reaction is localized in the tissues of the small intestine and results in a reduction of nutrient absorbing

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capacity (Kasarda, 1975; Kasarda et al., 1976a,b). The aggregatable 0 gliadins, or A-gliadins, which comprise about 4-5 polypeptides are among the gliadins active in celiac disease and may be the only fraction involved (Kendall et al., 1972), but this is not yet certain (D. D. Kasarda, personal communication, 1976). The A-gliadins are absent in the wheat cultivars ‘Justin’, ‘Anza’, ‘Ramona 50’, ‘Calidad’, ‘Atlas 66’, ‘Siete Cerros 66’’ ‘Warrior’, and ‘Omar’ (Platt et al., 1974). Some of these cultivars (Justin, Anza) have breadmaking potential similar t o Scout, Cheyenne, Ponca, and Inia 66, which produce A-gliadins. Although i t is not yet known whether the A-gliadin-deficient, cultivars elicit celiac disease, the absence of these proteins in cultivars having good processing properties suggests that such proteins of low nutritive value and toxic properties can be modified, deleted, or substituted by other component proteins without sacrificing use quality. Studies using stocks nullisomic (deficient) for the group 6 chromosomes should help to further identify the gliadin component (s) responsible for inciting celiac disease (D. D. Kasarda and C. 0. Qualset, personal communication, 1976). More recently, wheat gluten [component(s) not yet identified] has been implicated as a pathogenic factor also in schizophrenia (Singh and Kay, 1976). The same gliadin fraction(s) may prove to be causally associated with the two diseases (D. D. Kasarda et al., personal communication, 1976). AFFECTINGPROCESSING CHARACTERISTICS AND B. MAJORFACTORS NUTRITIONAL VALUEOF WHEATFLOUR I . Milling

Milling to separate the bran from the endosperm and reduce the endosperm to flour is an extremely important factor affecting the amino acid composition and processing properties of the flour (Pomeranz and MacMasters, 1968). Higher amounts of nutritionally better quality proteins are contained in the aleurone layer of the endosperm, but the aleurone is mostly separated with the bran (including also the seed coat and several other outer seed tissues) by the milling process. The bran is generally considered to be undesirable in flour for two reasons: (1) discoloration of the preferred white flour (Ziegler and Greer, 1971 ; Orth and Mander, 1975) and (2) components in the bran and aleurone, i.e., tannins, .and especially the nondigestible sugars raffinose and stachyose (Saunders and Walker, 1969; Stevens, 1970; Saunders et al., 1975a) often have undesirable effects on human digestion (Steggerda, 1968; Hickey et al., 1972; Turner, 1965). In contrast, the aleurone also contains proteins of high nutritional value (see Fig. 2 ) . The higher mineral content of the aleurone layer (see a comparable study on barley in Lin and Pomeranz, 1975)

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provides the basis of the method (flour ash content) used to test the efficiency of the milling process and the extraction rate. Even for the best milling wheats, flour ash content increases rapidly as the flour extraction rate exceeds 70% (Ziegler and Greer, 1971; Farrand, 1974a,b). The modern roller milling process itself involves the separation of the bran [and most of the aleurone layer(s) close to it] from the endosperm by a crushing, tearing, and shearing process, achieved by forcing the grains between cross-corrugated rollers moving a t different rates. The separation of the bran from the endosperm is improved by increasing the moisture content of the grain (especially of the bran) so that the bran is toughened and can be separated from the endosperm in relatively large flakes. Most commerical flour is produced from the separated endosperm after several sequential reduction and sieving processes (Ziegler and Greer, 1971). a. Milling Qua1it.y-Flour Yield. Grain hardness lias been associated with improved milling properties, higher flour yield, and high bran cleanup (Wrigley and MOSS,1968; Symes, 1969; Simmonds, 1972b; Simmonds et al., 1973; Mattern et al., 1973; B. Belderok and G. J. Doekes, personal communication, 1974). However, the degree of hardness typical of high quality durum wheats is not considered desirable in bread wheats (F. Fajersson and G. Rubenthaler, personal communication, 1974). The strong-gluten common wheats and durums (often with weak gluten) generally have hard endosperms, while weaker-gluten common wheats tend to have soft endosperms, even though the kernels of either type of wheat may be vitreous or opaque in appearance as the result of environmental influences. The harder wheats tend to mill a t faster rates and give higher flour yields a t any given ash content than do most weak-gluten “soft” wheats (Ziegler and Greer, 1971). The genetic basis for hardness seems to be relatively well established (Symes, 1969), with a t least one major hardness-controlling gene and two or more less effective genes contributing most to the differences in milling performance. One of these factors evidently is genetically associated with factors for higher protein content in Timstein and ‘Cheyenne’, as compared with Chinese Spring (B. Belderok and G. J. Doekes, personal communication, 1974). Grain hardness also has an influence on the susceptibility of the endosperm to granulation, on the particle size distribution, and on the amount of damaged starch (broken starch granules) obtained in milling. The biochemical basis for grain hardness has been investigated. Simmonds et al. (1973) found that composition of the protein matrix which holds the starch granules together in cells was similar for hard and soft isogenic lines, but that higher amounts of soluble material extractable from starch granules were associated with grain hardness. The soluble material was

GENETIC CONTROL O F PROTEINS I N WHEAT

465

30% protein carrying protease and a- and p-amylase activities, while the nonprotein material was mainly carbohydrate which on hydrolysis yielded glucose and traces of xylose, arabinose, and mannose. Hardness was believed to be due to the degree of adhesion between starch and protein. It should be noted, however, that soft endosperm “weak” gluten wheats need not be poor milling, as measured by the flour yield and rate of milling. The T. aestivum ssp. compactum, or “club” wheats, commonly grown in the Pacific Northwest area of the United States produce some of the highest yields of low-ash flour obtainable and also mill more quickly with less energy input than typical hard endosperm wheats (Reitz, 1964; G. L. Rubenthalcr, personal communication, 1974). Many club wheats, like some recent common soft white derivatives, evidently have thinner and less bran, since their bran clean-up is usually less complete than the better-milling hard endosperm wheats (C. F. Konzak and G. L. Rubenthaler, unpublished.) However, Larkin et al. (1951, 1952) found no influence of bran thickness on the millability of seven Pacific Northwest wheats. The two compressed cell layers of the central part of the seed coat on soft white wheats are of cellulose and are not suberized (MacMasters et al., 1971). Whether that difference is sufficient to account for the different milling properties of certain white wheats is not clear, but the “total inner endosperm extraction” technique of Vogel et al. (1976) should permit greater insight into the basis for flour yield differences. b. Effects of Milling on Starch Damage and Processing Characteristics of Flour. One study has associated the pentosan content of the grain with lower milling yield (Weswig et al., 1962). Pentosans have high waterabsorbing capacity and appear to be major constituents of cell walls of cereals (McNeil et al., 1974). Since the aleurone cell walls generally are thicker than those of endosperm cells (Buttrose, 1963), the separation of the aleurone layer with the bran and shorts fractions appears to be important for high milling yields of low-ash flour (Ziegler and Greer, 1971). The alcuroiie layer in different wheats varies in thickness (3256.8 pm) according to studies reviewed by MacMasters et al. (1971) and thus may account for some of the observed differences in milling properties. The thickness (number of cell layers and/or size of cells) of the bran (including aleurone and subaleurone tissues) differs among varieties and may affect flour yield. Recent work (Butcher and Stenvert, 1973a,b; Moss, 1973) also shows that poorer-milling soft-endosperm wheats absorb water more rapidly than do the hard-endosperm wheats and that a higher “detergent fiber” content is associated with lower-milling yield (Stenvert and MOSS,1974). Detergent fiber content may be influenced greatly by damage to aleurone cells in milling, hence comparisons are valid only with iden-

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tical conditions of grinding (Butcher, 1975). Differences in rates of water absorption between hard and soft wheats, and between good and poor milling wheats, were not considered in earlier studies (Everson and Seaborg, 1958) and still are not adequately reflected in milling procedures used in many experimental laboratories (G. L. Rubenthaler, personal communication, 1975). Differences in the rate of water uptake by hard and soft wheats appear to be of major importance to the ease and degree of endosperm separation in milling (Moss, 1973 ; Stenvert and Kingswood, 1976). Individual wheat kernels in a bulk sample may differ considerably in moisture contcnt before tempering, and may take up additional water a t different rates unless the grains are equilibrated to a pretempering moisturc of 1276, after which the final adjustment before milling can be made (G. W. Fisher, Technovators, Inc., personal communication, 1976). The degree of fragmcntation of thc bran may bc less in soft wheat than in hard or seinihard wheats, permitting a higher extraction rate to be achieved a t lower flour ash levels (Orth and Mander, 1975). However, bran fragmentation and bran clean-up both appear to be genetically controlled since these properties are variety dependent ( C . F. Konzak and G. L. Rubenthaler, unpublished, 1976). Grain hardness affects the percent starch damage (Symes, 1969; Simmonds, 1972b). Thus, flours milled from soft endosperm wheats have less starch damage and the flour particle size generally is smaller than those from hard endosperm wheats (Williams and McEwin, 1967). The alkaline water and water-retention tests (AWRC, WRC) widely used to predict pastry us breadmaking characteristics of wheat flours are based upon the high water-absorption capacity of damaged starch (Farrand, 1964, 1969, 1972; Yamazaki and Lord, 1971 ; Sollars, 1972; Greenwood, 1976). Flours of higher gluten content tolerate more starch damage and still perform satisfactorily in breadmaking (Pomeranz, 1971), but they are unsuitable for pastry (Yamazaki and Lord, 1971). Small differences in water absorption are apparently due to the water-soluble and starch fractions, but changes in the propcrtirs of the cornponcnts of hard us soft wheat flours occur during separation prior to reciprocal reconstitution (Sollars and Rubenthaler, 1975). Some damaged starch seems desirable for yeast breads since the increased water absorption may be beneficial and the damaged starch is more readily a\-ailable for dcgradation by amylases, providing sugar for fermentation during the dough-handling processes (see Wrigley and Moss, 1968; Pratt, 1971; Pomeranz, 1971; Bloksma, 1971; Kulp, 1972; W. Bushuk, personal communication, 1974). Chemical, biochemical, or physical damage may be necessary before amylolytic enzymes can degrade the starch granules (Greenwood, 1976). However, sugar or malt added

GENETIC CONTROL OF PROTEINS IN WHEAT

467

to the mix fully compensates for damaged starch in the breadmaking process. Recent work on “no-sugar” high-yeast short-time bread formulations also indicates that malt or malted wheat will compensate for sugar used in breadmaking with proof height another critical factor (Finney et al., 1977; Magoffin et nl., 1976). Schlesinger (1964) and Farrand (1969, 1972) showed that starch damage, while increasing water absorption and increasing gassing power, reduces tolerance to mixing, reduces loaf volume, and is generally deleterious to bread quality. If the wheat has been weathered, damage to starch by milling is undesirable because the increased activity of amylase enzymes induced during weathering causes excessive gassing and stickiness in the crumb structure of the baked products (Farrand, 1964, 1969, 1972). Different sizes of starch granules appear to have different contents of amylose us amylopectin types of starch, which may degrade differently during weathering or gelatinize differently in processing. Gelatinization, pasting behavior, and viscosity of starches are related, and are influenced by interactions with organic acids, especially linolenic acid (Goering et al., 1974, 1975). Recent evidence suggests that protease enzyme activity also increases during weathering and may be at least as important as amylase activity on the deterioration of Japanese noodle processing quality (Bean et al., 1974a,b). Protease activity also affects the bread-baking quality of wheat (Pomeranz et al., 1966a). The proportion of damaged starch in flour may be influenced by the control of tempering (moisture adjustment process) and mill grinding practices, such as varying roll-speed ratios and roll pressures in the reduction system (Ziegler and Greer, 1971 ; Farrand, 1972). Farrand (1972) obtained flours with three different levels of starch damage from the same wheat, using the same mill, yet keeping constant the homogeneity of the grist, extraction rate, and flour color. After studying the usual quality parameters, he concluded that the physical state of the starch component in flour has an influence on conventional rheological parameters and sedimentation values for estimating baking quality. The technological capability to effect any prescribed level of starch damage would provide a greater measure of flexibility in flour quality than would normally be encountered in conventional laboratory assessments ( Farrand, 1969). Thus, Farrand (1972) was able to achieve a greater distinction of the parameters controlled by the characteristics and content of the protein in wheat flours. The new short-time baking systems indicate possibilities for obtaining more accurate estimates of the processing properties and potential of different wheats via evaluation of several compensating and interdependent factors (Finney et al., 1977). The bread- or pastry-making properties of wheat flours may be in-

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CALVIN F. KONZAK

fluenced by the ability of the miller to control the compensating interrelationships between the protein and starch components (Farrand, 1972) or of the baker to control the dough properties by adjustments in the relative proportions of sugar, malt, and a-amylase in the dough (Pomeranz et al., 1966b; K. F. Finney et al., 1972; P. L. Finney et al., 1977; P. L. Finney, personal communication, 1976). The importance of milling t o wheat processing characteristics is suggested from (1) evidence for wide and complex variability in the proteins comprising gluten, (2) the evidence that part of this observed complexity may be an analytically induced artifact, (3) the evidence that major modifications in the physical properties and chemical composition of flours can be made in the milling process and in the final processing into food products, and (4) the evidence that the proportions of solubility fractions change during dough mixing (Kasarda et al., 1971, 1976a; Hoseney and Finney, 1971; Mecham et al., 1972; Farrand, 1972; Tanaka and Bushuk, 1973a,b,c; Orth and Mander, 1975). Better estimates of starch damage than AWRC may be needed (such as particle size distribution) to distinguish differences in the water-absorption capacity of the protein components, and different milling procedures may be needed to reveal the processing potential of future high-protein and high-lysine varieties. Selection for high water absorption properties of wheat gluten (independent of influences by starch damage) should be possible using semiautomatic gluten washing methods (Greenaway and Watson, 1975). The relationship of possible true differences in the relative water absorption capacity of gluten (wet:dry gluten ratio) to pastry or bread processing characteristics is not clear, but genetically controlled (varietal) differences apparently have been identified (Pollhamer, 1967, 1969). c. Eflects of MilZing on the Amino Acid Composition of Flour. White flour has a lower lysine content than the whole grain wheat from which it was milled (Toepfer et al., 1972). The wheat varieties tested by Simmonds (1962) differed with respect to the relative proportion of lysine in the wheat and extracted in the flour. Surprisingly, two varieties, ‘Gabo’ and ‘Broughton’, with slightly lower flour yields retained the highest proportion of lysine in the flour, indicating that the lysine-carrying proteins are better dispersed through the endosperm tissue in some wheats than is the case with others. The subaleurone cells of wheat generally have an appreciably higher protein content than the rest of the endosperm (Kent and Evers, 1969) ; the differences are even greater in high protein varieties (Barlow et al., 1974; Simmonds, 1974). Recently, V. A. Johnson et al. (1975a,b) also demonstrated that varieties and their cross progeny

GENETIC CONTROL OF PROTEINS IN WHEAT

469

may differ with relation to the proportion of lysine and protein present in the endosperm and extractable in white flour. Exploitation of these important genetic differences may improve the nutritional properties of wheat flour. 2. Nutritional Improvement by Supplements-Malt,

Wheat Malt Flour,

or Other Protein Sources

The nutritional value, including lysine content of cereal grains can be greatly enhanced on germination or malting. Cereal foods may be nutritionally improved when significant amounts of ground whole malted grain replaces white flour (P. L. Finney, personal communication, 1976). The nutritional value of bread can be easily and inexpensively increased between 20 and 60% by the inclusion of wheat malt as a large part of the dough. Until recently, it seemed impossible to substitute nutritionally significant amounts of malt in bread doughs. The major limiting factors were believed to involve sugar, low levels of yeast, and the long preparation times in the standard bread dough formulas used by bakers. The discovery of various compensating factors has led to the development of no-sugar, short-time bread formulations in which malts replace sugar. Studies show that a significant portion of the flour (up to 60%) can be replaced by wheat malt, resulting in an enhanced nutritional value and with improved dough processing characteristics. Strong, longer mix time wheats probably can be germinated longer with less loss of processing functionality with increased levels of malt (K. F. Finney, 1975 ; Pomeranz and Finney, 1975; P. L. Finney, personal communication, 1976) than standard all-purpose wheat blends. Soft endosperm wheats may be malted more easily (Pomeranz et al., 1975b), possibly owing to their faster wetting and the genetic association of germination inhibitors with red seed coat color. The “high-yeast,” “no-sugar” bread formulations under investigation also promise to shorten processing time and thus reduce cost. Studies t o achieve even more nutritional malts are in progress (Dalby and Tsai, 1976; P. L. Finney, personal communication, 1976). The development of high-lysine synthesizing yeasts might now be a useful approach to complement the nutritional potential of such formulations, since high-lysine yeasts should be inducible with mutagens. Such new approaches may also be more convenient and economically feasible worldwide than the use of special additives, protein concentrates, synthetic amino acids, or soy flour. Wheat protein concentrates (WPC), prepared by alkaline extraction of protein from the bran by-products of flour milling, may have promise as inexpensive supplements to improve the nutritional value of baked

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products (Fellers et al., 1966; Miladi et al., 1972; Saunders et al., 1975a). As with soy flours and other additives, only limited amounts of WPC can be added to bread formulations without significantly affecting the processing characteristics, although the amounts permissible may improve the amino acid balance of the products (Betschart et al., 1975). 3. Rheological Properties of Flour Proteins and Other Components

a. Thiols, Disuljide Linkages, Other Chemical Bonding Mechanisms. The contents of reactive thiol and disulfide groups in wheat flour and the rheological properties (viscosity, elasticity, strength) of dough correlate well with recognized baking quality characteristics (Pomerans, 1971, 1973c; Kasarda et al., 1971, 1976a; Redman and Ewart, 1971). The rheological properties of dough largely determine the volume and crumb structure of the baked loaf of bread (Wrigley and Moss, 1968; Bloksma, 1971; Pomeranz, 1971). It is a common practice to add oxidizing agents, such as iodate or bromate, and other flour improvers (ascorbic acid in Europe and more recently in the United States) to stiffen dough. The stiffening of dough occurs by the oxidative removal of thiol groups rather than by the formation of additional disulfide linkages between polypeptides and is explained by the reduction of thiol-disulfide interchange reactions in dough (Ewart, 1972a,b,c; Bloksma, 1975). An apparent anomaly in this regard is the similarity in effects of ascorbic acid and bromate as dough improvers. Grant (1974) found evidence that ascorbic acid acts via an enzymic mechanism related to a butanol-extracted lipid fraction of flour. Only a small portion of the thiol and disulfide bonds in isolated gluten (about 376, or about 20% of those that are reactive) are rheologically effective, i.e., restrict the elastic deformation (Bloksma, 1971, 1972a,b). The rheologically effective thiol groups are considered to be present in small molecules, such as glutathione and cysteine, since these compounds will diffuse much more rapidly in dough than do proteins and may participate in exchange and transfer reactions with protein disulfide groups, permitting a certain amount of slippage during the dough-mixing process (Archer, 1972; Jones et al., 1974). However, thiols also are chemically sensitive to inactivation by oxidation reactions, possibly yielding disulfide linkages, which may cause the dough t o stiffen (Ewart, 1972a,c; Bloksma, 1971, 1972a,b). Jones et al. (1974) indicate that less than 4% of the total disulfide is involved with determining the development time of dough during mixing; 11-13% of the total disulfide is involved with mixing tolerance. Differences in the rheological properties of doughs were directly related to the content of rheologically effective thiol and disulfide groups. G. Fabriani

GENETIC CONTROL OF PROTEINS I N WHEAT

47 1

(cited by Matsuo and Irvine, 1975) indicates that the ratio of reactive

SH to total SH is related to the cooking quality of spaghetti. The rheo-

logical properties of semolina doughs, measured on a Brabender Farinograph, show that higher dough consistency and strength estimated by farinogram band width are closely correlated with cooked spaghetti firmness or tenderness index (Matsuo and Irvine, 1975). b. Protein and Carbohydrates. The important rheological properties of wheat seem t o be more characteristic of the glutenins than of the gliadins (Hoseney et al., 1970a, 1972; Bietz et al., 1973; Greenwood and Ewart, 1975). Redman and Ewart (1971) suggest that the differences in rheological properties between glutelins of oats and barley from those of wheat may be due to the fact that glutenins of wheat have much less intermolecular disulfide cross-linking (and may have none, Greenwood and Ewart, 1975) but are intramolecularly linked by SS bonds. However, the complete lack of viscoelasticity of maize and oat flours may also be due in part to the influence of different amino acid composition and solubility properties of the proteins in those species. Barley, oats, and maize also lack certain alcohol-soluble proteins (Elton and Ewart, 1962), and the glutelin subunits of oats, maize, and sorghum are of lower MW than those of wheat, rye, and barley (Ewart, 1972b, 1975). The role of the protein in soft-wheat flours used for pastry products is not well understood. The carbohydrate components, particularly starch tailings (fine and damaged starch) and pentosans, are more important than protein (Yamazaki and Lord, 1971 ; Pomeranz, 1971 ; Bloksma, 1971; D’Appolonia et al., 1971; Wade and Elton, 1967; Sollars and Rubenthaler, 1975). However, recent studies on corn and barley starches suggest that pasting viscosity is due not only to the gelatinization and breakdown of starch granules (low viscosity is important to soft-wheat pastry-making potential) , but, rather, is controlled by the breakdown of an amylose-fatty acid (largely linolenic acid) complex (Goering et al., 1975). Many soft wheats have a low protein content, but some soft wheats with high protein, such as Atlas 66, prove to have both pastry and breadmaking properties. The high-protein flours are preferable for bread, whereas the low protein flours are suitable for pastries. The soft wheats generally have weak gluten traits and short-time mixing properties, quite unlike the hard-endospcrm, strong-gluten, bread wheats (Reitz, 1964). Soft wheats studied by Jones et al. (1974) had lower amounts of rheologically effective thiol and disulfide groups. Recent work a t Washington State University indicates that it may be possible to extend the mixing time and tolerance of soft common wheats, retaining the pastry-making properties while improving the bread dough properties (C. F. Konzak and G. L.

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Rubenthaler, 1974, 1975, unpublished). Also, it may be notable that some of the softer Australian wheats, ‘Sherpa’, ‘Ford’, and ‘Javelin’, have both pastry and general purpose breadmaking flour characteristics (Moss, 1973; G. L. Rubenthaler, personal communication, 1975). However, those cultivars still lack the mixing strength and stability of the standard hardendosperm bread wheats. The seeming absence of soft-wheat recombinant lines with more stable and stronger protein characteristics in crosses of wheats with weak vs strong protein properties may be due to genetic linkages. However, the selection against these recombinant types by breeders and technologists also may be a factor, due in part to assumptions about quality factors drawn by cereal technologists from their analyses of wheat quality “standard” varieties. Genetics and breeding experiments designed to isolate recombinant soft wheat genotypes with short us long mixing stability and low us high extensibility suggest that the doughmixing properties of wheat protein may not be the critical determinants of loaf volume, crumb characteristics, or pastry-making properties and that it may be possible to develop truly dual-purpose pastry and bread quality types of varieties (C. F. Konzak, G. L. Rubenthaler, and P. L. Finney, unpublished, 1976). Michigan State University recently has released to production a higher protein soft wheat variety, ‘Tecumseh,’ with good pastry-making properties. Tecumseh flours have large particle size even a t higher protein than normal soft wheats, providing positive evidence that even pastry products can contribute protein as well as calories to human nutrition (E. H. Everson, personal communication, 1976). c. Lipids. One other group of compounds, the glycolipids, present as only a small fraction of the total lipids, appear to be important to the rheological properties of bread dough. Synthetic glycolipids have considerable potentiql as dough improvers (Pomeranz, 1973b,c; Pomeranz and Finney, 1973a,b). The glycolipids have been extensively investigated by Pomeranz and colleagues (Pomeranz and Finney, 1969, 1973a; Wehrli and Pomeranz, 1970; Hoseney et al., 1970a,b; Pomeranz, 1971, 1973b,c). Lipids also seem to be important to the cooking properties of pasta made from durum wheat semolina (Irvine, 1971) and to bakery products made from soft wheats (Yamazaki and Lord, 1971). Many of the lipids are bound with protein as lipoprotein, but some are thought to be derived from the embryo and extracted with the flour during milling (Pomeranz, 1971).

4. Relation of Flour Protein Solubility Fractions to

Processing Characteristics Flour component fractionation and reconstitution experiments have established that the modified “Osborne” solubility characteristics of

GENETIC CONTROL O F PROTEINS I N WHEAT

473

wheat proteins relate directly to the functional role of these proteins in breadmaking (Hoseney et al., 1969a-d, 1970a,b ; Hoseney and Finney, 1971 ; Pomeranz et al., 1970). The gluten proteins are primarily responsible for the dough-forming properties of wheat flours, whereas the watersoluble components of wheat flours are important to maximum performance of the glutens in baking for all wheats used for bread (Pomeranz, 1971; Kasarda e t al., 1971, 1976a). However, it should be noted t h a t the albumin and globulin (water- and salt-soluble) fractions comprise a small proportion of the total protein (Bushuk and Wrigley, 1974) and that the percentage of these fractions decreases slightly with increasing protein content (Mitra and Bhatia, 1973). The “soluble” proteins are abundant in rye and triticale (Bushuk, 1972). High dough extensibility, a qualitative characteristic of the gluten proteins, may be preferred for breadmaking but not for the making of pastry or pasta (W. C. Shuey and G. L. Rubenthaler, personal communication, 1974). The quantity of gluten protein required for optimum product quality must be above a certain minimum (9-11%) for pasta, bread, and other leavened foods, but below that range for fine pastries (Reitz, 1964), because the quantity of protein directly relates t o the functional properties of dough. Several reports indicate that the gliadin: glutenin ratio is correlated with the rheological and bread baking properties of wheat flours and of the pasta-making properties of semolinas (Bushuk et al., 1969; Bushuk and Wrigley, 1971; Pomeranz, 1971; Wrigley, 1972a; Orth and Bushuk, 1972; MacRitchie, 1973; Matsuo and Irvine, 1975). The correlations ohtained, however, are not so high as to indicate an invariant relationship. Gliadins as a group contribute to dough stiffness, while glutenins, including high MW “residue” proteins, contribute to elasticity and stability during mixing (Hoseney and Finney, 1971 ; Finney, 1971; Wrigley, 1972a ; Bietz et al., 1973). The qualitative and quantitative composition of the proteins within the general gliadin and glutenin groups may vary within limits determined by their functional interactions. Results of Elton and Ewart (1962), Doekes (1968), Doekes and Hack (1971), and Wrigley and Shepherd (1974) suggest that wheats with similar functional properties may have distinctly different combinations of specific gliadins, and conversely, genetically closely related varieties may have similar gliadin spectra but different breadmaking properties. However, Tanaka and Bushuk (1973a,b,c) found the very strong gluten wheat, ‘Red River 68’, has more high M W proteins than ‘Thatcher’, which has medium-strength gluten, while Thatcher had more high MW proteins than Talbot, a weak gluten soft wheat. Similarly, quantitative and qualitative differences in specific glutenin proteins have been associated with the breadmaking properties of a number of wheat cultivars (Cole et al., 1976; Huebner and Wall, 1976).

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CALVIN F. XONZAK

Recent work, made possible as a result of the development of methods for solubilizing the “residue” protein of earlier extractions, has confirmed the suggestions that residue proteins were largely glutenins and that the glutenins possess viscoelastic characteristics important to the rheological properties of wheat gluten (Bietz and Wall, 1975; Bietz et al., 1975; Kasarda et al., 1976a). Glutenin proteins are not cross-linked by SS bonds. Different glutenin molecules are comprised of variable numbers of polypeptide chains not necessarily alike, which are joined end to end by SS bonds to form super molecules or concatenations possessing a linear, nonbranched configuration. The viscoelastic properties of glutenin are thought to be due t o complex conformations with water and interand intramolecular entanglements. The complex entanglements of gluten molecules provide the (loose) cross-links responsible for the properties of rubberlike elasticity. Stress causes the unfolding of individual polypeptide chains in a concatenation, and the natural tendency of the molecules to return to their compact or relaxed state of lower free energy accounts for the elastic responses. Intermolecular slip processes account for the viscous flow (Greenwood and Ewart, 1975). Genetic factors controlling important functional (breadmaking) properties of wheat have been identified with specific chromosomes. For example, studies using chromosome substitution lines (one pair of chromosomes from the test variety substituted for the same chromosome of a standard variety by backcrossing) show that dough-mixing strength in the variety ‘Itana’, as compared with ‘Kharkof MC22’, is associated with its chromosome 1D (Welsh and Hehn, 1964). The substitution of chromosome 1D from the variety Hope into the variety Chinese Spring increases loaf volume, indicating that genetic factors concerned also with loaf volume are located on chromosome 1D (B. Belderok and G. J . Doekes, personal communication, 1974). Glutenin subunits associated with breadmaking quality are coded by D genome chromosomes (Orth and Bushuk, 1973c; Bietz et al., 1975). The higher loaf volume capacities of Timstein, ‘Capelle Desprez’, and Cheyenne relative to Chinese Spring are associated with chromosomes 2D, 3B, and 1A but not with 1D (B. Belderok and G. J. Doekes, personal communication, 1974). Also, as indicated earlier, Kasarda et al. (1973) have demonstrated that the A-gliadin fraction of a-gliadins is absent from some wheats, such as Justin and Inia 66, which are known to have strong dough properties, as well as from wheats like Atlas 66 and Omar with weak dough properties. The converse is also true-A-gliadins are present in strong-gluten wheats like Cheyenne, and also in Capelle Desprez which has weaker gluten strength. Using the Cheyenne into Chinese Spring disomic substitution lines of Morris et al. (1966, 1968,

GENETIC CONTROL OF PROTEINS IN WHEAT

475

19731, Kasarda et al. (1973, 1974a,c, 1976a,b), and Platt et al. (1974) have established that the 4-5 aggregatable A-gliadin subfractions of the a gliadins are coded for by chromosome 6A. I n fact, most of the gliadin proteins are produced by chromosomes of groups 1 and 6 (Wrigley and Shepherd, 1973; D. D. Kasarda, personal communication, 1976). Baking quality, e.g., loaf volume, crust appearance and crumb grain and texture, are associated with chromosomes in group 1 of Cheyenne as compared to weak gluten Chinese Spring. Mixing strength is associated with chromosome 1 D in Cheyenne (Morris et al., 1966, 1968; Mattern et al., 1968a, 1973). Dough and baking properties are controlled also by factors on several other chromosomes including groups 2, 6, and 7, the specific relations depending on the variety (see below). The results suggest that certain specific gluten proteins may be relatively more important than others to the processing properties of wheats. The replacement of gliadin with high MW gliadin (MW 100,000) isolated by gel filtration in a synthetic dough system consisting of gliadin, glutenin, and starch caused a slight strengthening of the mixing properties, while the replacement of gliadin by lower MW gliadin (MW 44,000 and 27,000) caused a slight weakening of mixing properties. Replacement of gliadin by acetic acid-soluble glutenin or albumin fractions greatly weakened the mixing curves, suggesting that the quantitative MW distribution of gliadin components may be important in determining the functional properties of wheat flour dough systems. Interestingly, mixing of dough under conditions of accentuated breakdown did not change the proportion or characteristics of water- or salt-soluble proteins but increased the amounts of extractable alcohol-soluble and acetic acidsoluble proteins with a decrease of the “insoluble” fraction. Further analyses showed that the amounts of low MW gliadins and glutenins were increased by mixing (Preston eE al., 1975). Wasik and Rushuk (1975a) found that the quality of spaghetti made from durum wheat semolina was strongly correlated with glutenin :gliadin ratio, with correlations significant to 1% obtained with farinograph mixing tolerance index (-0.661), with gluten strength (0.845), and with tenderness index (-0.681) ; glutenin: gliadin ratio with farinograph mixing tolerance index (-0.666). The differences in sphaghetti-making quality were also found to be associated with the MW distribution and relative concentrations of the six largest glutenin subunits identified by SDS-polyacrylamide electrophoresis of reduced glutenin (see also Matsuo and Irvine, 1975). The proportion of residue protein (high M W glutenin) in gluten of herd endosperm wheats is closely correlated with baking and rheological properties (Tsen, 1967; Huebner 1970; Wall et al., 1971; Orth and Bushuk, 1972,1973b; Bietz et al., 1973; Huebner and Wall, 1975). The

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CALVIN F. KONZAK

correlation may result (at least in part) because the lower waterabsorbing ability of the high-MW glutenin compensates for increased starch damage caused by milling, with only the correct balance of water absorbing capacities of the gliadins, glutenins, starch, and lipid providing for satisfactory mixing properties. The results of Farrand (1969, 1972) suggest that modifications of the milling procedures to produce lower levels of damaged starch may greatly change estimates of the relative importance attached to several parameters used for processing quality estimation, e.g., water absorption, sedimentation, stiffness, extensibility, farinograph stability, AWRC and protein-loaf volume relationships (see also Pomeranz, 1971 ; Bloksma, 1971 ; Greenwood, 1976). The major function of gluten in making bread and other leavened products is to form a network able to hold gas bubbles formed by fermentation, and to keep starch granules separated, preventing them from growing together as they gelatinize during baking. Gluten is not absolutely essential for crumb structure, since completely protein-free bread with a reasonably good (albeit substandard) volume and crumb structure has been made of starches from wheat, rye, or potatoes using saturated monoglycerides (such as glycerol monostearate), vegetable gums, or gelatinized starch as binders (D’Appolonia et al., 1971), or using the microbial gum xanthan (J. A. Bietz, personal communication, 1975). IX. Genetic Variation for Protein Content and Lysine Composition i n Wheat

Many possible genetic sources of variation for improved protein content and lysine composition in common wheats are listed in reports by Lawrence et al. (1958), McDermott and Pace (1960), Tyuterev et al. (19731, Villegas et al. (1968), V. A. Johnson and Mattern (1972, 19751, and Vogel et al. (1973), but only a small number of these sources have been widely utilized (Table 4). Breeding experiments and other analyses indicate that a small number of major genes contribute to the high protein content in the Brazilian wheats ‘Frondoso’, ‘Frontiera’, and derivatives, especially Atlas 66, which have been used as parents in breeding wheats of higher protein content (Middleton et al., 1954; Davis et al., 1961; V. A. Johnson et al., 1963, 1968b, 1975b; V. A. Johnson and Mattern, 1975; Lofgren et al., 1968; Caepoiu et al., 1975; Balla and Gaspar, 1975). Improved protein mutants also have been induced (Dumanovib et d.,1970; Pepe and Heiner, 1975; Konzak, 1975, unpublished). Heritability estimates for grain protein content in wheat indicate in some cases important and variable influences of environment, with genes

GENETIC CONTROL OF PROTEINS IN WHEAT

477

for protein content showing largely additive genetic effects. Generally, heritability estimates have been sufficiently high to show that selection for higher protein content can be made with relative certainty of success. Davis et al. (1961) reported heritability estimates of 54% to 69% for crosses of soft red winter wheats (Frondoso and Frontiera sources) and Haunold et al. (1962) and Stuber et al. (1962) obtained heritability estimates of 56% and 83% respectively for crosses involving Atlas 66, while Sunderman el al. (1965) obtained heritability estimates of only 15-26% for protein content in the cross Atlas 66/Itana. Heritability estimates also for grain proteins calculated by Lebsock et al. (1964) for a hard red spring wheat cross varied from 37% to 70% based on the regression of F, and F, data, respectively, on F, line values. Flour protein content was inherited bimodally with little or no dominance for the spring wheat cross Selkirk/Gabo (Kaul and Sosulski, 1965). The lowest estimate was 66% for ffour protein content heritability based on analyses of flour from F, and F, plants, while heritability estimates of 76% to 89% were obtained for F, to F, lines, with F,/F,, to F,/F, correlations ranging from 92% to 102.6%. Heritability estimates for DBC value were only 27% in comparisons of the F,/F, and FJF, generations of 51 lines grown in six environments (Sharma et al., 1973). Protein content variation in F,, F,, and F, generations of crosses between Atlas 66 and derivatives with local hard red winter wheat varieties was found to show transgressive segregation for protein content. I n this study, thousand-kernel weight was negatively correlated with protein in one cross combination, but no relation was observed with another (Caepoiu et al., 1975). However, Jain et al. (1976), in a detailed analysis, consider the high heritability of TKW as an advantage in selecting for high protein content and high protein yield per acre. They observed strong to moderate negative correlations for protein content and TKW for four hard spring wheat crosses involving both Frontiera and Nap Hal as high-protein gene sources. The varieties Frondoso, Frontiera, ‘Frontana’, ‘Atlas 50’, Atlas 66, ‘Magnif 41’, ‘Aniversario’, and more modern derivatives of these cultivars probably carry the same gene(s) for high protein content (E. Favret and 0. Klein, personal communication, 1972; V. A. Johnson and Mattern, 1972, 1975; V. A. Johnson and Lay, 1974). Argentine wheats studied by Caepoiu et al. (1975) may have a largely similar gene base. The pedigrees of Frondoso and Frontiera trace to old Brazilian criollo (land) varieties (Silva, 1966). A factor (or factors) controlling the high endosperm protein content of Atlas 66 is genetically linked to leaf rust resistance and is located on chromosome 5D (Morris et at., 1973). Tolerance to acid soils (aluminum toxicity) and high protein content of

478

CALVIN F. KONZAK

TABLE 4 Some Useful or Potentially Useful Genetic Sources of Genes for High Protein and/or Lysine, Basic Amino Acids (BAA) in Common and Durum Wheats

Variety

Sel., P.I. or (3.1. No.

Source or origin

Vernalization response

Useful (high) trait ( 8 )

Common wheats

April Bearded Atlas 50 Atlas 66 Atlas 66-derived lines Kaw/Atlas 50 lines Frondoso Frontiera Aniversario Magnif 41 Magnif 41-ERT 1 Malans Nebr. Male Fert. Restorer Nap Hal Nap Hal Nap Hal Nugaines Mahratta Plainsman V

Hume*2//Nebred+/Agatha Hybrid English Pearl Fultz/Hungarian Fultz/Hungarian/Minturki/ Fultz Sel. H 189-29 Norin lO/Brevor. Sel. 14/ Sel. 27:15/Rioj/Rex, Sel. 53 Norin lO/Brevor, Sel. 14)/ (Brevor Sib, 50-3) Timgalen 22 A Induced mutants Chris Mutant MN6616M Marfed Mutant MJD720175 Rageni 15

England S I USA, N.C. USA, N.C. I D W USA, Neb. c W USA, Kansas Brazil CI 12078 S Brazil CI12019 S Argentina CI12578 S PI344466 Argentina S USA, Wash. WA6096 S S. Afr. CI6127 S NB542437 USA, Neb. W PI176217 India S and w PI 176221 India S PI176223 India S GI13968 USA, Wash. W Australia CIS500 S USA (Seed ReW search Associates, Scott City, Kansas) SD69103 USA, S.D. W CI16225 England W CI3285 S Sweden CI11849 USA, Ind. W CI12756 USA, Wis. W

Protein, Lysineasb Proteine Pr0teina.b Protein0.b Proteinbsc ProteinaJ Proteinaab Pr0teina.b Proteinb Proteinb Proteinnsb ProteinaSb Protein, lysineQLb Protein, BAAb Protein, BAAb Ly sine' Lysine0.b Proteinc

CI13447

USA. Wash.

W

Lysine0Bb

CI13449

USA, Wash.

W

Lysineaeb

CI15400 CI5484

Australia USSR

S S

Proteinb ProteinaJ

CI17241 WA6270 PI383308

USA, Minn. USA, Wash. Pakistan

S S S

Protein, Lysineb Proteinb

CI7337 CI12534 CI12561

Pr0teina.b Proteinneb Lysineanb Proteina Protein'

GENETIC CONTROL O F PROTEINS IN WHEAT

479

TABLE 4 (Continued)

Variety

Sel., P.I. or C.I. No.

Source or origin

Vernalization response

Useful (high) trait(s)

Durum whealsg Coll. F. E. Johnson Coll. If. V. Harlan Abyss. 27 Coll. H. V. Harlan 345 USSR 19284, Dickson 415 fulvo obscurum Obispado Zybicum, HN1 Anafil Claro Alonso Perez Jurado Dabat 119 Coll. ELS 3604-8D, Akaki COIL ELS 6404-2B, Akaki

CI3117 CI7499 CI7799 PI94712 P I 134919 PI136574 PI184542 PI 185760 PI 190992 PI192129 PI297852 PI297855

Tunisia Egypt Ethiopia Ethiopia Portugal Spain Portugal Portugal Spain Ethiopia Ethiopia Ethiopia

S S S S S S S S S S S S

Protein Protein Protein Lysine, BAA Protein Protein Protein Protein Protein Protein, lysine Protein Protein

V. A. Johnson ct al., 1972b; V. A. Johnson and Mattern, 1972, 1975. C. F. Konzak and G. Rubenthaler, 1974, unpublished. E. G. Heyne (personal communication, 1974, Kansas State University) ; Lofgren et al., 1968; C. F. Konsak and G. Rubenthaler, 1975, unpublished. Nap Hal, PI176217, is a mixture of lines having winter, semi-winter, or spring vernalization responses. Also, Bietz el al., (1975) detected variability for the presence versus absence of individual glutenin protein components in a PI176217 population. R. E. Allan and G. Rubenthaler, 197.5, unpublished. f J. F. Pepe and R. E. Heiner, 1975. Evaluations confirmed by cooperative research a t Washington State University and a t North Dakota State University (J. Quick, personal communication, 1976). a

Atlas 66 are genetically associated (Mesdag et al., 1970). At least one important gene for A1 tolerance also appears to be on chromosome 5D (C. F. Konzak and A. M. Prestes, unpublished, 1975). A gene for high lysine content may be present in chromosoine 1D of wheat (Mattern et al., 1968b), which may relate to the producer gene for the high lysine purothionin protein also located on that chromosome (Garcia-Olmedo et al., 1975). Riley and Ewart (1970) obtained evidence that King rye chromosome 11, which is homoeologous to group 5 wheat chromosomes, increases lysine in wheat protein by nearly 9%. Three induced mutants recently identified in advanced cultivars show promise as useful germ plasm sources for increasing protein content or improving protein composition. The male fertility restorer (for cytoplasmic sterile lines used in hybrid wheat breeding) and the Agropyron derivative (SD69103) may be distinct sources of high protein germ

480

CALVIN F. RONZAK

plasm. Atlas 66 appears to carry a different gene(s) for protein content from Nap Hal (PI176217), which also transmits higher lysine composition to its progeny (V. A. Johnson et al., 1973). Other Nap Hal accessions, PI176221 and PI176223, appear to be even more promising than PI176217 as protein and dibasic amino acid (DBAA) gene sources (C. F. Konzak, unpublished, 1975). The gene(s) from Atlas 66 appear(s) to provide capability for 2-4 percentage points more protein than that of ordinary wheats and is effective over a wide range of fertilizer levels. Atlas 66 and derivatives also have higher NRA than normal wheats and their foliage contains more N throughout the growing season. The high protein wheats also translocate more of the N taken up into grain protein (Croy and Hageman, 1970; Duffield e t al., 1972; V. A. Johnson et al., 1973; Schmidt et al., 1974; Dalling et al., 1975; Klepper, 1975), but take up no more soil N (V. A. Johnson et al., 1967). The genetic contribution to higher protein content is inherited independently from grain yield (Middleton e t al., 1954; V . A. Johnson et al., 1967; V. A. Johnson and Mattern, 19751, by way of increased N-use efficiency. The gene(s) for higher lysine in Nap Hal and (313449 also appear (s) to be different (V. A. Johnson e t al., 1975a,b; V. A. Johnson and Mattern, 1975). The variety ‘April Bearded’ (CI7337) appears to carry some of the same genes as Nap Hal for high protein and high lysine (V. A. Johnson and Mattern, 1975). Likewise, additivity of genes for high protein accompanied by increased DBAA values occurs in derivatives of Magnif 41 ert l/April Bearded, and high-protein genes of Magnif 41 ert 1 and CI5484 appear to be independent and additive in action (Nguyen Van Mung and C. F. Konzak, unpublished, 1976). Most important, recombinants with the high milling yield of Magnif 41 ert 1 appear to carry high protein content and DBAA value improvements in the milled white flour. Preliminary results also indicate that the high flour DBAA value of ‘Mahratta’ also is recombinable with the highprotein flour characteristic of Magnif 41 ert 1 (N. V. Mung and C. F. Konzak, unpublished, 1976). Indian wheats appear to be a major source of the high-lysine germ plasm. Nap Hal was obtained from India (possibly Nepal), while Mahratta (CI8500) from Australia, has the pedigree ‘Indian 17’ X ‘Federation’. Federation (CI4734) could also be a source of higher-lysine germ plasm, since it is a parent in the background of (2113447 and CI13449 and is a parent of ‘Marfed’ (CI11919) and numerous other Pacific Northwest wheats. Federation was selected from the cross ‘Purple Straw’ Sel. 14A/‘Yandilla’. Yandilla came from the cross ‘Improved Fife’TEtawah’, while the Etawah parent came from India (MacIndoe and WalkdenBrown, 1968). Some recent analyses of a series of Nepalese wheats sug-

GENETIC CONTROL O F PROTEINS I N WHEAT

481

gest that Nepal is in fact a geographic center of genetic variability for protein and DBAA composition in wheat (Table 5 ) . A. AMINO ACIDCOMPOSITION OF HIGH-LYSINE AND HIGH-PROTEIN WHEATS

No selection or mutant has yet been found in wheat which has lysine composition as high as those identified in barley, oats, or maize (V. A. Johnson et al., 1968a,b, 1972, 1975a; V. A. Johnson and Mattern 1972; Siddiqui and Doll, 1973; Doll, 1973; Munck et al., 1970; Mertz et al., 1974). Lysine values above 4.2 gm per 100 gm of protein have been obtained for Nap Hal, one of the promising sources of high-protein, highlysine germ-plasm (V. A. Johnson and Mattern, 1972). However, the environmental component of variation for lysine appears to be quite large (V. A. Johnson et al., 1973, 1975a,b; V. A. Johnson and Mattern, 1975; Gain et al., 1975). Recent amino acid analyses made a t the IAEA Laboratory a t Seibersdorf, Austria, on whole grain samples of Nap Hal, Mahratta, and several soft white wheat mutants generally confirm data of V. A. .Johnson et al. (1973) and Johnson and Mattern (1972, 1975) that Nap Hal has very high protein content (Table 6 ) . The lysine composition of Nap Hal is 3.17% in 19.7% protein, or about the same as Marfed mutant MJD720175 (3.21% in 13.7% protein grain). Grain from highprotein derivatives of Atlas 66 appears to contain more total lysine, but their lysine/protein ratio is the same as for normal wheats (Mattern el al., 1968b). However, sufficient variability in amino acid composition was present among Atlas 66 derivatives to suggest that selection might improve the balance of lysine and other amino acids (Mattern et al., 1968b, 1975). B. SITE OF THE HIGH-PROTEIN AND HIGH-LYSINE IN THE WHEATGRAIN COMPOSITION The embryo, and the aleurone layer of the endosperm, removed from the kernel by milling to produce white flour, are normally higher in protein and essential amino acids than the inner endosperm parts, but protein appears more concentrated near the aleurone tissues (Kent and Evers, 1969; Mattern et al., 1975), due largely to different size and ages of cells (Evers, 1970). It is not known if the same relations pertain for all wheats, since a larger portion of lysine is recovered in the flour from CI13449 than from Nap Hal, while the bran of both wheats retains proportionately more protein than does that of Atlas 66 (V. A. Johnson and Mattern, 1975). Recombinant derivatives with increased endosperm lysine and/or

TABLE 5 Analysis of Wheat vs Flour Protein Values for New Breeding Materials (1974 Crop, Pullman)

Variety or Sel. No.

Average flour yield

Wheat Udya DBC %

Wheat Kjeldahl

Flour Udya DBC %

Flour Udya protein

Flour Kjeldahl

CI 14995 subbarbarrosa CI14996 turcomanicum CI14997 lutinflalum CI14998 alborubrum CI14999 ferrugineum C115000 glaucolutescens CI15279 subrubruminjlatum C115001 graecum CIl5002 a2bidum CIl5003 lutescens CI15005 pseudoingrediens C115007 nigricolor CIl5008 milturum CIl5011 caesium

73.8 76.8 70.6 70.9 68.8 75.7 70.5 72.6 74.8 73.0 73.1 75.9 64.4 74.8

34.25 38.00 37.00 31.75 36.25 34.25 37.50 37.00 39.25 34.75 39.00 40.00 36.00 33.25

14.2 15.7 15.2 13.6 15.5 15.6 16.3 15.0 16.5 15.7 16.2 17.2 15.2 14.3

36.50 38.75 39.25 33.50 41.25 35.50 43.50 37.00 44.00 39.25 45.50 48.50 38.00 39.25

14.1 15.0 15.2 12.5 16.0 13.6 16.9 14.3 17.1 15.2 17.7 18.9 14.7 15.2

12.8 14.1 13.7 11.7 14.4 13.2 14.8 13.5 15.0 14.5 15.3 16.1 13.5 13.0

Difference U-K flour prot. 0.4 0.9 1.5 0.8 1.6 0.4 2.1 0.8 2.1 0.7 2.4 2.8 1.2 2.2

Udy (U) protein value is based on content of dibasic amino acid composition (DBAA), i.e., a dye-binding capacity (DBC). High deviations from Kjeldahl (K) protein values indicate a higher proportion of DBAA than for standard wheats. Analyses via cooperation of G. L. Rubenthaler, Western Wheat Quality Laboratory, USDA-ARS, Pullman, Washington.

Es

Z r

2 R

TABLE 6 Amino Acid Analyses of Wheats Grown at Pullman, Washington in 1972 and 1973 (Analyses IAEA Seibersdorf Laboratory, May 1974) Amino acid composition (gm/16 gm N)

Protein" content Crop year

Accession No.

1973 1973 1972 1973 1973 1973 1973 1973 1972 1973 1973 1973

MJD720 175 MJD720175 M JD720175 MJD720433 MJD720435 MJD720437 MJD720442 CI11919 CI11919 PI 176217 CI8500 CI4127

Name Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed mutant Marfed (check) Marfed (check) Nap Hal Mahratta

UdY (DBC) Kjeldahla 17.7

-

16.0 14.4 15.3 16.1 15.4 24.2 17.3 17.3

14.2 14.3 14.0 13.3 13.5

14.3 14.8 14.0 14.3 19.7 13.7 14.3

% Lysa protein

mg Lys/gm sample

Lysine

3.16 3.19 3.19 3.06 2.93 2.87 2.93 3.04 3.00 3.17 3.20 2.87

4.49 4.56 4.47 4.07 3.95 4.11 4.33 4.26 4.28 6.24 4.39 4.11

2.90 2.90 2.90 2.80 2.68 2.63 2.67 2.78 2.74 2.88 2.93 2.62

Glutamic acid Arginine 29.8 28.7 30.6 29.9 30.9 31.0 30.4 30.0 31.2 32.0 28.6 29.8

Dry weight basis; all values other than dye-binding capacity (DBC) are based on Kjeldahl N (protein

=

5.07 5.01 5.03 4.66 4.62 4.46 4.59 4.72 4.96 4.85 5.23 4.92

N X 5.7).

Proline 9.8 9.5 9.9 10.0 10.2 10.2 10.1 9.8 10.2 11.0 9.43 10.1

0 3

w

T1Y

M

2

484

CALVIN F. KONZAK

protein have been recovered from crosses Atlas 66/Nap Hal, and Atlas 66/CI13449 (V. A. Johnson et al., 1975b). Important differences have been observed in milling and protein analyses of several spring wheat sources of high protein and high lysine grown under two Washington State environments in 1974 (Table 7). The crop year rainfall a t Pullman was 719 mm, but only 351 mm a t Lind. The temperature and evapotranspiration conditions a t Lind also were higher than those a t Pullman during the period of grain development and maturation. In the analyses, differences between the Kjeldahl protein and Udy protein values were observed for both whole grain and flour samples of Nap Hal and Mahratta, but not for Magnif 41 ert 1. Magnif 41 is genetiTABLE 7 Protein Content of Selected Wheats (1974 Crop)O

Name Seed coat color Yield Bu/A Pullman Lind

TKWb

Pullman Lind Protein content Pullman: Wheat Kc Wheat U d Flour K Flour U Lind: Wheat K Wheat U Flour K Flour U AWh + F protein Pullman K Lind K AK -+ U protein, flour Pullman Lind

Mahratta CI8500

Nap Hal PI176217

Magnif 41 ert. 1 WA6096

Marfed CI11919

April Bearded CI7337

Wared CI15926

W

R

R

W

R

R

29.4 27.5

23.9 11.2

3.5.1 9.6

32.2 29.8

28.5 17.5

34.8 -

34.5 39.0

21.8 23.0

38.5 35.3

32.5

33.5 31.3

29.5 -

12.3 14.2 10.3 12.0 13.7 14.9 12.3 14.3

15.7 16.7 13.6 14.1 18.0 18.2 15.5 16.5

16.0 15.7 15.8 16.2 20.0 18.4 19.8 20.4

12.1 14.4 10.5 11.7 14.3 15.8 12.8 13.3

1.5.5 16.4 13.7 14.0 17.8 17.7 16.3 17.0

13.8 12.6 13.6 -

2 .Q 1.4

2.1 2.5

0.2 0.2

1.6 1.5

1.8 1.5

-

1.7 2.0

0.5 1.0

0.4 0.6

1.2 0.5

0.3 0.7

1.0 -

1.2

Analyses via cooperation of G. L. Rubenthaler, Technologist in Charge, Western Wheat Quality Laboratory (U.S.Department of Agriculture, Agricultural Research Service), Pullman, Washington. TKW = thousand-kernel weight. < K = Kjeldahl protein (N x 5.7). d U = Udy protein.

GENETIC CONTROL O F PROTEINS I N WHEAT

485

cally related to Atlas 66. Higher Udy or DBC protein values (based on acilane orange dye-binding capacity) , indicate relatively higher amounts of DBAA, including lysine (Mossberg, 1969). The higher protein content grain of Magnif 41 ert 1 demonstrated from two different cultural environments may involve a better genecontrolled distribution of protein throughout the endosperm than is the case for Nap Hal. The flour yield for Nap Hal was low compared with Magnif 41 ert 1 and Wared, but similar to the poorer milling Marfed check. However, a comparison of the Udy vs Kjeldahl protein values for the whole grain vs flour fractions indicates that the grain of Nap Hal has above normal DBAA content and shows that relatively more of the DBAA are extracted with the bran than with the flour in the milling process. I n striking contrast, the grain from Mahratta, while lower in protein than Nap Hal st both Pullman and Lind, loses about the same amount of Kjeldahl as Udy protein to the bran fractions, retaining the highest Udy-Kjeldahl protein in the flour for the group of wheats tested. Among the remaining group, CI3285 has what might be a significant Udy-Kj eldahl protein difference, but only from the Pullman location, whereas CI6127 shows a similar relation, but only from the Lind location. The results suggest that the protein composition of GI3285 and GI6127 has a greater sensitivity to environmental influences than is the case for Magnif 41 ert 1, Mahratta, or Nap Hal. The differences in the UdyKjeldahl values for whole grain and flour suggest that the distribution of protein in the endosperm of wheat may be an important factor affecting the contribution of lysine and protein to the flour. The protein (and lysine) of wheats like Nap Hal may be more concentrated in and near the aleurone region, while that of varieties like Mahratta and Magnif 41 ert 1 may be better distributed in the cells away from the aleurone region. These differences suggest that both the location and distribution of proteins in the wheat endosperm may be genetically independent from the protein content and composition. The milling characteristics (flour yield under comparable conditions, including ash content) may also limit the extraction of the protein and lysine into the flour fraction. However, Nap Hal and Mahratta both have poor milling properties. Some of the observed differences may simply reflect the relative ease by which the high protein cells near the aleurone may be separated with the inner endosperm vs the bran, since Evers (1970) found the same protein content per cell in hard and soft wheats, but more protein was extracted from the hard wheats because of their better endosperm separation. Mattern et al. (1975) has obtained similar results with Nap Hal and derivatives. Preliminary inheritance studies conducted at Pullman, Washington, indicate genetic transmission of the high milling yield by Magnif 41 ert 1 permits the

486

CALVIN F. KONZAK

isolation of recombinant lines with higher flour protein and DBAA from crosses involving CI3285, CI5484, CI6127, CI7337, and CI8500 (N. V. Mung and C. F. Konzak, unpublished, 1976). The higher protein content of grain from Atlas 66 and derivatives is contributed entirely to the endosperm (white flour) fraction, while that of Nap Hal is divided between the bran and flour fractions (V. A. Johnson ot al., 1975a,b). The poor bran cleanup and low flour yield characteristics of Nap Hal may be responsible for the main loss of protein and lysine to the bran fraction. The higher lysine content of Nap Hal separates partly into the bran and endosperm fractions, with the endosperm fraction containing no more than the normal lysine content observed for Atlas 66 and ‘Centurk’. Relatively more protein, as well as more lysine, is extracted in the endosperm fraction from the lower protein soft white winter wheat CI13449 and derivatives, which yield more flour than Nap Hal, but still have relatively poor cleanup compared with wheats like Centurk (V. A. Johnson et al., 1975a). However, some selections from Nap Hal/ Atlas 66 show increased endosperm lysine content (V. A. Johnson and Mattern, 1975). Genetic factors regulating protein content are recombinable by breeding and show high heritability (V. A. Johnson et al., 1972; Middleton et al., 1954; Lofgren et al., 1968). Difficulties were not encountered in recombining high protein factors in high-yielding lines. Lofgren e t aE. (1968) estimated that Kaw and Atlas 50 differed by three to four genes affecting protein content, by a t least two genes affecting mixing time, and by two to four genes controlling flour yield. Satisfactory breadmaking properties were recovered in high-protein derivatives of triparental crosses (V. A. Johnson et al., 1975a,b). One hard red winter wheat selection, named ‘Lancota’ (CI17389, formerly NE701132) from the cross Atlas 66/ ‘Comanche’//’Lancer’ was released in 1975 by the Nebraska and South Dakota Agricultural Experiment Stations. In tests conducted recently at Pullman, Lancota did not have the full protein content potential shown by weaker gluten derivatives from crosses of Atlas 66 parentage (C. F. Konzak and G. L. Rubenthaler, unpublished, 1974) and appeared to show strong effects of environment on protein content (E. Donaldson and G. L. Rubenthaler, personal communication, 1975). However, high-protein hard red spring wheat derivatives with stronger gluten characteristics have been recovered from crosses made at Pullman with other weak-gluten but high-protein Atlas 66/Comanche hard red winter wheat lines from Nebraska (C. F. Konzak, unpublished). Schmidt e t al. (1974) and Mattern et al. (1975) also note that high protein content factors appear to be inherited independently of protein quality for breadmaking.

GENETIC CONTROL OF PROTEINS IN WHEAT

487

X. Chromosomal location of Genes Affecting Protein Content, Composition, and Use Properties

The establishment, by aneuploid techniques, of genetic near-isolines in the wheat Chinese Spring (Sears, 1954, 1969; Morris and Sears, 1967) has permitted a considerable expansion in knowledge of the genetics of wheat proteins. Near-isolines include intervarietal and intergeneric chromosome substitutions, ditelosomics, monosomics, nullisomics, tetrasomics, nullitetrasomics, and synthetic polyploid series. Some protein differences have been identified by monosomic analyses. Induced mutant isolines have figured relatively little in the analyses conducted to date, but can be expected to add to the expansion of knowledge for wheat, as has been the case for other organisms. With the genetic near-isolines, the genotypeenvironment interactions and genotypic differences can be markedly reduced, permitting the identification of genetic differences in a wide range of properties, including protein content and composition, enzyme activity, baking properties, mixing characteristics, etc. The identification of proteins by electrophoresis and electrofocusing techniques has added considerably to the base of information already available, because there is a low or negligible environmental component t o the variation observed, and the differences identified seem to be the products of specific genes or gene interactions. A summary of data on the chromosome locations of genes controlling the synthesis, composition, and properties of proteins and of processing quality-related factors shows that the biochemical genetic analysis of wheat is one of the most rapidly developing areas of modern plant science (Table 8 ) . In many cases the chromosomal location of genes controlling specific proteins have been identified and their homoeologous relationships in the three wheat genomes have been established (Mclntosh, 1973). Genetic associations of quality-related components are common: particular chromosomes often carry concentrations of particular kinds of genes. The evolution of genes as identified by their products, the genetic buffering and intergenomic chromosome compensation proved by cytogenetic and other analyses, and the effects of deleting, adding, or substituting chromosomal units or sets provide new insight into the genetic variability that may yet be revealed by genetic recombination or induced by mutagens. An excellent illustration both of the homoeology and genetic associations of wheat gliadins has been obtained by Wrigley and Shepherd (1973). The chromosomes responsible have been identified by combined electrofocusing and electrophoresis of gliadins from the nullitetrasomic

TABLE 8 Chromosomal Association of Genetic Factors Controlling Proteins, Protein Quality, and Related Characteristics in Wheat Chromosome and arm( ) 1A

Trait Protein content: Total protein (flour, Prolamines

Material0

+ effect)

Gluten (+ effect) or Gliadin

Gliadin Non-gliadin proteolipid (component 21 of RodriguezLoperena et al., 1975) Miscellaneous: &Purothionin (apoprotein producer gene) Processing properties: Loaf volume (+ effect) Loaf volume, crust appearance (+ effect, minor factor) Protein content: Gliadin (3 components)

Gluten (fractions AI, BI, CS, and D, = gliadins?)

Method(s)*

b P

00 00

Reference

TST subst. in CS TC subst. in CS, CS N-T

1, 2 3

CNN subst. in CS X CS (Fi hyb.), CNN, CS CNN subst. in CS CNN subst. in CS CS N-T

4

Siddiqui, 1972 Manghers et al., 1973; Solari and Favret, 1970 Sasaki et al., 1973

36, 3 3 5, 37

Kasarda et al., 1974c Kasarda et al., 1976b Aragoncillo el al., 1975s

d 9

F

s3 r fl

CS N-T

5

Garcia-Olmedo et al., 1975; Fernandez de Caleya el al., 1976

CNN subst. in CS

6, 7, 20

CNN subst. in CS

4

B. Belderok and G. J. Doekes, personal communication, 1974 Morris et al., 1966

CS N-T

3, 5

CS D T

3

Shepherd, 1968; Wrigley and Shepherd, 1973; K. W. S h e p herd (R. Morris, personal communication, 1975) Khrabrova el ul., 1973

z Pi

0

Gliadin (band KI) Enzyme(s): a-Amylase ( effect) Peroxidase (- effect = inhibitor?) Miscellaneous: Nucleolar organizer

+

1B

CS DT, N-T

3

Shepherd, 1968

CS D T CS D T

11 11

Gale-and Spencer, 1974 Gale and Spencer, 1974

CS Mono, Null, D T

8

Nucleolar activity (regulator, RNA synthesis) RNA synthesis (regulator, f effect) Nitrate reduction (+ effect)

CS DT, alien subst., 6X/4X derivatives CS D T

8, 9

8, 9

Crosby, 1957; Crosby, Lmgwell, and Svihla 1960; Sears, 1959b Viegas and Mello-Sampayo, 1975 Jain et al., 1968a

CS DT, N-T

10

Edwards, 1973, 1974

Protein content: Total protein (kernel) Total protein (flour, - effect)

TST subst. in CS, Hope subst.

CS D T

12 1, 2

Nods and Tsunewaki, 1972 Siddiqui, 1972

4

Sasaki et al., 1973

36, 3 3 3

Kasarda et al., 1974c Kasarda et al., 1976b Manghers el al., 1973; Solari and Favret, 1970

3

Garcia-Olmedo el al., 1975; Fernandez de Caleya el al., 1976

13, 4

Welsh and Watson, 1965

4

Morris et al., 1966

Gluten a-Gliadin Gliadins Prolamines (+ effect)

in CS C N N subst. in CS x CS (F1 hyb.), CNN, CS C N N subst. in CS CNN subst. in CS T C subst. in CS, CS N-T

Miscellaneous: a-Purothionin (apoprotein pro- CS N-T ducer gene) Processing properties: Dough properties ( f effect, TST subst. in CS farinograph stability). Loaf volume, crust appearance, CNN subst. in CS crumb grain and texture (+ effect)

(Continued)

lb

TABLE 8 (Continued) Chromosome and arm( ) 1B( S )

Trait

Material'

Protein content: Gluten (fractions AI, BI, Cg, CS DT and D4 = gliadins?) Gliadin (bands K,, K4, and Mz) CS N-T Gliadins CS DT; 6X and 4X RSC, TC and PLD Gliadins (6 components) CS DT, N-T

(0

0

Method (s)*

Reference

3

Khrabrova et al., 1973

3

Shepherd, 1968 Wrigley, 1970a

12, 3

Shepherd, 1968; Wrigley, 1972a; Wrigley and Shepherd, 1973; K. W. Shepherd (R. Morris, personal communication, 1975)

3

Enzyme: Peroxidase (leaf, Per 1BS1) Miscellaneous: Nucleolar organizer

CS DT, N-T

3, 14

May et al., 1973

CS Mono, Null, DT

8

Nucleolar organizer (structural gene)

CS DT, alien subst., 6X/4X derivatives

8, 9

Crosby, 1957; Crosby Longwell and Svihla, 1960; Sears,J1959b Viegas and Mello-Sampayo, 1975

Protein content: Glutenin (subunits 2, 3) CS DT, N-T Enzyme(s): Malate dehydrogenase (Mdh-I) CS DT, N-T, LDN subst. in a-Amylase (+ effect) Miscellaneous: Nitrate reduction (+ effect)

3, 14

Bietz et al., 1975

3, 14

Bergman and Williams, 1972

CS DT

11, 14

Gale and Spencer, 1974

CS DT, N-T

10

Edwards, 1973, 1974; Deckard el al., 1975

cs

c)

2 w

2!

5

1D

Protein content: Total protein (kernel, Total protein (kernel, Gluten (+ effect)

+ effect) + effect)

KKF-MC22 Mono CS Mono X SN64 and X LR CNN subst. in CS X CS (F1 hyb.), CNN, CS T C subst. in CS, CS N-T

2 1 4

Welsh and Watson, 1965 Jha et al., 1971 Sasaki et al., 1973

3

6X and 4X RSC, T C and PLD 6X and 4X C T H and PLD CNN subst. in CS CNN subst. in CS LDN, CNN subst. in CS LDN, SN64, L R

3, 5 3 36, 3 3 3 3

Manghers et al., 1973; Solari and Favret, 1970 Wrigley, 1970a Konarev et al., 1972 Kasarda et al., 1974c Kasarda et al., 1976b Bietz et al., 1975 Joppa et al., 1975

CS Mono X SN64 and X LR CNN subst. in CS

15 15

Jha t-t al., 1971 Mattern et a/., 1968a

CS N-T

3

Garcia-Olmedo et al., 1976; Fernandez de Caleya et al., 1976

CS Mono X SRV 29 and X SRV 210 CNN subst. in CS

23

Maystrenko et a/., 1973b

4

TST subst. in CS

4

Imf volume (+ effect)

Hope subst. in CS

6, 7, 20

Dough properties (- effects, strength)

K K F MC22 Mono, TST subst. in CS

2, 13, 16

R. Morris et a/., personal communicat.ion, 1974 B. Belderok and G. J. Doekes, personal communication, 1974 B. Belderok and G. J. Doekes, personal communication, 1974 Welsh et al., 1968

Gliadins (2 prolamine components) Gliadins Gliadins (2 components) a-Gliadin Gliadins Glutenin (subunits 1, 4) Glutenin (subunits 1, 4) Amino acid: Tryptophan content (+ effect) Lysine content (possible 15 % increase) Miscellaneous: u-Purothionin (apoprotein producer gene) Processing properties: Gluten quality (+ effects, strength) Dough properties (mixing strength, effect) Loaf volume (+ effect)

+

(Continued)

TABLE 8 (Continued) Chromosome and arm( ) 1D

Method(s)b

Reference

Dough properties (mixing tolerance, gas retention, effect) Dough properties (strength)

KKF MC22 Mono X Itana

13, 17, 18

Welsh and Hehn, 1964

LDN, CS 1D addition, D T

3

Joppa el al., 1975

Protein content: Gliadins (4 components)

CS DT, N-T

5, 3

+

WS)

Material-

Trait

Gliadins (bands J 2 and 5 3 ) Gliadins (bands Jf and 5 3 ) Gluten (fractions A,, BI, C,, and D4-gliadins?) Gluten (2 components, urea extract of kernels) Enzyme: Peroxidase (leaf, Per-1 DS-1, Per-1 D S d ) Miscellaneous: Nitrate reduction (+ effect) Peroxidase activity ( - effect = inhibitor) Processing properties: Dough properties (enhancer)

CS DT, N-T Wheat-rye chr. transloc lD(L)-E(S) CS DT

3 3

Wrigley and Shepherd, 1973; K. W. Shepherd (R. Morris, personal communication, 1975) Shepherd, 1968; Wrigley, 1972a Shepherd, 1973

3

Khrabrova et al., 1973

CS DT

3

Boyd and Lee, 1967; Boyd el al., 1969

CS DT, N-T

3, 14

May et al., 1973

CS DT, N-T CS DT

10 11

Edwards, 1973, 1974 Gale and Spencer, 1974

CS DT

19, 13

Maystrenko et al., 1973a

E

2 s

4

0

2* w

Protein content: Glutenin (high M W subunits 1, CS DT, N-T, extracted tetraploids PLD C T H RC, T C 4; 2 lower MW subunits of Orth and Bushuk 1974, not observed) CS DT, N-T, extracted tetraGlutenin (4 subunits; high ploids PLD, CTH, R C MW = subunits 1, 4 of Bietz et al., 1975) Processing properties: Flour quality (dough strength) CS DT, extracted tetraploids, PLD 2A

Protein content: Total protein (kernel, effects)

+

Gluten (+effect) Total protein (kernel,

+ effect)

Gliadins? (joint control with 6-41 Enzyme(s): Phenolase complex activity (+effect, reported as t yrosinaee) Phenolase complex activity

3

Bietz el al., 1976

3

Orth and Bushuk, 1974

20, 13

Kaltsikes el al., 1968b

Rye chr. subst. in CS

21,22

CS Mono X SRV 210 CNN subst. in CS X CS (FI hyb.), CNN, CS CS N-T

23 4

N. Darvey, personal communication, 1974; Jagannathan and Bhatia, 1972 Maystrenko el al., 1973b Sasaki et al., 1973

3

Waines, 1973

Hope and TST subst. in CS

25

Zeven, 1972

p-Glucosidase

25 Koga I1 X subst. of Peko, Hope, and T S T in CS 25 CNN, Hope, KF, MQ, TC, T S T and 2R(2A) subst. in CS 3, 14 2R(2A) subst. in CS

Miscellaneous: Inhibitor (of 6D gliadins)

CS N-T, tetrasomics

Phenolase complex activity

3

Larsen 1974 Wrigley and McIntosh, 1976

C. E. May, personal communication, 1975 Shepherd, 1968 (Continued)

TABLE 8 (Continued) Chromosome and arm( ) 2A

2A(L)

I&

W

lh

Trait

Material"

Processing properties: Baking absorption, loaf volume TC subst. in CS (+ effects) Baking absorption (+ effects) Hope subst. in CS Milling properties (+ effect) CNN subst. in CS Protein content: Total protein (- effect, major CS D T factor) Enzyme (s): Phenolase complex activity CS dimonotelo 2A X TST 2A (kernel phenol reaction; subst. in CS 36.8 5.8% linkage to centromere) Acid phosphatase (+ effect) CS D T a-Amylase (+effect) CS D T Miscellaneous: Regulator? (gluten protein CS D T fractions B, and B, merge, joint control with group 6 chr.) Nucleolus organizer (regulator) CS DT, alien subst. in CS 4X/6X Nitrate reduction (regulator or CS DT, N-T inhibitor) Protein component ratio CS D T (+effect, albumins/globulins) Processing properties: Dough properties (inhibitor) CS D T

Method ( s ) b

Reference

4

Welsh et al., 1968

4 33, 24

Welsh et al., 1968 Mattern et al., 1973

22

Bozzini and Giacomelli, 1973

25

Wrigley and McIntosh, 1975

E2

1:

kl

11 11

Gale and Spencer, 1974 Gale and Spencer, 1974

3

Khrabrova et al., 1973

8, 9 10

Viegas and Mello-Sampayo, 1975 Edwards, 1973, 1974

26

Bozzini and Giacomelli, 1973

19, 13

Maystrenko et al., 197313

z

5

Protein content: Total protein (flour,

+ effect) Total protein (kernel, + effect)

Gluten ( - effect) Gliadin? (joint control with 2 D and 6D) Miscellaneous: “Regulator or inhibitor” of gluten subunits Processing properties: Gluten quality (weak inhibitor)

Dough strength, baking absorption (+ effects, major factor) Loaf volume Enzyme: a-Amylase (+ effect) Miscellaneous: Regulator? (gluten protein fractions B3 and B4,gliadins merge, joint control with group 6 chr.) Processing properties: Dough strength (enhancer)

TST, S61.5, and Hope subst.

in CS Rye chr. subst. in CS

1, 2

Siddiqui, 1972

21, 22

CNN subst. in CS X CS (FI hyb.), CNN, CS CS N-T

4

N. Darvey, personal communication, 1974; Jagannathan and Bhatia, 1972 Sasaki et al., 1973

3

Waines, 1973

CS N-T

3

Orth and Bushuk, 1974

CS Mono X SRV 210

23

Hope subst. in CS

4

Maystrenko et al., 1973b; 0. I. Maystrenko (R. Morris, personal communication, 1975) Welsh el al., 1968

Hope and T S T subst. in CS

4

Welsh et al., 1968

CS D T

11

Gale and Spencer, 1974

CS D T

3

Khrabrova et al., 1973

CS DT

19, 13

Maystrenko et al., 1973a ~

(Continued)

I&

TABLE 8 (Continued) Chromosome and arm( ) 2D

Materiala

Trait Protein eontent: Total protein (kernel, effect) Gluten ( effect) Gliadin (35 mm band, joint control with 2B and 6D) Enzyme (s): Peroxidase Per 5C (leaf blade, stem, root, glume)

+

+

Phenolase complex activity (reported as tyrosinase, effect) Phenolase complex activity (kernel phenol reaction) Processing properties: Gluten properties (+ effect dispersion) Dough properties (strength, effects) Loaf volume ( f effects)

+

+

Enzyme: Phenolase complex activity (kernel phenol reaction) Protein content: Gluten (fractions B, and B4

CD

Q,

Method(s)b

Reference

Rye chr. subst. in CS CS Mono x SRV 29 CS N-T

21,22 27 3

Irani and Bhatia, 1972 Maystrenko et al., 1973b Waines, 1973

CS N-T

3, 14

TST subst. in CS

25

Kamagata and Nishikawa, 1973; Nishikawa and Kamagata, 1974 Zeven, 1972

E

3 4

3

CS N-T; KF, RE, TST subst. in CS

25

Wrigley and McIntosh, 1975

TC subst. in CS

28

Favret et al., 1969

Hope subst. in CS

13, 17

Welsh et al., 1968

TST subst. in CS

7

B. Belderok and G. J. Doekes,

CS N-T, D T

25

Wrigley and McIntosh, 1975

CS D T

3

Khrabrova et al., 1973

personal communication, 1974

z8

$

gliadins merge, joint control with group 6 chr.) Enzyme: Peroxidase ( - effect = inhibitor?) Processing properties: Dough properties ( - strength, = inhibitor) 3A

Protein content: Total protein (kernel, Total protein (flour, Total protein (kernel,

+ effect) + effect) + effect)

Albumins (bands 1, 2, and 6) Amino acid: Tryptophan content (feffect) Enzyme(s): Esterases (3 bands) Esterase (plant development) Peroxidase, Per 5C, root, empty glume Esterases (leaf and root, 3 slow bands) Processing properties: Loaf volume (+ effect, minor factor) Baking absorption (+ effect, minor factor) Kernel hardness, water absorption (+ effects, major factor)

CS DT

11

Gale and Spencer, 1974

CS DT

13, 19

Maystrenko el al., 1973a

CS Mono X SN64 and X L R TST subst. in CS CNN subst. in CS X CS (FI hyb.), parents CS N-T

1 1, 2 4

J h a et al., 1971 Siddiqui, 1972 Sasaki et al., 1973

3, 14

Cubadda, 1975

CS Mono X SN64 and X LR

15

J h a el al., 1971

CS N-T CS aneuploids CS N-T

3, 14 3, 14 3, 14

Boeeini et al., 1973 Nakai and Tsunewaki, 1974 Kamagata and Nishikawa, 1973

CS DT, N-T, D genome subst. L D N

3, 14

Bergman and Williams, 1972

Hope subst. in CS

4

Welsh et al., 1968

TST subst. in CS

4

Welsh et al., 1968

TST subst. in CS

29, 30

B. Belderok and G. J. Doekes, personal communication, 1974 (Continued)

TABLE 8 (Continued) Chromosome and arm( ) 3A(a

=

L)

3A(B = S)

Trait Enzyme(s): Esterases (bands 3Aa-1, 3Aa-2, 3Aa-3) Esterases (bands 1, 2, and 6) Esterase (kernel, band 9) Acid phosphatase (+effect) a-Amylase (+ effect) Glutamate oxaloacetate transaminase Got-AS Miscellaneous: Nitrate reduction (- effect) Protein content: Total protein (kernel) Gluten (fractions B,, B?, Cgand C6 = non-gliadins?) Enzyme (s): Esterases (3 bands) Esterase (fast-moving dimer) Peroxidase ( - effect = inhibitor?) Processing properties: Dough properties (enhancer)

Material.

Method(s)b

Reference

CS DT, N-T

3, 12

CS DT, N-T

CS DT CS DT CS DT, N-T

3, 12, 14 3, 14 11 11 3, 14

C. E. MayJ personal communication, 1975 Cubadda et al., 1973, 1975 Nakai, 1973 Gale and Spencer, 1974 Gale and Spencer, 1974 Hart, 1975

CS DT, N-T

10

Deckard et al., 1975

CS DT CS D T

12 3

Noda and Tsunewaki, 1972 Khrabrova et al., 1973

CS Null, DT CS DT, N-T CS D T

3, 14 3, 14 11

Barber et al., 1968a,b Bergman and Williams, 1972 Gale and Spencer, 1974

CS D T

13, 19

Maystrenko et al., 1973a

CS DT, N-T

3B

Protein content: Total protein (kernel, Gluten ( + effect)

+ effect)

Gluten ( - effect) Albumin (76mm band) Amino acid: Tryptophan content (+ effect) Enzyme (s): Esterase (6 bands, kernel) Esterase (kernel, bands 3, 8, 11, 13) Esterase (plant development) Peroxidase, Per 4C, root, stem Peroxidase (culm, root)

Processing properties: Loaf volume (+ effect) Dough strength, baking absorption (+ effects) Loaf volume (+ effect)

CS Mono X SN64 and X L R CS Mono X SRV 210 CNN subst. in CS X CS (PI hyb.), CNN, CS CS N-T

1 23

Jha et al., 1971 Maystrenko et al., 1973b

4

Sasaki et al., 1973

3

Waines, 1973

CS Mono X SN64 and X LR

15

Jha et al., 1971

CS Null, N-T CS N-T

3, 14 3, 14

Barber el al., 1968s Cubadda et al., 1975

CS aneuploids CS N-T CS N-T

3, 14 3, 14 3, 14

Nakai and Tsunewaki, 1974 Kamagata and Nishikawa, 1973 Nishikawa and Kamagata, 1974; K. Nishikawa (R. Morris, personal communication, 1975)

CD subst. in CS

7

Hope and TST subst. in CS TST subst. in CS

Protein content: Total protein (kernel) CS D T Non-gliadin proteolipid (CM CS DT, N-T components 6, 7 = protein band 4 and components 14 and 15 of Rodriguez-Loperena et al., 1975 = protein band 3 of Noda and Tsunewaki, 1972)

0

8m

2 8

2

r

s hj

g

4

B. Belderok and G. J. Doekes, personal communication, 1974 Welsh et al., 1968

4

Welsh et at., 1968

2

3, 12, 14 5, 37

Noda and Tsenewaki, 1972 Aragoncillo el al., 1975a

L2

ZJ

dF r3

is (Continued)

E

TABLE 8 (Cuntinued) Chromosome and arm( ) 3B (S)

3B (L)

3D

Trait

Materide

Enzyme: Esterase (fsst-moving dimer) Processing properties: Dough propreties (inhibitor) Enzyme(s): Esterase (leaf, band Est-SBL-1) Esterase (kernels, ban& Est S B-6, Es~-SBL-?') Esterases (2 bands) Acid phosphatase a-Amylase (+ effect) Glutamate oxdoacetate transaminase GotBS Miacellaneuus: Nitrate reduction (+effect) Protein content: Total protein (kernel, effect) Albumins Albumin (P.C.S.) Albumins (bands 4,6, 7) Albumins (82,91 and 97 mm bands) Enzyme (s) : Esterases (kernel, 2 bands, regulator?) Esterase (bands 10 and 13) Esterase (kernel, bands 5 and 12)

+

Method(s)b

Reference

CS DT, N-T

3, 14

Bergman and Williams, 1972

CS D T

13, 19

Maystrenko et al., 19738

CS DT, N-T CS DT, N-T

3, 12, 14 3, 12, 14

May et al., 1973;Nakai, 1973 Nakai, 1973

CS DT, N-T CS D T CS DT CS DT, N-T

3, 12, 14 11 11 3, 14

Cubadda et al., 1975 Gale and Spencer, 1974 Gale and Spencer, 1974 Hart, 1975

CS DT, N-T

10

Edwards, 1973, 1974

TC subst. in CS CS Mono X T. durum CS Mono X T . durum CS N-T CS N-T, Ae. squarrosa

2

31, 32 31, 32 3 3

Kuspira and Unrau, 1957 Borzini et al., 1970 Boarini et al., 1971 Cubadda, 1975 Waines, 1973

CS N-T

3, 14

CS Mono X T. durum CS N-T

3, 14 3, 14

,

Bosaini et al., 1973; Cubadda et al., 1975 Nagayoshi et al., 1974 Cubadda et al., 1975

sE

Z

r

t2 3 R

CS aneuploids Esterase (plant development) Esterases (root, 2 slow-moving CS N-T bands) Processing properties: Hope and TST subst. in CS Milling properties (flour yield effects, minor factors) Loaf volume TST subst. in CS Gluten quality (weak inhibitor) CS Mono X SRV 29

+

3, 14 3, 14

Nakai and Tsunewaki, 1974 Bergman and Williams, 1972

24

Welsh et al., 1968

4 23

Welsh et al., 1968 Maystrenko et al., 1973b; 0 . I. Maystrenko (R. Morris, personal communication, 1976)

3 D ( a = L)

Enzyme(s): Esterase (leaf, 1 slow-moving CS DT, N-T band) Esterases (kernels, bands EstCS DT, N-T SDa-1 , Est-3Da-2) Esterases (leaf, EstSDa-1, CS DT, N-T Ed-3Da-2) Glutamate oxaloacetate trans- CS DT, N-T aminase Got-D3 (location approximately 4.3 C.O. units from centromere) Glutamate oxaloacetate trans- CS DT, Trd-Agro. elong aminase Got-AgS transloc. Miscellaneous: Nitrate reduction (leaf extracts, CS DT, N-T in vivo, - effect)

3D(@= S = Left) Protein content: Total protein (kernel) Total protein (kernel)

CS D T Triticum-Agropyron transloc

3, 14

Bergman and Williams, 1972

3, 12, 14

Nakai, 1973

3, 12, 14

May et al., 1973

12

Hart, 1971; Hart et al., 1976

12

Hart et al., 1976

10

Deckard et al., 1973

3, 5 3, 5

Noda and Tsunewaki, 1972 Rodriguez-Loperena et al., 1976 (Continued)

cn

0,

TABLE 8 (Continued) Chromosome and arm( ) 3D(P = S = Left)

Trait

Materiala

Gluten (fractions B,, BI, Ca, and Cg = gliadins?) Non-gliadin proteolipid (CM component 5 = protein of Noda and Tsunewaki, 1972 = 1 compound of Waines, 1973) Enzyme(s): Esterase (fast-moving band) Esterase Peroxidase ( - effect = inhibitor?) Processing properties: Dough properties = enhancer, modifier; effect dough strength, - effect extensibility

+

4A

Protein content: Total protein (flour, effect) Gluten (kernel, effect)

+

+

Method(s)b

Reference

CS DT

3

Khrabrova et al., 1973

CS DT, N-T

5, 37

Aragoncillo et al., 1975a

CS DT, N-T CS N-T CS DT

37 3 11

Bergman and Williams, 1972 Cubadda et al., 1975 Gale and Spencer, 1974

E

9

Z

1

R

0

CS DT

19, 13

Maystrenko et al., 1973a

Hope, and S-615, subst. in CS CNN subst. in CS X CS (F1 hyb.), CNN, CS CS N-T

1, 2 4

Siddiqui, 1972 Sasaki et al., 1973

3

Waines, 1973

3, 14 3

Nagayoshi, 1973 Avivi et al., 1972

Albumins (83 and 69 mm, joint control with 4B) Enzyme(s): 8-Amylase CS Null Alcohol dehydrogenase CS N-T

3 L

Alcohol dehydrogenase

Rye chr. additions in HFT, Triticale CS N-T

Acid phosphatase (reported as alk. phosphatases; see Hart, 1973a; Torres and Hart, 1976) Processing properties: CNN subst. in CS Milling properties ( effect, minor factor) Dough properties (J'alorimeter, TST subst. in CS effect) Doughproperties (+effect, bak- Hope subst. in CS ing absorption, minor factor)

+

+

3, 14

Irani and Bhatia, 1972

3, 14

Brewer et al., 1969

24, 33 13

Mattern et al., 1973; Sasaki et al., 1973 Welsh et al., 1968

4

Welsh et al., 1968

z%

0

Enzyme(s): Alcohol dehydrogenase, Adh-Al, a subunit Lipoxygenase Lpx-A1 (Coleoptile) a-Amylase (+ effect) Protein content: Total protein (kernel) Non-gliadin proteolipid (components 12, 13= part of CM3 proteinof Aragoncillo 1973 and 83mm band of Waines, 1973; component 16 = part of 69mm band of Waines, 1973) Enzyme(s): Acid phosphatase (+ effect) Acid phosphatases (Acph 4, Acph 8) 8-Amylase (component C)

0

CS DT, N-T

3, 14

CS DT, N-T

3, 14

Hart, 1970, 1971, 1973a,b; Tang and Hart, 1975 Hart and Langston, 1975

CS D T

11

Gale and Spencer, 1974

CS D T CS DT, N-T

12 5, 37

Noda and Tsunewaki, 1972 Aragoncillo et al., 1975a

TIr 0

cd

s L-d

3

2

2 8

3> r3

CS D T CS DT, N-T

11 3, 14

CS D T

3, 14

Gale and Spencer, 1974 Hart, 1973a,b; Torres and Hart. 1976 Joudrier and Cauderon, 1976 (Continued)

en

0 W

TABLE 8 (Cmtinued) Chromosome and arm( ) 4-4(8)

4B

Materiala

Trait a-Amylase (+ effect) Processing properties: Dough properties (inhibitor)

Reference

CS DT

11

Gale and Spencer, 1974

CS DT

13, 19

Maystrenko et al., 1973a

4

1, 2

Siddiqui, 1972 Sasaki et al., 1973

23 3

Maystrenko et al., 1973b Waines, 1973

3, 14 3, 14 3, 14

Avivi et al., 1972 Kobrehel and Feillet, 1975 Brewer et al., 1969

4

Welsh et d.,1968

24, 33

Mattern et al.. 1973

4

Morris et al., 1966

23

Maystrenko et al., 1973b; 0. I. Maystrenko (R. Morris, personal communication, 1975)

Protein contend: Total protein (flour, - effect) Gluten (+effect)

S-615 subst. in CS CNN subst. in CS X CS (FI hyb.), CNN, CS Gluten (+ effect) CS Mono X SRV 29 Albumins (83 and 69 mm, joint CS N-T control with 4A) Enzyme(8): Alcohol dehydrogenase CS N-T CS N-T Peroxidase (band C) CS N-T Acid phosphatases (originally reported alk. phosphatases; see Torres and Hart, 1976) Processing properties: Loaf volume, baking absorption TST subst. in CS (+effects, minor factor) CNN subst. in CS Milling properties (+ effect, tentative) CNN subst. in CS Dough propertiea (strength), loaf volume, crust appearance, crumb grain and texture (+ effects, major factors) Gluten quality (weak inhibitor)

Method(@

CS Mono X SRV 210

E

d

:T’

E

5 k

Protein content: Total protein (kernel) Enzyme(s): Acid phosphatases (Acph 2, Acph 3) Acid phosphatase (+ effect) a-Amylase (+ effect) Processing properties: Dough properties (major inhibitor) Enzyme (s): Alcohol dehydrogenase, Adh-B1, 0 subunit Lipoxygenase Lpx-B1 (Coleoptile) 4D

Protein content: Total protein (kernel, effect) Total protein (flour, - effect) Total protein (crude protein, effect) Gluten (+ effect)

+

+

Gluten (+ effect) Albumin (Mb 0.19)

CS D T

12

Nods and Tsunewaki, 1972

CS DT, N-T

3, 14

CS D T CS D T

11 11

Hart, 1973a,b; Torres and Hart, 1976 Gale and Spencer, 1974 Gale and Spencer, 1974

CS D T

13, 19

Maystrenko et al., 1973a

0 H

33 Q

CS DT, N-T

3, 14

CS DT, N-T

3, 14

Hart, 1973a,b; Torres and Hart, 1976 Hart and Langston, 1975; G. E. Hart, personal communication, 1976

81: ;3

0 F

o

r

'd

z5

td

0

TC subst. in CS Hope and S-615 subst. in CS CNN subst. in CS X CS (FI hyb.), CNN, CS CNN subst. in CS X CS (FI hyb.), CNN, CS CS Mono X SRV 29 CS D-genome X durum hybrids

2 1, 2

4

Kuspira and Unrau, 1957 Siddiqui, 1972 Sasaki et al., 1973

4

Sasaki et al., 1973

8

23 32

Maystrenko et al., 1973b Bozrini et aZ., 1971

?i

(Continued)

G

01

%

TABLE 8 (Continued) Chromosome and arm( ) 4D

Trait Albumin (65-mm band) Non-gliadin proteolipid (CM 3 = component 17 of Rodriguez-loperena et al., 1975; = 65 mm band of Waines, 1973) Enzyme (9): Alcohol dehydrogenase Acid phosphatases (reported as alk. phosphatases; see Torres and Hart, 1976) Processing properties: Loaf volume, crumb grain and texture (+ effects, minor factors) Loaf volume (+ effect) Milling properties (flour yield, effect, minor factor)

+

Enzyme(s): Alcohol dehydrogenase, Adh-Dl, 6 subunit @Amylase ( f effect) Lipoxygenask Lpx-Dl (Coleoptile)

Materialm

method(^)^

Reference

CS N-T, Ae. squarrosa CS DT, N-T

3, 14 5, 37

Waines, 1973 Aragoncillo et al., 1975s

CS N-T CS N-T

3, 14 3, 14

Avivi et al., 1972 Brewer et al.. 1969

CNN subst. in CS

4

Morris et al., 1966

TST subst. in CS Hope subst. in CS

4 24

Welsh et al., 1968 Welsh et al., 1968

CS DT, N-T

3, 14

CS DT CS DT, N-T

11 3, 14

Hart, 1973a,b; Tang and Hart, 1975 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1976

zT: R

Protein content: Glutenin (high MW subunit 5 ) Enzyme(s): Acid phosphatases (Acph 5, Acph 6) 8-Amylase (2-Ca components)

5A

Protein content: Total protein (kernel, effect) Total protein (flour, - effect) Total protein (kernel, effect) Total protein (kernel, effect)

+ +

+

CS DT, N-T

3, 14

Bietz et al., 1975

CS DT, N-T

3, 14

CS D T

3, 14

Hart, 1973a,b;Torres and Hart, 1976 Joudrier and Cauderon, 1976

TC subst. in CS TST subst. in CS CSMono XSN64 , CNN subst. in CS X CS (FI hyb.), CNN, CS CS Mono X SRV 210

2 1, 2 1

Kuspira and Unrau, 1957 Siddiqui, 1972 Jha et al., 1971

!3 M

4

Sasaki et al., 1973

3 0

23

Maystrenko et at., 1973b

0

15

Jha et al., 1971

5rJ 0

Gluten (+effect) Amino acid: Tryptophan content (+ effect, CS Mono X SN64 major factor) Miscellaneous: Purothionin lipid moiety (least CS N-T effective gene) Processing properties: Baking absorption (+ eff eet, Hope and TST subst. in CS minor factor) Gluten quality (weak inhibitor) CS Mono X SRV 29 Protein content: Gluten protein (fractions BI, B4, and D4) Miscellaneous: Peroxidase activity ( - effect = inhibitor) Nitrate reduction (- effect, leaf ext.)

2

xe r 0

34

4

Garcia-Olmedo et al., 1975;Fernandez de Caleya et al., 1976

'd

4

Welsh et al., 1968

2

23

Maystrenko et al., 197313; 0. I. Maystrenko, (R. Morris, personal communication, 1976)

CS DT

3

Khrabrova et al., 1973

CS DT

11

Gale and Spencer, 1974

CS DT, N-T

10

Deckard et al., 1973

ge

5

4

ie2

01 0

(Continued)

-3

TABLE 8 (Continued) Chromosome and arm( ) 5 W )

5NL)

Trait

Materiala

Processing properties: Dough properties (major inhibitor) Protein content: Albumin Enzyme: Lipoxygenaae Lpx-A& (Coleoptile) Miscellaneous: Nitrate reduction (+effect)

5B

Reference

CS D T

13, 19

Maystrenko el al., 1973a

CS DT, N-T

3

Shepherd, 1971, 1973

CS DT, N-T

3, 14

Hart and Langston, 1975; G. E. Hart, personal communication, 1976

CS DT, N-T

10, 42

Deckard et al., 1975

2 1, 2 4

Kuspira and Unrau, 1957 Siddiqui, 1972 Sasaki et al., 1973

4

Welsh el al., 1968

4

Welsh et al., 1968

34

Garcia-Olmedo et at., 1975

CS D T

3

Khrabrova et al., 1973

CS D T

13, 19

Maystrenko et al., 1973a

Protein contat: Total protein (kernel, effect) TC subst. in CS Total protein (flour effect) TST subst. in CS Gluten ( - effect) CNN subst. in CS X CS (F1 hyb.), CNN, CS Processing properties: Baking absorption (minor Hope subst. in CS factor) Loaf volume (+effect) TST subst. in CS Miscellaneous: Purothionin lipid moiety CS N-T (moderate effectiveness)

+ +

Protein content: Gluten (fractions Bl, Be, and D4) Processing Properties: Dough properties (inhibitor)

method(^)^

Protein content: Albumin Enzyme: Lipoxygenase Lpx-BB (Coleoptile)

5D

Protein content: Total protein (flour,

+ effect)

Total protein (flour, - effect) Total protein (kernel) Total protein (kernel, effect) Gluten (kernel, effect)

+

+ Gluten (kernel, + effect)

Gluten ( - effect) Non-gliadin proteolipid (component 1 of RodriguezLoperena et al., 1975) Miscellaneous: Purothionin lipid moiety (most effective locus) Processing properties: Milling properties (flour yield, effects), kernel hardness

+

CS DT, N-T

3

Shepherd, 1971, 1973

CS DT, N-T

3, 14

Hart and Langston, 1975; G. E. Hart, personal communication, 1976

TST and C D subst. in CS

2

Hope subst. in CS Hope subst. in CS ATL 66 subst. in CS CNN subst. in CS X CS (FI hyb.), parents CS Mono X SRV 29 CS Mono x SVL CS DT, N-T

1, 2 3 2 4

B. Belderok and G. J. Doekes, personal communication, 1974 Siddiqui, 1972 Halloran, 1975 Morris et al., 1973 Sasaki ct al., 1973

23 28 5, 37

Maystrenko et al., 1973b Avila and Favret, 1966 Aragoncillo et al., 1975a

CS N-T

34

Garcia-Olmedo et aZ., 1975; Fernandez de Caleya et al., 1976

CNN subst. in CS

33, 24

B. Belderok and G. J. Doekes, personal communication, 1974; Mattern et al., 1973; M. Sasaki et aZ., personal communication, 1974 (Continued)

z

0

TABLE 8 (Continued) Chromosome and arm( ) -

5D

Reference

Trait

Materida

Method (s)~

Milling properties (flour yield, effect; hardness?)

Hope and TST subst. in CS

24

Baking (water) absorption (+effect) Baking (water) absorption (+effect) Dough strength (major factor) Loaf volume, crumb grain and texture (+ effects, minor factor)

CNN, Hope and TST subst. in CS Hope and TST subst. in CS

4

4

B. Belderok and G. J. Doekes, personal communication, 1974; Welsh et al., 1968 B. Relderok and G. J. Doekes, personal communication, 1974 Welsh et al., 1968

CNN subst. in CS CNN subst. in CS

13, 35 4

Morris et al., 1966 Morris et al., 1966

~~

+

Protein content: Gluten (fractions B,, Bq, and D4) Total protein (genes Prol, Pro2, assoc. with nucleolus organizer region) Miscellaneous: Nucleolus organizer (structural gene) Nitrate reduction (+effect) Processing properties: Dough strength properties (major inhibitor) Milling properties (- effect, flour yield)

? CS D T

3

Khrabrova et al., 1973

Hope subst. in CS, CS D T

8

C. N. Law, personal communication, 1976

CS DT, alien subst. 6X/4X derivatives CS DT N-T

8, 9

Viegas and Mello-Sampayo,

10

Edwards, 1973, 1974

CS DT

13, 19

Mayitrenko et al., 1973s

CS DT

6

Maystrenko et al., 1973a

1975

2 k

Protein content: Albumin Enzyme ( 9 ) : Lipoxygenase Lpx-D8 (Coleoptile) Miscellaneous: Nucleolus organizer (regulator) Nitrate reduction (+effect) 6A

Protein content: Gluten (+effect) Gluten (+ effect)

CS DT, N-T

3

Shepherd, 1971, 1973

CS DT, N-T

3, 14

Hart and Langston, 1975; G. E Hart, persond communication, 1976

CS DT, alien subst. 6X/4X derivatives CS DT, N-T

8, 9 10, 42

Viegas and Mello-Sampayo, 1975 Deckard et al., 1975

CS Mono X SRV 29

23

Maystrenko el al., 197313

4

Sasaki el al., 1973

CNN subst. in CS X CS (FI hyb.), CNN, CS Gliadins (joint control by regu- CS N-T lator in 2A?) Gliadins (5 components) Gliadins (4 components, aggregatable A-gliadins)

Enzyme(s): a-Amylase Esterase (flag leaf) 6A(a = S)

CS N-T CNN subst. in CS

CS Null CS aneuploids

Protein content: A-gliadins (bands 17,19,21,22) CS DT, N-T Enzyme(s): Aminopeptidase ( A m p A I ) CS DT, N-T

n

n

2 3

n n

0

2

!a

0

3

Shepherd, 1968; Waines, 1973

3, 5 3

Wrigley and Shepherd, 1973 Kasarda el al., 1973, 1974a, 1976b

r q

3, 14 3, 14

Nsgayoshi, 1973 Nakai and Tsunewaki, 1974

3

Kasarda et al., 197613

3, 14

G. E. Hart, personal communications, 1973, 1976

(Continued)

+d

m 0 Y

E

5

z d

9Y

z Y

TABLE 8 (Continued) Chromosome andarm( ) 6 A ( a = S)

6A@ =L)

Trait

Material.

Glutamate oxaloacetate transaminase Got-A3 (1st leaf blade) Miscellaneous: Nitrate reduction (+effect) Protein content: Gliadins (5 components)

+

Total protein (kernel, effect) Gluten Gliadins (bands Me, M7) Gluten (fractions Al, D4 = gliadms?, joint control of fractions Be B4, with group 2 chr.) Enzyme (s) : Esterase (band Est-6A-I) a-Amylase (band 2)

*Amylase Glutamate oxaloacetate transaminase Got-Ad (1st leaf blade)

Method(s)b

Reference

CS DT, N-T

3, 14

Hart, 1975

CS DT, N-T

42

Deckard et aE., 1975

CS DT, N-T

3, 4

I(.W. Shepherd (R. Morris,

CS D T CS D T CS DT, N-T CS D T

12 4 3 3

CS DT, N-T CS D T

3, 14 3, 14

CS D T CS DT, N-T

11 3, 14

personal communication, 1975); Wrigley, 1972a; Wrigley and Shepherd, 1973 Noda and Tsunewaki, 1972 Maystrenko et al., 1973a Shepherd, 1968, 1973 Khrabrova et al.. 1973

May et al., 1973 Nishikawa and Nobuhara, 1971; K. Nishikawa and M. Nobuham, personal communication, 1975 Gale and Spencer, 1974 Hart, 1975

Miscellanems: Nitrate reduction (structural gene) Nitrate reduction (+effect) Protein component ratio (+ prolamines) Processing properties: Dough properties (enhancer) 6B

Protein content: a-Gliadin Gliadin L4, “regulator”? Gliadins (10 components)

CS DT, N-T

10

Edwards, 1973, 1974

CS DT, N-T CS D T

10 26

Deckard el al., 1975 Bozzini and Giacomelli, 1973

CS D T

13, 19

Maystrenko et al., 1973a 0 M

CNN subst. in CS CS N-T CS N-T

Total protein (kernel, - effect) CNN subst. in CS X CS @‘I hyb.), CNN, CS Enzyme: Peroxidase CS and transfer from Ae. umbellulata Miscellaneous: “Regulator or inhibitor” (of CS N-T glutenin subunit) Protein component ratio CS D T (+prolamines) Processing properties: Gluten quality (weak inhibitor)

Milling properties (flour yield, effect) Loaf volume ( f effect) Dough strength (+ effect)

+

3, 36 3 3 4

Kasarda et al., 1974a, 1976b Shepherd, 1968 Wrigley, 1972a; Wrigley and Shepherd, 1973 Sasaki et al., 1973

2 3

0 0

0

3

!a

0

r

3

0

Upadhya, 1968

crl +d

3

Orth and Bushuk, 1974

26

Bozzini and Giacomelli, 1973; A. Bozzini, personal communication, 1974

CS Mono X SRV 210

23

TST subst. in CS

24

Maystrenko et al., 1973b; 0. I. Maystrenko (R. Morris, personal communication, 1975) Welsh et al., 1968

TST subst. in CS TST subst. in CS

24 24

Welsh et al., 1968 Welsh el al., 1968 (Continued)

e

E?

5

E

d H

bJ

TABLE 8 (Continued) Chromosome and arm( ) 6B (S)

Trait

Materiala

Protein content: Non-gliadin, proteolipid (CM components 2 and 10 of Rodriguez-Loperena et al., 1975) Gluten (+effect) Enzyme(8): Peroxidase (leaf, band C-2; root, band C-3; "suppressor" peroxidase (root C-9); peroxidase C-2 located distally to root peroxidase C-3 and root peptidase A-3) Acid phosphataee ( effect) Aminopeptidase, Amp-B1

+

a-Amylase (+ effect) Glutamate oxaloacetate transaminase Got-B1 (1st leaf blade) Peptidase (root band A-3)

Reference

CS DT, N-T

5, 37

Aragoncillo et al., 1975a

CS D T

4

Maystrenko et al., 1973a; 0 . I. Maystrenko (R. Morris, personal communication, 1975)

CS DT; Ae. umbelluhta, and chr. addition and translocation lines; T. dicoceum

3, 14

MacDonald and Smith, 1972

11

CS D T CS DT, N-T

3, 14

CS DT CS DT, N-T

3, 14

Gale and Spencer, 1974 Hart, 1973a; G. E. Hart, personal communication, 1976 Gale and Spencer, 1974 Hart, 1975

3, 14

MacDonald and Smith, 1972

CS DT; Ae. umbellulata and chr. addition and translocation lines; T. dimecum

11

Miscellaneous: Nucleolus organizer (structural gene) Nitrate reduction ( +effect) Protein content: Gliadins (10 components) Gluten (fractions A,, Da = gliadins, joint control fractions Ba and B, with group 2 chr.) Enzyme(s): Peroxidase (suppressor, band C-9 root) a-Amylase Amy-GB (19.5 2.7 map units from centromere) a-Amylase (band 3)

6D

+

a-Amylase (+effect) Esterase (leaf, band Est-GBL-I) Esterase (leaf) Glutamate oxaloacetate transaminase Got-Bb Processing properties: Dough strength properties (enhancer) Protein content: Gliadins (joint control by 2B and 2D) Gluten (+ effect, dispersion index)

CS DT, alien subst. 6X/4X derivatives CS DT, N-T

8, 9

Viegas and Mello-Sanipayo,

10

Deckard et al., 1975

CS DT, N-T CS DT

3, 5 3

Wrigley and Shepherd, 1973 Khrabrova et al., 1973

CS DT, addition and translocation lines of CS and Ae. umbellulato; T. dicoccum CS DT 6B(@)X PLD

3, 14

MacDonald and Smith, 1972

3, 14

K. Nishikawa and M. Nobu-

CS DT

3, 14

CS DT CS DT, N-T CS aneuploids CS DT, N-T

11 3, 14 3, 14 3, 5

hara, personal communication, 1975 Nishikawa and Nobuhara, 1971; Nishikawa, 1973 Gale and Spencer, 1974 May et al., 1973 Nakai and Tsunewaki, 1974 Hart, 1975

CS D T

13, 19

Maystrenko et al., 1973a

CS N-T

3

Shepherd, 1968; Waines, 1973

T H subst. in CS

28

Solari and Favret, 1970

1975

~~

-

(Continued)

TABLE 8 (Continued) Chromosome and arm( ) 6D

6D(a

=

S)

Q,

Trait

Material.

method(^)^

Reference

Gliadin (bands 18, 20, part of band 16) Enzyme(s): Amylase

CS N-T

3

Kasarda ei al., 1976b

CS Null, CS Mono X 7'. durum

3

Nagayoshi, 1973

Esterase (flag leaf) Protein content: Gluten

CS ancuploids

3, 14

Nakai and Tsunewaki, 1974

CS D T

4

Khrabrova et al., 1973; 0 . I. Maystrenko (R. Morns, personal communication, 1976)

CS DT, N-T CS DT, N-T

3 3, 14

Hart, 1973a Hart, 1975; Hart et al., 1976 (G. E. Hart, personal communication, 1976)

Enzyme(s): Aminopeptidase, A m p D l Glutamate oxaloacetate transaminase Got-Dl (1st leaf blade) Miscellaneous: Nitrate reduction (+ effect) 6D@ = L)

E!

Protein content: Gliadins (bands M , and La, regulated also by 2A) Gliadins (4 components)

Gliadin (fractions AI, Dd, joint control fractions BJ and B, with group 2 chr.)

CS DT, N-T

10

Deckard et al., 1975

CS DT, N-T

3

Shepherd, 1968

CS N-T, D T

3, 5

CS D T

3

Shepherd, 1968: K. W. Shepherd (R. Morris, personal communication, 1975); Wrigley, 1972a: Wrigley and Shepherd, 1973 Khrabrova et al., 1973

s2 r

0

?w

Enzyme ( s ): a-Amylase (band l ) , Amy 6D-I, CS DT, CS 6D(@ X PLD, 1 1 . 9 k 3 map units from tetra CTH, T . durum, centromere Aegilops spp.

a-Amylase (+ effect) a-Amylase Amy 6D-8 Esterase (leaf; bands Est6DL1, Est-GDL-8) Glutamate oxaloacetate transaminase Got-DI (1st leaf blade) Miscellaneous: PEP carboxylase activity ( - effect) Processing properties: Dough strength properties (inhibitor or regulator) 7A

Protein content: Total protein (kernel, effect) Total protein (kernel, gluten, effect) Total protein (flour, - effect) Gluten Amino acid: Tryptophan content (strong effect) Enzyme(s): Esterase (slow moving band)

+

+

+

3, 14

Nishikawa et al., 1976; K. Nishikawa and M. Nobuhara, personal communication, 1975,

CS DT CS DT CS DT, N-T

11 3, 14 3, 14

CS DT, N-T

3, 14

Gale and Spencer, 1974 Nishikawa et al., 1976 May el al., 1973; C. E. May personal communication, 1975 Hart, 1975

1976

CS DT

38

Fortini el al., 1973

CS D T

13, 19

Maystrenko et al., 1973a

0

2

1 0

0 ?¶

‘d

z

0

CS Mono X SN64 CNN subst. in CS X CS (F, hyb.), CNN, CS TST subst. in CS CS Mono X SRV 210

23

CS Mono X SN64 D chr. subst. in LDN

1

Jha et al., 1971 Sasaki et al., 1973

4

Siddiqui, 1972 Maystrenko et al., 1973b

4

15

Jha et al., 1971

cj

3, 14

Bergman and Williams, 1972

11

2

(Continued)

3>

z -J

TABLE 8 (Continued) Chromosome and arm( )

Trait

Materials ~~

Peroxidase (band d) Miscellaneous: Nucleolus activity (regulator)

~

~~

a-Amylase (+ effect) Endopeptidase Ep-A1 (seedling) Miscellaneous: Protein component ratio Nitrate reduction (inhibitor or regulator)

Method(s)* ~

Reference ~

CS N-T

3, 14

Kobrehel and Feillet, 1975

CS DT, alien subst. 6X/4X derivatives

8, 9

Viegas and MeUo-Sampayo, 1975

11

Gale and Spencer, 1974

10

Edwards, 1973, 1974

13, 19

Maystrenko et al.. 1973a

CS D T

11

Gale and Spencer, 1974

CS DT

3, 14

CS DT CS DT, N-T

3, 14

Nishikawa and Nobuhara, 1971; Nishikawa, 1973; Nishikawa et al., 1976 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1976

CS D T

26

CS DT, N-T

10

Enzyme: a-Amylase (+effect) CS D T Miscellaneous: Nitrate reduction (regulator or CS DT, N-T inhibitor) Processing properties: Dough properties (strength, CS D T minor inhibitor) Enzyme ( 8 ): Peroxidase ( - effect = inhibitor?) a-Amylase (band 13), Amy-7A

E 00

11

A. Bozzini, personal communication, 1974 Edwards, 1973, 1974

d

3 ?

w

7B

Protein content: Total protein (kernel, Total protein (kernel, Total protein (kernel,

+ effect) + effect) + effect)

Gluten (+ effect)

T C subst. in CS CS Mono X SN64 CNN subst. in CS X CS (F1 hyb.), parents C N N subst. in CS X CS (F1 hyb.), CNN, CS CS Mono, DT

CM 2 protein (chloroformmethanol extract) Amino acid: Tryptophan content CS Mono X SN64 and X L R Miscellaneous: Inhibitor (of C M 1 component CS N-T 2, structural gene in 6B) Processing properties: Loaf volume (+ effect, minor TST subst. in CS factor) Gluten quality (weak inhibitor) CS Mono X SRV 29 Milling properties (major gene) Dough properties (strength, loaf volume, crust appearance, grain and texture; effect) Protein content: Non-gliadin proteolipid (components 8 and 9 of Rodriguez-Loperena et al., 1975 = albumins of Waines, 1973)

2 1 4

Kuspira and Unrau, 1957 J h a et al., 1971 Sasaki et al., 1973

4

Sasaki el al., 1973

37

Garcia-Olmedo and Carbonero, 1970

15

J h a et al., 1971

5, 37

Aragoncillo et al., 1975a

4

Welsh et al., 1968

23

CNN subst. in CS CNN subst. in CS

4

4

Maystrenko et al., 1973b; 0 . I. Maystrenko (R. Morris, personal communication, 1975) Mattern et al., 1973 Morris et al., 1966

CS DT, N-T

5, 37

Aragoncillo et al., 1975a

+

7WS)

(Continued)

TABLE 8 (Continued) Chromosome and arm( )

7B(S)

Trait

Material"

Enzyme(s): a-Amylase (+ effect) Esterase (intermediate moving band) Processing properties: Milling properties (flour yield, effect)

Reference

CS D T CS DT, N-T

11 3, 14

Gale and Spencer, 1974 Bergman and Williams, 1972

CS DT

6

Maystrenko et al., 1973a

CS D T

22

Fortini et al., 1973

CS D T

3, 14

a-Amylase (+ effect) Endopeptidase, E p B l , Epl (seedling)

CS D T CS DT, N-T

11 3, 14

RuDPCase activity (+ effect) Miscellaneous: Protein component ratio

CS D T

38

Nishikawa and Nobuhara, 1971; Nishikawa et al., 1976 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1975 Fortini et al., 1973

CS D T

26

A. Bozzini, personal communication, 1974

3, 39

Favret et al., 1970

1 1, 2

Jha et al., 1971 Siddiqui, 1972

+

Protein content: Total protein (seedling leaf, - effect) Enzyme(s): a-Amylase (band 15), Amy-7Rl

7D

method(^)^

Protein content: Total protein (kernel, effect, TC subst. in CS 4 bands = regulator gene) Total protein (kernel, effect) CS Mono X LR Total protein (flour, - effect) Hope subst. in CS

+ +

Total protein (kernel,

- effect)

CNN subst. in CS X CS (Fj hyb.), parents Albumin CS N-T, Ae. squarrosa Proteolipid C M 3 protein (com- CS N-T ponent 11) Enzyme ( s ): Peroxidase (band a) CS N-T Esterase (intermediate band) CS DT, N-T Esterase (slow band) D chr. subst. in LDN Glutamate oxaloscetate trans- CS DT, N-T aminase Got-I Processing properties: Milling properties (+effect; TST subst. in CS flour yield, minor factor) Kernel hardness (water absorp- TST subst. in CS tion, effects, major factor) Loaf volume, crumb grain and CNN subst.. in CS texture (+effect) Loaf volume (+ effect, minor TST subst. in CS factor)

+

Prolein conlent: Non-gliadin proteolipid (CM 1 CS DT, N-T components 3 and 4 of Rodriguez-Loperena et al., 1975 = albumins of Waines, 1973) Miscellaneous: Nucleolus activity (regulator) CS DT, N-T, 6X/4X derivatives

Esterification of sterols of endosperm

CS DT, N-T

4

Sasaki et al., 1973

3 5, 40

Waines, 1973 Rodriguez-Loperena et al., 1975

3, 3, 3, 3,

Kobrehel and Feillet, 1975 Bergman and Williams, 1972 Bergman and Williams, 1972 Hart, 1975

14 14 14 14

24

Welsh et al., 1968

4, 6

4

B. Belderok and G. J. Doekes, personal communication, 1974 Morris et al., 1966

4

Welsh et al., 1968

5, 37

Aragoncillo et al., 1975a

8, 9

W. S. Viegas and T. MelloSampayo, personal communication, 1974 Torres and Garcia-Olmedo, 1974

41

(Continued)

TABLE 8 (Continwd) Chromosome andarm(

cn

to

Trait Enzyme(s): a-Amylase (band ll), Amy-TD

Materials

Method (sY

Reference

CS DT, Aegilops spp., TetraCTH

3, 14

a-Amylase (+ effect) Acid phosphatase (+ effect) Endopeptidase Ep-D1 (seedling)

CS DT CS D T CS DT, N-T

11 11 3, 14

Peroxidase (+ effect) Miscellaneous: RuDPCase activity (- effect) Processing properties: Dough properties (extensibility, “enhancer”)

CS D T

38

Nishikawa and Nobuhara, 1971; Nishikawa, 1973; Nishikawa et al., 1976 Gale and Spencer, 1974 Gale and Spencer, 1974 Hart and Langston, 1975; G. E. Hart, personal communication, 1976 Gale and Spencer, 1974

CS D T

38

Fortini et al., 1973

CS D T

13, 19

Maystrenko et al., 1973a

a Variety abbreviations: ATL 66 = Atlas 66; CD = Capelle Desprea; CNN = Cheyenne; CS = Chinese Spring: CTH = Canthatch; HFT = Holdfast; KKF MC22 = Kharkof MC22: LDN = Langdon (durum); LR = Lerma Rojo; PLD = Prelude; RE = Red Egyptian: RSC = Rescue; SN64 = Sonora 64;SRV 29 = Saratovskaya 29; SRV 210 = Saratovskaya 210; TC = Thatcher; TST = Timstein. Genetic stock abbreviations: D T = ditelosomics; Mono = monosomics; N-T = nullitetrasomics; Null = nullisomics; subst. = substitutions; chr. = chromosome. * Code for method(s): 1 = dye-binding capacity; 2 = Kjeldahl; 3 = electrophoresis: 4 = AACC; 5 = electrofocusing: 6 = quadrumat Jr. Mill; 7 = experimental baking; 8 = cytology; 9 = autoradiography; 10 = in vivo NRA analyses; 11 = enzyme yield; 12 = isoelectrofocusing; 13 = farinograph; 14 = chemical tests; 15 = amino acid analyses; 16 = sedimentation; 17 = doughball; 18 = fermentation test; 19 = alveograph; 20 = mixing; 21 = micro-Kjeldahl; 22 = Lowry N ; 23 = gluten swelling; 24 = Buhler Mill; 25 = phenol reaction; 26 = solubility fractionation; 27 = flour fractionation; 28 = gluten dispersion index; 29 = starch damage; 30 = water absorption; 31 = immunochemical tests; 32 = immunoelectrophoresis; 33 = quadrumat; 34 = petroleum ether extract; 35 = mixograph; 36 = pH separation, aggregation; 37 = electrophoresis ETOH extract; 38 = in vitro enzyme activity analysis; 39 = Coleman nitrogen analyzer; 40 = electrophoresis of chloroform-methanol extracts; 41 = ether extract, 4 kernels mc, fluorometry; 42 = i n vitro NRA analyses.

to

*F

0

2 J

p

P4

GENETIC CONTROL OF PROTEINS I N WHEAT

523

stocks of the hexaploid wheat variety Chinese Spring (Fig. 6). Even better illustrations of homoeology and genetic relations have been shown in isoenzyme studies (Hart, 1970, 1973a,b, 1975). May et al. (1973) have distinguished four ways by which homoeologous genes of hexaploid wheat may function in accordance with the Jacob and Monod (1961) concept of structural (producer and conformation) genes and regulator genes: (1) The structural and regulatory genes on homoeologous chromosomes are identical. The biosynthetic products of such genes are identical and do not change in concentration in the nullisomictetrasomic lines, indicating complete compensation, i.e., triplicated genes (Morris and Sears, 1967). Duplicated genes controlled in a similar way would produce a constant amount of product independent of the number of gene loci present in the genome. (2) The structural genes differ, but the regulatory genes on homoeologous chromosomes are identical. The products of such genes are distinguishable in various ways, e.g., as doublet or triplet sets of isozymes, and compensation in the nullisomic-tetrasomic lines occurs by increase of an homoeologous gene product. Example: group 6 esterases, and possibly the a-amylases in groups 6 and 7. (3) The structural genes are identical but the regulator genes are different in homoeologous chromosomes. The genetic relations involved are distinguishable by the occurrence of different amounts of identical products and thus only partial compensation occurs in nullisomic, tetraso-

0

FIG.6. Chromosomal control of gliadin synthesis in wheat endosperm as detected using combined electrofocusing and electrophoresis techniques in the analysis of proteins of aneuploid genetic stocks of Chinese Spring wheat. (Reproduced from Wrigley and Shepherd, 1973. Courtesy of the authors and New York Academy of Sciences.)

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CALVIN F. KONZAK

mic and disomic substitution lines. Minor genes differ from major genes as regards regulatory functions. (4) The structural genes and regulatory genes are in each of the homoeologous chromosomes. In this case the genetic relations involved are distinguishable by the absence of genetic compensation. As for the “diploidized” genes described by Morris and Sears (1967), the absence of a particular homoeologous chromosome or segment results in the absence of the gene product. Biochemical genetic analyses offer to reduce to more simple terms the evident complexity of protein differences among wheat varieties. For example, several prolamine (gliadin) proteins which distinguish the strong us weak gluten varieties, respectively, Thatcher (TH) and Chinese Spring (CS), prove to be controlled by genes located in chromosomes of the A, B, and D genomes of homoeologous group 1 (Fig. 7). Electrophoretic analyses (Manghers et al., 1973) using nullitetrasomic stocks and disomic substitutions of group 1 Thatcher chromosomes into Chinese Spring demonstrate that of the gliadins by which the two varieties differ: (1) Three are controlled by genes present in TH 1A. (2) Another three proteins, which are structurally evolved from those of TH lA, are controlled by the homoeologous chromosome TH 1B. (3) Two other proteins are structurally identical (duplicated) and controlled by genes present in TH 1A and 1B. (4) CS carries the null alleles for all 8 of these proteins. ( 5 ) Two gliadins common to TH and CS are shown to be controlled by chromosome lD, from their absence in nulli 1D tetra 1A and nulli 1D tetra 1B. (6) One protein controlled by chromosome 1B is present in CS;

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FIQ.7. Prolamine patterns for slow-moving bands (buffer pH = 3.2) in Thatcher and Chinese Spring wheats, substitution lines of Thatcher 1A and 1B of Chinese Spring. Arrows on the left show deletion, and those on the right show addition of proteins. Arrows with a circle indicate a probable case of duplicated proteins. (Reproduced from Manghers et a l , 1973.)

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the null allele is in TH 1B. Differences in the genetic regulation of proteins also are evident by the changes in genetic compensation shown in the electrophoretograms : (a) Thatcher 1B carries a regulator gene which increases the amount of one protein common to both varieties. I n this case the homoeologous locus for the structural protein is identical in the two varieties but the regulator genes differ. (b) I n the second case, genetic compensation by TH 1B fails because TH 1B carries the null allele for the structural gene of a homoeologous protein, controlled also by chromosome 1A in both varieties. However, the regulator gene for this protein is present in TH 1B. (c) The third protein is deleted from the TH 1B substitution because TH 1B lacks the structural and regulator genes present in CS 1B. I n similar ways, a considerable number of structural and regulator loci controlling protein synthesis have been identified (Table 8 ) . Certain general conclusions relative to the genetics of protein content, amino acid composition, various processing factors, and specific proteins are now apparent from the experimental results described earlier and those listed in Table 8. 1. Homoeologous, structurally evolved genes which code for proteins are located in several chromosome groups of each of the three genomes. 2. The majority of gliadin proteins (prolamines) are controlled by genes in chromosomes of groups 1 and 6. 3. Specific proteins are absent when arms of chromosomes are deleted. The concentrations of these same proteins are usually increased when the dosage of the chromosomes carrying the genes is increased. The concentrations of specific proteins may depend on whether there are 0, 1, 2, 4,or 6 chromosome doses. 4. Monomer subunits of dimer enzymes, such as alcohol dehydrogenase, are produced in amounts proportional to the dosage of the homoeologous chromosomes carrying the structural genes. 5. Evolutionary changes often have modified the structural and sometimes the functional characteristics of the gene products coded by different homoeologous chromosomes (presumably the gene structure has been altered), but regulation may remain similar in each genome for the homoeologous proteins (e.g., gliadins, alcohol dehydrogenases) . 6. Hybrid enzymes (isozymes of phosphodiesterase) occur in hexaploid wheat that are comprised of components identified in A . squarrosa (D genome) and components present in several tetraploids (probably B genome), but are not present in a number of diploids. A component similar to one in tetraploids occurs in several diploids. New hybrid dimer enzymes (alcohol dehydrogenases) occur in wheat-rye hybrids and rye chromosome substitution lines.

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7. Genes controlling the production of unique proteins are present in each of the three wheat genomes. 8. Homoeologous alleles may have different levels of activity or “strength” with relation to other alleles (regulatory genes evolved) ; this relation is exemplified by the different production of the lipid moiety for purothionins by group 5 homoeologs. Some specifics relative to proteins identified in the seven chromosome groups are summarized below :

Chromosome group 1 1. Genomes A, B, and D each may carry homoeologous, evolved, producer (structural) genes for gliadins, purothionins, peroxidases. 2. Chromosome arms 1A(S), l B ( L ) , and 1D(S) are implicated in the process of nitrogen reduction and probably carry structural genes. 3. Chromosome 1D codes for two unique subunits of high MW glutenin, proteins that are important to dough elasticity in hexaploid wheat. 4. Differentiation occurs in gliadins of different varieties, possibly also relating to differences associated with regulation or inhibition by factors in group 2 chromosomes. 5. Chromosome 1D of Cheyenne may carry ( a ) gene(s) increasing lysine over 15%. Chromosome group 2 1. Chromosome 2A and probably chromosomes 2B and 2D carry regulators (or inhibitors) of nitrate reductase activity (NRA). The alien chromosome substitutions with high protein content and the inhibition of 2B and 2D of gliadin synthesis in 6D indicate a homoeologous series of inhibitor loci. 2. Chromosome 2A in ‘Saratovskaya 210’, Hope, Timstein and ‘S-615’, and 2D in Saratovskaya 29 carry genes for high crude protein content, indicating the possibility that the NRA “inhibitor” factors have been modified in these varieties. 3. Group 2 chromosomes apparently carry genes affecting a-amylase enzyme content. 4. Phenolase complex activity is associated with chromosomes 2B and 2D, suggesting that a homoeologous series will be found. Chromosome group 3 1. Genomes A, B, and D may carry homoeologous esterase, peroxidase (Per), a-amylase ( A m y ) , glutamate-oxaloacetate transaminase ( G o t ) , and possibly for acid phosphatase (Acph) loci. 2. Genomes A and D carry a homoeologous esterase seemingly absent from B. 3. Genome A carries a unique esterase locus.

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4. Chromosome arm 3B(L) is implicated in the process of nitrate reduction. 5. Genomes A and D carry loci controlling several “gliadinlike” proteins. 6. Genomes B and D carry loci controlling proteolipids.

Chromosome group 4 1. Genomes A, B, and D may carry evolved homoeologous acid phosphatase, alcohol dehydrogenase (Adh), and lipoxygenase ( L p z ) genes as well as genes affecting a-amylase content. 2. Genomes A, B, and D carry genes affecting milling properties (flour yield). 3. Chromosome 4D controls the formation of a glutenin subunit. Chromosome group 5 1. Genomes A, B, and D in Chinese Spring may carry homoeologous genes controlling the production of the lipid moiety for the purothionins, with the gene on 5 D most effective. 2. Genomes A, B, and D carry homoeologous structural genes for lipoxygenase and for “gliadinlike” proteins. 3. Chromosome 5 D of Atlas 66 carries gene (s) for high protein content. This factor may be concerned with translocation of N into the grain. 4. Chromosomes 5D in Cheyenne, Timstein, and Hope carry a kernel hardness gene affecting milling properties. 5. Chromosomes 5 D in Capelle Desprez, Hope, Timstein, and ‘Sinvalocho’ carry gene (s) for higher protein or gluten content. 6. Chromosome 5 D carries nucleolus organizer, regulator, and structural genes and a factor affecting nitrogen reduction (a single cistron?). Chromosome group 6 1. Genomes A, B, and D may carry homoeologous structural gene loci controlling the synthesis of gliadins, esterases ( E s t ) , Amy, aminopeptidases (Amp),Got, and are of major importance for gliadin production. 2. Chromosome arm 6A(p) carries a structural gene concerned with N reduction. 3. Chromosome 6B carries unique Amy and Per loci. 4. Chromosome 6D carries an inhibitor (or regulator) of PEP carboxylase activity. Chromosome group 7 1. Genomes A, B, and D carry possibly homoeologous, structurally evolved loci controlling a-amylases ( A m y ) ,and endopeptidases ( E p ). 2. Genomes A and B carry possibly homoeologous esterase loci. 3. Genomes B and D carry possibly homoeologous esterase loci.

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4. Chromosome arms 7A(S) and 7A(L) carry regulators or inhibitors of NRA. 5. Chromosomes 7D carry gene(s) affecting kernel hardness and milling properties of Timstein, a regulator of nucleolar activity in Chinese Spring, and an inhibitor of RuDPCase activity. 6. Chromosome 7B carries a gene affecting dough mixing characteristics in Cheyenne, and a promotor of RuDPCase activity in Chinese Spring. 7. Chromosome 7A of Cheyenne carries a dominant gene having a negative effect on protein content. XI. General Discussion

Compared with the other main cereals, wheats differ in two important ways that have genetic consequences: (1) All important cultivated wheats are polyploids, i.e., tetraploid or hexaploid, while maize, barley, rice, and sorghum are all diploids. (2) I n wheats, as in barley, corn, rice, and sorghum, the genetically controlled protein components present in highest proportion have the lowest nutritional value. Yet many protein components, mainly prolamines, are evidently vital to the unique properties of wheats. I n all the high-lysine sources of maize, barley, and sorghum identified to date, the proportion of prolamines in the proteins is sharply reduced. The main uses of wheats are now determined by the suitability (quality) of flours (or semolina) milled from them for often rather specialized baked products, e.g., breads, pasta, cakes, and cookies. Through intense selection and breeding over centuries, classes or genetic races of wheats adapted for those end-product uses have been isolated, and in many cases have displaced from cultivation those wheats with other quality properties. The main unique features of wheats are concerned with the doughforming properties of gluten, fundamental to the structure of leavened and unleavened bakery products. Processing technology, constantly changing, can exploit or reduce the importance of protein compositional differences. Current trends are toward stronger gluten wheats, with strength mainly due to high MW glutenins. Wheat gluten (once thought to be simple) has proved t o be comprised of polypeptide chains which are distinct in many ways-molecular size, charge, and solubility in various polar and nonpolar solvents, detergents and other chemical compounds (mercuric chloride, mercaptoethanol, etc.) . The subcomponents of gluten show variability in amino acid composition and in their associations with lipids, thiols, and carbohydrates. Certain

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of the “insoluble” (glutenin) components of wheat gluten are undoubtedly remnants of cellular organelles and the endoplasmic reticulum of the cytoplasm. Protein bodies are observed in developing endosperm, but their identity is virtually lost in the mature endosperm and in flour. The laminar structure of protein bodies observed in the immature endosperm is disturbed in the mature grain, and subjected to considerable further modification by interactions with other cellular components upon wetting and mixing of flour components during the formation of dough. According to a recent hypothesis, many of the characteristics of wheat gluten proteins from flour may be artifacts (Bernardin and Kasarda, 1973a,b; Mecham, 1973; Kasarda, 1970; Bernardin, 1975). Flour proteins released by breaking open endosperm cells during milling or by the crushing forces of dehydration processes during maturation are free to interact with other cellular constituents, such as salts, sulfhydryls (glutathione), etc., and do so instantly upon wetting. The solubility characteristics of the gluten a t high protein concentrations (considered mainly gliadins) change as water absorption (and mixing in dough) proceeds (Kasarda et al., 1976a). There still is some uncertainty about the nature, MW, and properties of the glutenins; hence the very large molecular size and low water-absorbing capacity of some glutenins also may be artifacts reflecting the state of gluten protein components in dough, but not in the intact endosperm cells. However, after reduction, alkylation, and other treatments, a portion of the high-MW proteins remain insoluble in alcohol but soluble in dilute acid. The latter are considered glutenins. Conformational and other alterations can be induced in proteins by changes in pH and ionic strength, and by chemical bonding due to oxidative reactions, once the protein subunits are free (Bernardin and Kasarda, 1973a). The relationships between the individual gliadin components and glutenin subunits are not yet clear, but it should now be possible to investigate them, since virtually all of the protein in wheat can be made soluble without denaturing any one component. The rapid wetting ability of proteins observed by Bernardin and Kasarda (1973a) suggests that the slower water absorbing (long mixing time) ability of certain wheat flours may be due to differences in their composition of hydrophilic us hydrophobic chemical groups, which eventually are modified during dough mixing. The complexity of wheat proteins seems to present a formidable obstacle to the nutritional improvement of wheat proteins via plant breeding, while maintaining the unique processing and product-use characteristics currently important to man. Nevertheless, the prospects for improving the nutritional composition of wheat proteins are not so limited as they may seem.

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Essentially all wheat protein components and quality factors are controlled by genes, but genetic linkages may impede recombination, and limit progress in selection for quality improvements. The useful germplasm sources already identified indicate that a basis for significant improvements (although yet smaller than those in other cereals) is available in wheat. If the higher lysine factor on Cheyenne 1D is confirmed, the 15% increase in lysine associated with that chromosome is equal to the advantage of Hiproly over normal barley. I n fact, the gene duplication resulting from the polyploid nature of wheat may endow wheats with greater, rather than less, genetic potential for the desired changes in protein composition: (1) Only minor adjustments in protein composition may be necessary to achieve important nutritional improvements. (2) Those proteins containing the highest concentrations of lysine (especially globulins and albumins) normally are present as a small proportion of the total wheat protein and evidently are of minor importance to doughforming properties. (3) Any negative effects of increases in the soluble proteins on the functional properties of dough might be compensated by introducing genes for greater gluten strength, for which considerable variability is available. Certain components in the glutenin fraction also may have appreciably higher lysine than others, and although the lysine composition of glutenin is lower than in albumins and globulins, the glutenin fraction is a much greater part of the total wheat protein. Furthermore, the evident complexity of wheat proteins may not have the significance that seems apparent: (1) All gliadin proteins (a, p, y , and 0 ) separated by electrophoresis differ only slightly in amino acid composition and amino acid sequence, although the differences evidently are sufficient to alter some physical properties in solutions differing in pH and ionic strength. (2) All gliadins appear to be compactly folded globular proteins with similar functions in dough (Bernardin, 1975; Kasarda et al., 1976a). (3) Certain glutenin proteins may be far more important than others (Bietz et al., 1975; Orth and Bushuk, 1974). Hence, it may be possible to genetically reconstruct other equally effective protein complexes, e.g., increasing or decreasing some gliadins and increasing some glutenins. Some specific proteins a t lower concentrations may be rheologically more effective than others per unit of protein, as is suggested from recent studies on cy-gliadins by Bernardin (1975). The basis for wheat quality may, in fact, be much more simple than might be supposed from detailed analyses (Wrigley and Moss, 1968; Orth et al., 1972; Bushuk, 1972). Only four measurements of grain and flourmixing properties may determine the quality of sound normal wheats-

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kernel hardness, dough stiffness, dough strength, and dough stability. New breadmaking processes, among them the Chorleywood process (highspeed mixing), a new nonfermentation process (Chamberlain e t al., 1962; Kilborn and Tipples, 1972a,b; J . A. Johnson and Sanchez, 1972; Bushuk, 1972), and the short-time baking systems (Finney et al., 1977; Magoffin et ul., 1976) also have a bearing on the importance assigned to particular protein components, especially in technologically advanced countries. However, it appears that wheat flours with stronger gluten properties may become increasingly important because of their use in blends with weaker flours (wheat, rye, oats, etc.) as well as with materials added to bolster the nutritional properties of bakery products, e.g., malt, soy flour. Stronger gluten wheats also appear to retain dough strength longer during malting, possibly permitting greater development of nutritionally improved protein components (P. L. Finney et al., personal communication, 1976). Genetic linkages, as indicated in the studies by Shepherd (1968), can be expected t o limit the facility with which protein composition and characteristics can be manipulated by breeding (Orth and Bushuk, 1972; Manghers et al., 1973; Wrigley and Shepherd, 1973; Bietz et al., 1975). However, genetic variability in proteins coded by individual chromosomes does exist and can be induced (Avila and Favret, 1966), permitting the reconstruction of varieties with newly designed protein complexes. A rather wide genetic variation in molecular composition of gliadins is possible, but is of questionable consequence to rheological and other measured properties of doughs, even though the proportion of gliadin present in the flours often influences the rheological properties of doughs by shortening mix time. Starch damage incurred during milling, especially of hard endosperm wheats, may confound data from sedimentation, mix time, loaf volume, and other measurements, such as water absorption, so that the true effects of some protein differences are obscured (Farrand, 1964, 1969, 1972). In barley, the high lysine content of Hiproly and several mutants may be due in part to a higher-lysine glutelin and in part t o a marked increase in the proportion of a particular saline-soluble fraction that is high in lysine. I n barley, as well as in wheat, the saline-soluble (globulin) purothionins have an exceptionally high lysine content (9.8 to 15 gm of lysine per 100 gm of protein). Purothionins are bound with lipid as lipoproteins, but are separable from the lipid (Nimmo et al., 1968; Garcia-Olmedo et al., personal communication, 1974; 1975). This protein may be one of those increased in the high-lysine barley variants. However, the potential nutritional value of purothionins is dubious owing

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to their antibiotic properties and possible toxicity to animals. Recent evidence that the purothionins may be extraction artifacts suggests that the native proteins involved could still have nutritional importance. Perhaps more significant is the evidence that the lysine content of the prolamine fraction in a high-lysine barley mutant, Bomi 1508, has been increased by about 300% (albeit with a reduction in proportion of prolamines from 29 to 9%) , with a lower, but significant, lysine composition increase occurring also in the glutelin fraction. The increased lysine in the glutelin fraction of Bomi 1508, though relatively small, is of significance because of the high proportion (39%) of the glutelin protein. I n some maize mutants, a glutelin fraction is increased both in proportion and in composition of lysine. Considerable variation in lysine composition among different gliadins and lox higher lysine composition for some soluble proteins has been observed by Booth and Ewart (1970). Since the evolutionary relationships generally indicate similar potential for genetic variability among all Gramineae, it should be possible to identify and increase high-lysine components among those glutenins of wheat that are genetically related to the glutelins of barley and maize. The relatively higher lysine composition of some wheat glutenin fractions compared with gliadins suggests that some subcomponents in glutenins may contain above-average amounts of lysine as is the case for rice glutelin. The amino acid composition may also be influenced by the addition (and presumably by substitution) of rye chromosomes to wheat. Riley and Ewart (1970) found the addition of chromosome I of ‘King 11’ rye to ‘Holdfast’ wheat increased the lysine content of the endosperm by 8.7%. Since rye chromosome I is homoeologous to group 5 chromosomes of wheat, they suggested that group 5 wheat chromosomes may be important in the determination of the lysine content of wheat proteins. The King I1 rye chromosome I also increases the cysteine content by 10.70J0,while rye chromosome I1 increases proline by 9.1% and reduces aspartic acid by 8.676. Chromosome IV increases, but VI reduces, arginine by 11.7 and 8%, respectively. Rye chromosome VI also increases proline by 11.8% while chromosome VII reduces threonine by 10.4% (Riley and Ewart, 1970). Resolution of the many protein complexes by combinations of electrophoretic, electrofocusing, and chemical separation techniques using aneuploid stocks is clearly illustrated in the identification of the genetic basis for the synthesis and regulation of the lipoprotein purothionins (GarciaOlmedo and Carbonero, 1970; Garcia-Olmedo et al., 1975). The use of different extraction methods for analyses of a wide variety of wheats and wheat relatives (in addition to the aneuploid material) revealed structural genes coding the two purothionin analogs evolved prior to the for-

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mation of tetraploid wheats. The additive contributions t o the total purothionin content from genes in each of the homoelogous chromosomes has been demonstrated, while differences in apparent concentration of the two purothionin analogs in different wheats is a consequence of the different production of the lipid moiety by genes in different chromosomes of the three genomes. Many others have observed differences in the density of proteins on electrophoretograms which suggest additive contributions of duplicate and triplicate genes coding for the same protein (Shepherd, 1973; Bozzini et al., 1973; B. L. Johnson et al., 1967; Cuhadda et al., 1975; GarciaOlmedo et al., 1975; Hart, 1970, 1975; Tang and Hart, 1975; H a r t et al., 1976). However, additive epistatic and complementary types of gene action are known for many genetic traits; hence the genetic control of proteins can be expected to be similar. In fact, Riley and Ewart (1970) reported that the effects of rye chromosome additions on amino acid content in triticale are independent of the presence of wheat genomes; instead, the wheat genomes appear to be epistatic to individual rye chromosomes in their effect on the content of threonine, glutamic acid, proline, alanine, valine, and lysine. Similarly, the effects of individual rye chromosomes on amino acid composition may be reversed or concealed by additive or interactive effects of other rye chromosomes (Riley and Ewart, 1970). Changes in these genetic relationships may improve nutritional value. Recent work using scanning EM demonstrates genetically controlled differences in the ultrastructure of wheat proteins, and indicates that this new technique can be usefully applied to the resolution of the genetic basis for differences in protein structure. This work has revealed variability in the ultrastructure of protein fractions which was not detected or predicted by previous chemical and physical methods of analysis. Likewise the new work relating the conformational structure of individual proteins to physical-chemical properties and interactions in dough has suggested new hypotheses about t.he nature of native proteins and their reactions to solvents. Sequencing of individual proteins and subunits has only begun, but should answer important questions. New extraction methods have achieved nearly complete dissolution of all wheat proteins. The use of HgC1, and mercaptoethanol, reveals heterogeneity in the subunit composition of the high M W glutenins (Mecham et al., 1972; Bietz et al., 1973; Bietz and Wall, 1975; Huebner and Wall, 1975, 1976; Cole et al., 1976). These glutenins were previously found to be present in a residue insoluble in acetic acid, alcohol, or salt water, but are of greatest importance to the strength and elasticity properties of wheat gluten (Orth and Bushuk, 1972; Hoseney et al., 1970a; Bietz et al., 1973). The new methods of Bietz et al. (1975) almost totally ex-

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tract glutenins, and may be more suitable for genetic and breeding studies, since the absence of a band in electrophoretograms must be related to a genetic difference, and not to possible differences in the assembly of subunits into native glutenin molecules. The new evidence revealing the complex genetic regulation of ratelimiting steps in the N reduction process (especially the identification of inhibitors of these processes and the increased protein content resulting from their deletion or substitution by alien chromosomes) has already provided a new insight into ways to increase protein content and enhance N use efficiency of wheat by breeding. Further knowledge about the enzymological and genetic aspects of N reduction, N utilization, and the translocation of amino acid and protein subunits to the developing endosperm should provide the basis for removal by breeding of factors that limit protein accumulation in the endosperm and which affect the chemical and physical characteristics of the proteins stored therein. The existence of genes responsible for inibiting such processes as N reduction is evidence that energetics calculations comparing the ability of wheats to produce protein us carbohydrates cannot provide realistic estimates of the possibilities for improvements by genetic means. Clearly, our existing wheats do not synthesize proteins, or carbohydrates for that matter, as efficiently as they might upon removal of these inhibitors, or by modifying such energy-wasting processes as photorespiration. Genetic variation with respect to most protein traits investigated seems to be detectable if a sufficient search is made using modern methods of extraction and analysis (Doekes, 1968, 1973; Khrabrova e t al., 1973; Wrigley and Shepherd, 1974). This suggests that the potential for recombination breeding to improve protein nutritional value may be enhanced by electrophoretic studies and chemical analysis of specific proteins. Use of these laboratory screening methods may assist the breaking of genetic associations which may be required to achieve the desired goals. Chromosomes of the same or different homoeologous groups share in the genetically controlled synthesis of protein dimers, and may carry genes which regulate the production of proteins structurally determined by loci on other chromosomes in different genomes. The complex interactions possible in the hexaploid wheat system make the analytical work especially difficult, hence several approaches and methods of analyses often must be used. The introduction of alien species chromosomes, induction of mutations, transfer of nuclear material to different cytoplasms, and the resynthesis of new tetraploid and hexaploid combinations from wild diploid wheat relatives and other grass species further expand the opportunities for biochemical genetic analyses and for improvements by

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breeding. The use of the two-dimensional electrofocusing-electrophoresis method may also distinguish more variations than the standard onedimensional procedure. The higher resolution possible with this method has been demonstrated with gliadin proteins by Favret et al. (1970) and by Wrigley and Shepherd (1973). XII. Role for Induced Mutations

Several lines of evidence suggest ways in which induced mutation methods can be employed to improve wheat protein, and may explain some mutations already identified in induced and natural genetic resource materials. A. PRINCIPLES 1. Removal of “Inhibitors” Affecting the Quantity, Composition, and Physical Properties of the Endosperm Proteins

Maystrenko et al. (1973a) have shown that the rheological properties of ditelosomic stocks 5A(L), 7A(L), 4B(L), 2D(L), 5D(L), and SD(L), each lacking one arm of the respective chromosome, were improved over those of the standard euploid Chinese Spring. Whether any of the improvements were due to increased protein content or changed composition has not been determined. They found, however, that several other ditelosomic lines had significantly poorer rheological properties than Chinese Spring. Ditelosomic 5D (L) had a significantly higher flour yield, suggesting that the grain hardness factor, which is responsible for higher starch damage and important to the milling and baking properties of hard wheats like Cheyenne, may be a gene inactivation or deletion in the short arm of chromosome 5D. Edwards (1974) and A. Bozzini and M. Giacomelli (personal communication, 1974) observed that Chinese Spring ditelo 2A(S) has higher NRA than the euploid. Edwards (1974) noted also that similar “inhibitors” or “regulators” identified using the ditelo and nullitetrasomic stocks are present in each arm of chromosome 7A of Chinese Spring, and he observed that several other chromosomes were concerned with N reduction. Obviously, where it can be determined by genetic or other tests that useful wheat varieties carry inhibitors of N metabolism, these inhibitors could be inactivated or deleted by the use of mutagen treatments, resulting in improved genotypes. The variety Cheyenne, for example, appears to carry the 2A inhibitor of protein content, since Cheyenne chromosome 2A substitutions into Chinese Spring have no higher protein

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content than Chinese Spring (B. Belderok, personal communication, 1974;

M. Sasaki et al, personal communication, 1975) and the NRA of these lines is similar to Chinese Spring (R. L. Warner, personal communica-

tion, 1974). On the other hand, genetic tests show that Saratovskaya 210 carries a high protein gene on chromosome 2A suggesting that this cultivar may already carry a deletion or inactivation of the 2A inhibitor. Other lines of evidence suggest a possible basis for the inhibitor US enhancer interactions observed. One of these concerns the roles and interaction of nuclear and cytoplasmic organelle “genes” on the various synthetic processes : (a) Nuclear genes and cytoplasmic organelle DNAs may code different subcomponents of a protein polymer, such as RuDPCase, reported by Wildman and associates (see Kawashima and Wildman, 1971, 1972; McFadden and Tabita, 1974). (b) Electrophoretically different acid phosphatases are produced when genomes of wheat-rye hybrids or amphiploids are introduced into rye as compared to wheat cytoplasm (J. Rimpau and G. Robbelen, personal communication, 1974). (c) The number of proteins made on 80 S ribosomes is directly related to chromosome number in tetraploid and hexaploid wheats which have similar cytoplasm (Nagabhushan and Zalik, 1974). These lines of evidence suggest that in certain genotypes various alternative and functionally different (enzyme protein) subunit components may be produced by the action of nuclear genes in each of the three wheat genomes ; or, functionally different regulators which interact with cytoplasmic organelles may be produced by genes in each of the three genomes. Thus, genes of three genomes must interact in a cytoplasm with semiautonomous organelles controlled by, and originally evolved for, interaction with genes of one genome. Sears (1972) has suggested a similar basis for the action of certain nuclear genes that cause chlorophyll deficiency in wheat (see also Khush, 1973). Arnason (1956) has reported a maternally inherited striata chlorophyll deficiency mutation in wheat, presumably due to an induced alteration of plastom DNA. Thus, similar genetic phenomena also can occur as the consequence of evolutionary mutations in the plastom or nuclear counterparts of nuclear gene-cytoplasmic organelle interactive systems. A second line of evidence suggests that the inhibitor us enhancer gene relationship could be due to different intergenomic gene (or gene-product) interactions in the formation of polymers, which may function differentially as enzymes (or as important subunit components of gluten) : (a) New alcohol dehydrogenase (ADH) dimers are produced upon substitution of the homoeologous rye chromosome for wheat chromosomes in group 4; the different isozymes of ADH present in hexaploid wheat prove to be homo- and heterodimers of components produced by

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genes in each of the three genomes (Hart, 1970, 1971, 1973, 1975; Irani and Bhatia, 1972). Similarly, the glutamate oxaloacetate transaminase (Got) isozymes of homoeologous groups 3 and 6 may be homo- or heterodimers of components produced by genes in the different genomes (Hart, 1975; Hart et al., 1976). Evidence from Nishikawa and Nobuhara (1971) suggests that isozymes of a-amylase may be similarly constructed. (b) Two or more chromosomes from the same or different genomes are involved in the production of specific gliadin polypeptides (Shepherd, 1968; Waines, 1973). (c) Different component density (quantitative) changes for specific gliadins are found in different nullisomic-tetrasomic combinations (Shepherd, 1968, 1973 ; Wrigley and Shepherd, 1973). New “gliadinlike” proteins occur in an amphiploid of A . caudata and A . umbellulata (Waines and Johnson, 1971). A third line of evidence suggests that one gene-controlled component may affect the function of another, e.g., the activity of an enzyme, by changing the cell pH, by directly interfering with a regulating system, or by functioning as an inducer: (a) an organic acid produced in maize cells inhibits NRA in vitro (Ku, 1973) ; (b) a low-hlW protein in maize stimulates NRA (Ku et al., 1973) ; (c) an enzyme, possibly a protease, degrades nitrate reductases (Wallace, 1974) possibly a t faster rates in different genotypes. Each of these possibilities seems compatible with the theoretical considerations about gene regulation in higher plants proposed by Britten and Davidson (1969). The various evolutionary processes undoubtedly have modified the nuclear vs cytoplasmic “gene” and gene-gene interrelations associated with the construction of enzymes or other active (functional) components in dough. Evolutionary modification, with selection toward better balance and greater efficiency of biosynthetic systems, is still a basis for improvements by breeding. This evolutionary modification may be akin to diploidization, which has proceeded only a relatively small degree in polyploid wheats (see MacKey, 1968a,b, 1970). In Triticale, an artificially synthesized species, the genetic systems controlling protein synthesis and accumulation appear to be the sum of those from the two parents functioning independently in the cells throughout endosperm development (Dexter and Dronzek, 1975a,b). In some triticales, however, the genetic systems responsible for amylase control appear to be out of phase, resulting in some degradation of starch and, hence, poor endosperm development upon maturation (Simmonds, 1974; Dedio et al., 1975). Some genetic systems in polyploid wheats may become similarly out of phase by evolutionary changes or may be unnecessarily redundant because evolutionary changes have not yet occurred. Important functional and compositional improvements in wheat proteins might be achieved by

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adjusting such “gene”-controlled relationships via recombination breeding, induced mutations, and by cytoplasm changes, perhaps followed by mutation induction. 2. Inactivation of Deletion of Genes Controlling the Production of

Antimetabolites, Toxic, or Otherwise Undesirable Components Genetically controlled differences in the content of specific proteins occur among cereal varieties, species or genera. Albumin proteins known to inhibit animal, human, and insect, but not wheat amylases have been identified in different varieties (Silano et al., 1973, 1975; Petrucci et al., 1974). Protease (trypsin) inhibitor(s), more abundant in some rye and triticale varieties than in wheat or other triticales, may function as antimetabolites by lowering feeding efficiency of the grain or flour (Madl and Tsen, 1974a,b), Moreover, in certain instances the antimetabolite components appear to be rather heat stable (Madl and Tsen, 1974b). Certain gliadin components (including a-gliadins) appear t o have a causal relationship to celiac disease in man (Kasarda, 1975; Kasarda et aZ., 1976a,b), but may be present or absent in varieties with similar processing quality characteristics (Kasarda et al., 1973). The fact that quantitative and/or qualitative differences in antimetabolites, etc., occur among wheat varieties and related forms indicates that these components are not essential in modern cultivars. The genetic removal of antimetabolites would likely improve the nutritional value of many cereal varieties (Kakade, 1974). 3. Modifications to the Amino Acid Composition of Storage Protein

Data relating the effects of genetic variations in storage protein composition are yet limited. However, some gliadin fractions and glutenin subunits may contain distinctly more lysine than others (see Kasarda et al., 1976a). Cereal chemists, wheat geneticists, and breeders know that varieties may differ markedly with regard to the stiffness, extensibility, and as well as the water absorption speed and capacity of their proteins. The amino acid components and their order in the protein molecules are important determinants of these and other physical properties. Thus, the properties of these proteins (or protein subunits in the case of glutenins) may be influenced by the interaction of the amino acid components in determining the net molecular charge, balance of hydrophobic us hydrophilic groups, etc. (Ewart, 1975; Kasarda et al., 1976a). An increase in the amount of lysine, for example, might be expected to increase the water solubility and negative charge balance of the protein in which it is located. Such changes could have important effects on the processing characteristics of wheat proteins. Genetic variations of this type un-

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doubtedly occur to some extent already among existing genetic resource stocks. The variety Mahratta, and Nap Hal accessions are possible examples. These wheats have extremely weak, rapid-wetting flours. Similarly, the slightly higher lysine mutant MJD720175 induced in Marfed spring wheat apparently has weaker, more wettable flour traits. However, reductions in protein strength and increased wettability are no serious problem to the breeder since he has available a great diversity of genetic sources for even excessive protein strength and/or with slower flour wetting properties. Most surely he can recombine the various traits to achieve compensation for any effects due to increased lysine by adjustments in other proteins, and thus recover progeny with satisfactory flour processing properties. Consequently, the probability that the processing properties of storage proteins might be altered by changes in amino acid composition need not deter efforts to induce or select genetic variants with significantly improved protein nutritional value. Theoretically, all genes are mutable, possibly differentially so with different mutagenic agents, and the relative frequencies of different types of genetic alterations also can be expected to differ (Nilan e t al., 1976a). The polyploid nature of wheats endows them with considerable genetic buffering capacity to withstand alterations severe enough to be lethal in diploids. But, it is likely that the storage proteins have no vital function except to provide nutrition for the developing seedling during germination. Thus, viability should be unaffected by mutagen-induced structural or regulatory changes in storage proteins. Furthermore, such alterations may not lead to the defective endosperm conditions encountered in high lysine corn, barley, and sorghum. In fact, all higher-lysine wheats identified so far have normal endosperms.

B. MUTATIONS IN PROTEIN STRUCTURE AND

THE

GENETICCODE

The genetic codes for lysine, as an example, as compared with nutritionally poorer amino acids in storage proteins, may also be favorable for improving protein nutritional quality. According to Yanofsky et al. (1964a,b, 1966), Garen (1968) , and Schaap (1971), the RNA codons GAA or GAG code for glutamic acid, which is the most abundant amino acid in wheat proteins, while the codons for lysine, AAA or AAG, can be derived from GAB or GAG by mutation of G to A, which requires one DNA transition mutation from C to T. Gene transitions are among the most common base substitution mutations induced by chemical mutagens. Likewise, RNA arginine codon AGA, can be converted to a lysine codon by a similar base transition. The change from arginine to threonine is also possible, but requires a transversion, which is a less common base

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substitution. Transversions evidently may be inducible by mutagens like sodium azide (A. Kleinhofs, personal communication, 1976). Codon changes from GAA or GAG (both of which code for glutamic acid) would require two base substitutions to achieve the AUG codon for methionine, whereas the change from the leucine codons AUU or AUC to that for methionine would require only one transition type of base substitution. In these instances, the cereal storage proteins should be responsive to alterations, especially toward increased lysine composition. Another type of mutation might involve alteration of the regulator or operator site on a gene responsible for the synthesis of lysine or other desired amino acid in such a way that the feedback-inhibition control might be impaired. This type of change would result in a higher pool of the free amino acid, possibly to its incorporation into proteins, but more likely to an increase in free amino acids in the endosperm tissues. Base substitution mutations of the type described are brought about by errors induced by damage to DNA which cause DNA polymerase to incorporate the wrong bases (Saffhill, 1974). However, simple amino acid substitutions in specific storage proteins may have less, if identifiable, impact on the total AA composition than mutations of regulatory genes.

C. EFFICIENT INDUCTION OF IMPROVED PROTEIN MUTANTS Two general approaches offer promise for inducing mutations for improving the content or amino acid composition of wheat protein. These approaches may not be as efficient as might be possible via application of cell culture techniques, but they are practical in view of the fact that methods using cell culture have not been devised for isolating plants carrying gene-controlled changes in storage proteins. The first, or more conventional, approach simply involves mutagen treatments to seed of a selected cultivar, growing the M, generation under conditions to avoid contamination, then screening the M, endosperms or M, endosperms from M, plants for protein content and/or compositional variations. The chemical mutagens, ethyl methanesulfonate (EMS), diethyl sulfate (dES), isopropyl methanesulfonate (iPMS), methyl and ethyl nitroso urea (MNH, ENH), ethyleneimine (EI), and possibly sodium aeide (AZ) are among the more effective and efficient agents for inducing mutations in wheat. It should be noted, however, that EI, MNH, and ENH, are potent carcinogens as well as mutagens, whereas the others except for A 2 are weak carcinogens. Efficient methods for azide treatments to polyploid wheats have not been fully developed; this mutagen, however, is among the most potent agents for barley but is inactive as a chromosome-breaking agent or as a carcinogen (Nilan et al., 1973, 1976a,b; C. F. Konzak, A. Kleinhofs, and R. A. Nilan,

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unpublished). The second method, using monosomic lines, is much more efficient from the standpoint of selection, but has the disadvantage that any mutations isolated will likely be valuable only as gene resources since monosomic series are seldom if ever available in currently successful cultivars. Both approaches require efficient preliminary screening or selection methods, but it should be noted that Doll (1972) isolated several high-lysine barley mutants from analyses of only about 6000 M, lines. The monosomic method as outlined by Tsunewaki and Heyne (1959) involves treatment of seed derived from proved monosomic plants, growing the MI in isolation to avoid cross contamination, then screening seed from M, plants for the desired trait. The advantage is that nullisomics are few and often abnormal and most of the nearnormal progeny derived from an MI-treated monosomic individual will carry the treated chromosome, either in a mono- or disomic condition. Since all individuals will be essentially “homozygous” for an induced change in the critical chromosome, the realized efficiency is about 9 or 10fold, while the probability of recovering a mutation in the noncritical chromosomes of the population is the same as for an euploid cultivar. Mutagen treatments (usually radiation-UV or X- or 7-rays) to pollen can also be made to inactivate an inhibitor, etc. Pollen of a normal plant is treated either in the spike or in collected anthers and applied to emasculated spikes of identified monosomics or nullisomics (when viable and sufficiently female fertile). Seeds grown on the first-generation plants could be individually analyzed or samples of their progeny tested for any induced change. New developments in screening methods may greatly improve the efficiency of mutagen techniques for the production of useful genetic variation. These new developments include near infrared protein analyzers (Pomeranz and Moore, 1975; Williams, 1975; Pomeranz, 1976; Watson et al., 1976), and single-seed test techniques (Brunckhorst e t al., 1974a,b; Mertz et al., 1974). Recent research indicates that the infrared system may be able to simultaneously screen samples for protein and lysine, as well as certain other amino acids, ash (mineral) content, and possibly other components all at one time on the same unweighed sample; the data can be computed and registered electronically, and may require only 11 seconds per sample (K. A. Norris, personal communication, 1976). XIII. Directions for Future Research

Considerable progress already has been made by cereal chemists and geneticists toward an understanding of wheat quality parameters and of the nature, variability, and genetic basis for specific endosperm (protein and other) components of wheat. Until recently, genetic tools have played

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a minor part in the analysis of wheat protein and processing quality components. To date induced mutants have had essentially no roIe in the analyses, but offer unique advantages for future investigations. There is a sound basis for the more conventional or empirical approaches commonly practiced in wheat research. The discovery and synthesis of bread flavor by Wiseblatt and Zoumut (1963) is a striking example of the success of an empirical approach by which tests of only a few concoctions of dough components saved perhaps decades of systematic research to the same end. Likewise, the development of the so-called high-yielding wheat and rice varieties exemplifies the success of conventional plant breeding. More systematic approaches often provide the important new leads for exploitation, as evidenced by the discovery of the inhibitors of nitrate reductase and RuDPCase activity in wheat. Also, new analytical methods are continually being developed (see Novacky and Wheeler, 1971; Lein et al., 1973; Concon, 1972; Brunckhorst et al., 1974a,b; Merts et al., 1974; Pomeranz and Moore, 1975; Pomeranz, 1976; Williams, 1975; Dohan et al., 1976) , and more efficient laboratory equipment is now generally available to obtain information for saving valuable time and resources in practical research programs. Of particular significance are new developments in near-infra-red spectroscopy that permit simultaneous rapid screening for protein content and for several specific amino acids on the same unweighed sample (K. A. Norris, personal communication, 1976). Certain improvements in the nutritional value of wheat protein may require genetic engineering, including the induction of mutations. These genetic tools can be used to isolate new germplasm sources and to better define approaches likely to be most successful and exploitable by more conventional techniques. The genetic data already available have great value for both practical application and basic research. These data suggest several lines of investigation that should contribute new knowledge and/or useful resource materials toward the development of wheats with satisfactory processing qualities and with more nutritionally improved protein : 1. Develop caryopsis (kernel) composition ideotypes for the important quality classes of wheats. Although the scientific basis for ideotype concepts of wheat quality types is still relatively weak, enough evidence is now available to suggest several (including alternative) lines of investigation. 2. Increase research into the genetic basis of processing quality differences, preferably by means of reciprocal chromosome substitution series. 3. Identify additional genetic resources for improving protein content and nutritional composition by (a) more intensive screening of available genetic resource collections, including induced mutations, and (b) routine

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screening of segregates from intercrosses among wheat varieties, as well as from crosses between wheat species utilizing as necessary special genetic stocks to enhance interspecific and intergenomic chromosome pairing (see Sears, 1974). 4. Determine the location of high protein and high lysine components in promising genetic stocks by milling and other kernel fractionation studies. 5. Investigate the detailed protein and amino acid composition of the proteins in the higher protein and higher lysine sources, especially of induced mutants, aneuploids, and substitutions (since these genetic stocks may be more nearly isogenic) to estimate the potential for shifts in the amounts of high-lysine components in the albumin-globulin and glutenin proteins of wheat. 6. Conduct classical genetic studies of possible high-protein and highlysine sources to distinguish genetic differences, recover transgressive segregates, and determine the protein composition in endosperms of hybrid genotypes including recombinants, especially to determine whether separate genes control the distribution of protein and lysine fractions contained in the aleurone, subaleurone, and interior endosperm cells. 7. Increase the use by wheat breeders of identified high-protein and higher-lysine resource materials to combine higher protein and lysine composition into varieties with acceptable processing characteristics. 8. Encourage breeding and biochemical genetic studies to increase NRA of wheats, identify sources of genes controlling NRA and relationships t o specific proteins. 9. Investigate further the role of the cytoplasm us nuclear components in the synthesis of wheat endosperm proteins. Several different cytoplasm sources have been incorporated in a t least two varieties of HRS wheat and one of durum, and many lines isocytoplasmic for T. timopheevi L. us T. aestivum L. cytoplasm, have been developed in hybrid wheat breeding programs. Investigations of these materials should include (a) the protein composition, solubility groups, and amino acid distribution, including analyses of components within solubility groups, and (b) the number and characteristics of ribosomal proteins. Several kinds of investigations utilizing the various chromosome substitution series and the Chinese Spring aneuploids should be done to provide further background for mutation and other genetic experiments. Among these are: 1. Analyze the amino acid composition and protein fractions of all available relatively stable aneuploid stocks, including tetrasomics, nullitetrasomics, ditelosomics, chromosome addition and untested substitution series. 2. Study the number of 80 S ribosomal proteins associated with the

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above stocks to assess the roles of the nucleus us the cytoplasm in protein synthesis, and to identify specific genetically controlled relationships. 3. Synthesize additional tools for investigating the role of the cytoplasm in the genetic control of biosynthetic processes, e.g., the transfer of Chinese Spring and at least one other contrasting use-quality “base” variety (possibly Cheyenne, because of existing substitutions, etc.) into a different cytoplasm; transfer monosomic series in Cheyenne to this different cytoplasm and into the original Cheyenne cytoplasm (the series now has Chinese Spring cytoplasm). The selection of “foreign” cytoplasm(s) for this purpose might be based on analyses of existing hexaploid and tetraploid materials in those cytoplasms. 4. Synthesize “new” tetraploid and hexaploid combinations from diploid forms (including induced mutants) selected for lysine composition. 5. Induce specific mutations using the “monosomic” approach to expand the utility of aneuploid series, while maintaining desired linkages. For example, (a) the induction of spring habit and short straw traits in Cheyenne would adapt this variety for a wider range of research, (b) the inactivation of “inhibitors” of particular processes, as NRA in chromosome groups 2 and 7, may provide useful genetic testers and possibly useful germ plasm for breeding, (c) induction of mutations affecting the structural and/or regulator functions of genes on chromosomes known to control proteins of nutritional and/or processing quality would greatly assist analyses of these traits, and enhance the scientific basis for breeding developments, (d) isolation of a wider base of genetic sources for improved lysine composition would add greatly to the likelihood of success in breeding nutritionally better wheats. 6. Develop efficient methods for inducing specific gene duplications (possibly via acridines) using the “monosomic” approach to induced mutations. 7. Identify high lysine protein components, especially among gliadin and glutenin, then use the various aneuploid series to identify chromosome (9) responsible for their production and regulation; determine the effect of duplicating the chromosome(s), and of induced duplications or deficiencies, as appropriate. 8. Identify and remove by genetic means antagonistic or toxic factors limiting the nutritional utilization of wheat proteins and carbohydrates. XIV. Summary and Conclusions

1. The genetic basis for the control of protein content and composition appears to be more complex in polyploid wheats than in diploid species,

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but changes in a specific gene may produce large and important effects. Only four main parameters (kernel hardness, dough strength, dough stiffness, dough stability) appear to be concerned with wheat quality. The extent of starch damage incurred during milling is a prime factor in pastry flour quality. However, the possible role of organic acids needs investigation. 2. The number and variety of the proteins in the endosperm of polyploid wheat is essentially the sum of those in their diploid progenitors. This offers opportunities rather than limitations for the selection or induction of genetic variations affecting endosperm protein composition. 3. Gene interactions appear to be especially important in polyploid wheats. The basis for some of the interactions, which are concerned with protein synthesis, evidently also involves relations with cytoplasmic organelles. 4. The biochemica1 systems of current wheat varieties may not be as efficient or harmoniously functioning as those theoretically achievable by genetic means. This is indicated from the discovery of genetically controlled inhibitors of the important biosynthetic processes, NRA, and RuDPCase activity. Removal of these inhibitors by genetic means may increase the biosynthetic efficiency of wheats for protein production and yield. 5. The analytical potentialities of aneuploid and related genetic materials, including induced mutants, are yet relatively unexploited. Exploitation of the potentials of these materials in research on wheat quality and nutrition should lead to important new discoveries. 6. Newly developed isocytoplasmic lines in wheats offer unusual opportunities to investigate the roles of the cytoplasm and nuclear systems controlling endosperm protein composition in wheat. 7. Model induced mutation studies conducted using monosomic lines offer the potential for increased efficiency for detecting mutations in specific chromosomes, without any loss of efficiency for the production of mutations in other chromosomes. The efficiency is, however, dependent on easy (morphological) or rapid biochemical distinction of monosomic individuals or expression of the desired trait in the hemizygous condition. 8. Based on existing evidence, experiments to induce mutations (delete inhibitors) , especially of loci affecting NRA and RuDPCase activity, may be warranted to improve varieties in which monosomic series are available (and where the inhibitors still exist). 9. Given the genetic variability in resources for improving the nutritional composition of wheats, breeders can be expected to develop cultivars which are not only high yielding, agronomically successful and widely adapted, but which also produce more and nutritionally better

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protein while retaining or even improving upon those processing characteristics required for making traditional foods.

ACKNOWLEDGMENTS The helpful suggestions generously made by Drs. J. A. Bietz (Cereal Properties Laboratory, USDA-ARS, Peoria, Illinois 61604), J. S. Wall (Cereal Properties Laboratory, USDA-ARS, Peoria, Illinois 616041, Rosalind Morris (Department of Agronomy, University of Nebraska, Lincoln, Nebraska 68503), P. Mattern (Department of Agronomy, University of Nebraska, Lincoln, Nebraska 68503), and D. D. Kasarda (Western Regional Research Center, 800 Buchanan Street, Albany, California 94710) are greatly appreciated. Special thanks are due also to Dr. C. J. Peterson (USDA-ARS Wheat Investigations), and Drs. R. L. Warner and J. A. Vigue (Department of Agronomy and Soils, Washington State University), Dr. P. L. Finney (USDA-AM), Mr. G. L. Rubenthaler (Western Wheat Quality Laboratory, Pullman, Washington), and Dr. W. J. R. Boyd, Visiting Professor in Agronomy and Soils on professional leave from the University of Western Australia, Perth, Australia, for their reviews; and particularly to Mrs. Dorothy Sander for editorial and other suggestions on the manuscript. Sincere appreciation is also expressed to Drs. M. Fried, B. Sigurbjornsson, C. Lamm, A. Micke, and R. Rabson (Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, Vienna) for stimulating this review and for making much of the work possible. Analytical data provided through the cooperation of Ms. Helga Axmann, Drs. S. C. Hsieh, and H. Perschke (IAEA Laboratory, Seibersdorf, Austria) and through the cooperation of Dr. Stephen Muench and Mr. Kurt Jackson (Department of Agronomy and Soils, Pullman, Washington) and Mr. G. Rubenthaler (Technologist in Charge, Western Wheat Quality Laboratory, USDA-ARS, Pullman, Washington) are greatly appreciated. The dedicated secretarial assistance of Ms. Dorothy Larson, Susan Jones, Catherine Smith (Washington State University), and Marilyn Muller (IAEA) during the development and completion of the manuscript is gratefully acknowledged.

REFERENCES Adam, C. A., and Novellie, L. 1975. Acid hydrolases and autolytic properties of protein bodies and spherosomes isolated from ungerminated seeds of Sorghum bicolor (Linn.) Moench. Plant Physiol. 55, 7-11. Adam, C. A., Novellie, L., and Liebenberg, N. V. W. 1976. Biochemical properties and ultrastructure of protein bodies isolated from selected cereals. Cereal Chem. 53, 1-12. Andrews, T. J., Lorimer, G. H., and Tolbert, N. E. 1973. Ribulose diphosphate oxygenase. I. Synthesis of phosphoglycolate by Fraction I protein of leaves. Biochemistry 12, 11-18. Aragoncillo, C., Rodriquez-Loperena, M. A., Carbonero, P., and Garcia-Olmedo, F. 1975a. Chromosomal control of non-gliadin proteins from the 70% ethanol extract of wheat endosperm. Theor. A p p l . Genet. 45, 322-326. Aragoncillo, C., Rodriquez-Loperena, M. A., Carbonero, P., and Garcia-Olmedo, F.

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1975b. Nigrosine staining of wheat endosperm proteolipid patterns on starch

gels. Anal. Biochem. 63, 603-606.

Aranyi, C., and Hawylewicz, E. J. 1969. Application of scanning electron microscopy to cereal specimens. Cereal Sci. Today 230-233. Archer, M. J. 1972. Relationship between free glutathione content and quality assessment parameters of wheat cultivars (Tm’ticum aestivum L.). J . Sci. Food Agric. 23, 485-491. Amason, T. J. 1956. Maternal inheritance of leaf variegation in hexaploid wheat. Can. J. Bot. 34, 801-804. Aslam, M., Huffaker, R. C., and Travis, R. L. 1973. The interaction of respiration and photosynthesis in induction of nitrate reductase activity. Plant Physiol. 52, 137-141. Auricchio, S., De Viria, B., Carta De Angelis, L., and Silano, V. 1974. Alphaamylase protein inhibitors from wheat in coeliac disease. Lancet 1, 98. Avila, A., and Favret, E. A. 1966. Inheritance of gluten quality in wheat. Proc. Int. Wheat Genet. Symp., Znd, 1963 Hereditas, Suppl. 2,55-61. Avivi, L., Efron, Y., and Feldman, M. 1972. Effect of somatic chromosomal association on the zymograph of alcohol dehydrogenase (ADH) in common wheat. Genetics 71, Suppl. 3, Part 2, 52. Axtell, J. D., Oswalt, D. L., Mertz, E. T., Pickett, R. C., Jambunathan, R., and Srinivasan, G. 1974. Components of nutritional quality in grain sorghum. In “High-Quality Protein Maize,” pp. 374-385. Dowden, Hutchinson and Ross, Stroudsburg, Pennsylvania. Balla, L., and Gaspar, L. 1975. Some results and problems in wheat breeding for higher protein content and improved quality a t Martonvbsbr. Proc. Int. Winter Wheat C o n f , 2nd, 1976 pp. 367-371. Balls, A. K., Hale, W. S., and Harris, T. H. 1942. A crystalline protein extracted from a lipoprotein of wheat flour. Cereal Chem. 19,279. Barber, H. N., Driscoll, C. J., and Vickery, R. S. 1968a. Enzymic markers for wheat and rye chromosomes. Proc. Znt. Wheat Genet. Symp., 3rd, 1968 pp. 116-121. Barber, H. N., Driscoll, C. J., Long, P. M., and Vickery, R. S. 196813. Protein genetics of wheat and homoeologous relationships of chromosomes. Nature (London) 218, 450-452. Barlow, K. K., Simmonds, D. H., and Kenrick, K. G. 1973a. The localization of water soluble proteins in wheat endosperm as revealed by fluorescent antibody techniques. Experientia 29, 229-231. Barlow, K. K., Buttrose, M. S., Simmonds, D. H., and Vesk, M. 1973b. The nature of the starch-protein interface in wheat endosperm. Cereal Chem. 50, 443454. Barlow, K. K., Lee, J. W., and Vesk, M. 1974. Morphological development of storage protein bodies in wheat. In “Mechanisms of Regulation of Plant Growth” (R. L. Bielleski, A. R. Ferguson, and M. M. Cresswell, eds.), pp. 793797. Royal Society of New Zealand, Wellington. Bean, M. M., Keagy, P. M., Fullington, J. G., Jones, F. T., and Mecham, D. K. 1974a. Dried Japanese noodles. I. Properties of laboratory prepared noodle doughs from sound and damaged wheat flours. Cereal Chem. 51, 416-427. Bean, M. M., Nimmo, C. C., Fullington, J. G., Keagy, P. M., and Mecham, D. K. 1974b. Dried Japanese noodles. 11. Effect of amylase, protease, salts and p H on noodle doughs. Cereal Chem. 51,427433. Beck, J. C.,Parker, J. H., Balcavage, W. X., and Matson, J. R. 1971. Mendelian

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genes affecting development and function of yeast mitochondria. In “Autonomy and Biogenesis of Mitochondria and Chloroplasts” (N. K. Boardman, A. W. Linnane, and R. M. Smillie, eds.), pp. 194-204. North Holland Publ., Amsterdam. Beckwith, A. C., Nielsen, H. C., Wall, J. S., and Huebner, F. R. 1966. Isolation and characterization of a high-molecular weight protein from wheat gliadin. Cereal Chem. 43, 14-28. Bedetti, C., Bozzini, A., Silano, V., and Vitoazi, L. 1974. Amylase protein inhibitors and the role of AegiEops species in polyploid wheat speciation. Biochim. Biophys. Acta 360, 299307. Bennett, M. D., and Smith, J. B. 1973. Genotypic, nucleotypic and environmental effects on meiotic time in wheat. Proc. Int. Wheat Genet. Symp., 4th, 1979 pp. 637-644. Bergman, J. W., and Maan, S. S. 1973. Genetic control of isozymes in wheat rye addition lines with rye or wheat cytoplasm. Proc. Int. Wheat Genet. Symp., 4th, 1979 pp. 329-335. Bergman, J. W., and Williams, N. D. 1972. Isozyme variants of esterase and malate dehydrogenase among wheat aneuploids. Agron. Abstr. p. 23 (by permission). Bernardin, J. E. 1974. Wheat protein fibrils and their relationship to dough properties. In “Proceedings of the 12th Western Wheat Worker’s Conference” (W.E. Kronstad and F. H. McNeal, eds.), pp. 15-16. Agricultural Research Service, USDA Publ. Boseman, Montana (by permission). Bernardin, J. E. 1975. The rheology of concentrated gliadin solutions. Cereal Chem. 52, 136r-145r. Bernardin, J. E., and Kasarda, D. D. 1973a. Hydrated protein fibrils from wheat endosperm. Cereal Chem. 50,829-536. Bernardin, J. E., and Kasarda, D. D. 197313. The microstructure of wheat protein fibrils. Cereal Chem. 50,735-745. Betschart, A. A., Saunders, R. M., Bean, M. M., and Kohler, G. 0. 1975. Effects of processing on the baking quality of wet alkaline process wheat protein concentrate. Cereal Chem. 52, 812-822. Bhatia, C. R.. 1968. Electrophoresis of analogous enzymes in Triticinae. Proc. Znt. Wheat Genet. Symp., Srd, 1968 pp. 111-115. Bhatia, C. R., and Rabson, R. 1976. Bioenergetic considerations in cereal breeding for protein improvement. Science 194, 1418-1421. Bietz, J. A., and Rothfus, J. A. 1970. Comparison of peptides from wheat gliadin and glutenin. Cereal Chem. 47, 381392. Bietz, J. A., and Rothfus, J. A. 1971. Differences in amino acid sequences of gliadin and glutenin. Cereal Chem. 48,677-690. Bietz, J. A,, and Wall, J. S. 1972. Wheat gluten subunits: Molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cereal Chem. 49, 418-430. Bietr, J. A., and Wall, J. S. 1973a. Glutenin variability among wheats with known genetic differences. Cereal Sci. Today IS, 301 (Abstr. No. 136). Bietr, J. A., and Wall, J. S. 1973b. Isolation and characterization of gliadin-like subunits from glutenin. Cereal Chem. 50, 537447. Bietz, J. A., and Wall, J. S. 1975. The effect of various extractants on the subunit composition and associations of wheat glutenins. Cereal Chem. 52, 145-155. Bietz, J. A., Huebner, R. F., and Wall, J. S. 1973. Glutenin-the strength protein of wheat flour. Baker’s Dig.47, 26-31, 34-35, and 67. Bietz, J. A., Shepherd, K. W., and Wall, J. S. 1975. Single-kernel analysis of glutenin: Use in wheat genetics and breeding. Cereal Chem. 52, 513-532.

GENETIC CONTROL OF PROTEINS I N WHEAT

549

Bloksma, A. H. 1971. Rheology and chemistry of dough. In “WheatChemistry and Technology” (Y. Pomeranz, ed.), 2nd ed., pp. 523-584. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Bloksma, A. H. 1972a. The role of thiol and disulphide groups in dough rheology. Ztz “Working and Discussion Meeting, 7th Meeting of the International Association for Cereal Chemists (ICC), June 8-10, 1972” (ICC Secretariat, ed.), pp. 100-107. Int. Assoc. Cereal Chem., Vienna. Bloksma, A. H. 197213. The relation between the thiol and disulfide contents of dough and its rheological properties. Cereal Chem. 49, 104-118. Bloksma, A. H. 1975. Thiol and disulfide groups in dough rheology. Cereal Chem. 52, 170r-183r. Booth, M. R., and Ewart, J. A. D. 1969. Studies on four components of wheat gliadins. Biochim. Biophys. Acta 181, 226-233. Booth, M. R., and Ewart, J. A. D. 1970. Relationship between wheat proteins. J. Sci. Food Agric. 21, 187-192. Boulter, D., Ellis, R. J., and Yarwood, A. 1972. Biochemistry of protein synthesis in plants. Biol. R e v . Cambridge Philos. SOC.47, 113-175. Bourque, D. P., and Wildman, S. G. 1972. Evidence that nuclear genes code for several chloroplast ribosomal proteins. Biochem. Biophys. Res. Commun. 50, 532-537.

Bowden, W. M. 1959. The taxonomy and nomenclature of the wheats, barley, and ryes and their wild relatives. Can. J. Bol. 37, 657-684. Bowes, G., and Ogren, W. L. 1972. Oxygen inhibition and other properties of soybean ribulose 1,5-diphosphate carboxylase. J. Biol. Chem. 747, 2171-2176. Bowes, G., Ogren, W. L., and Hageman, R. H. 1971. Phosphoglycolate production catalysed by ribulose diphosphate carboxylase. Biochem. Biophys. Res. Commun. 45, 716-722.

Boyd, W. J. R., and Lee, J. W. 1967. The control of wheat gluten synthesis a t the genome and chromosome levels. Experientia 23, 332-333. Boyd, W. J. R., Lee, J. W., and Wrigley, C. W. 1969. The D-genome and the control of wheat gluten synthesis. Erperientiu 25, 317319. Bozzini, A., and Giacomelli, M. 1973. Endosperm composition of Chinese Spring wheat ditelocentrics. Proc. Znt. Cong. Genet., 13th, 19Y3 Genetics 74, Suppl. 2, Part 2, s29. Bozzini, A., Cantagalli, P., Piazzi, S. E., and Sordi, S. 1970. An immunochemical approach to species relationship in Tn‘ticum and some related species. Theor. Appl. Genet. 40, 300-304. Bozzini, A,, Cantagalli, P., and Piazzi, S. E. 1971. Chromosomal location of the genetic control of two proteins specifically attributed to hexaploid wheat by immunochemical methods. E.W.A.C. Newsl. 3, 16-17 (by permission). Bozzini, A., Cubadda, R., and Quattrucci, E. 1973. Esterases in Triticum and some related species. Proc. Int. Wheat Genet. Symp., 4th, l9Y3 pp. 783-787. Brewer, G. J., Sing, C. F., and Sears, E. R. (1969). Studies of isozyme patterns in nullisomic-tetrasomic combinations of hexaploid wheat. Proc. Natl. Acud. Sci. U S A . 64, 1224-1229. Britten, R. J., and Davidson, E. H. 1969. Gene regulation in higher cells: A theory. Science 165, 349-357. Brohult, S., and Sandgren, E. 1954. Seed proteins. In “The Proteins” (H. Neurath and K. Bailey, eds.), 1st ed., Vol. 2, Part A, pp. 487-512. Academic Press, New York. Brown, L. R. 1975. The world food prospect. Science 190, 1053-1059.

550

CALVIN F. KONZAK

Brunckhorst, K., Lein, K. A., and Schon, W. J. 1974a. Bestimmung des Lysingehaltes und Selektion des Hiproly-Merkmales nach RuTkkreuzung bei Gerste. I. Prufung und Entwicklung analytischer Methoden. Z . Pflanzenzuecht. 73, 269-283. Brunckhorst, K., Robbelen, G., and Zosche, M. 1974b. Bestimmung des Lysingehaltes und Selektion des Hiproly-Merkmals nach Ruckkreuzung bei Gerste. 11. Vererbungstudien und Auslese auf Lysin in ‘Asse’ x ‘Hipro1y’-Kreuaungen. 2. Pflanzenzuecht. 73, 1-12. Busch, R. H., and Maan, S. S. 1974. Possible use of cytoplasmic variability in wheat improvement. Wheat Newsl. 20, 163-166 (by permission). Bushuk, W. 1972. Ultrastructure related to wheat processing and products. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 193-211. Avi Publ., Westport, Connecticut. Bushuk, W., and Wrigley, C. W. 1971. Glutenin in developing wheat grain. Cereal Chem. 48, 448-455. Bushuk, W., and Wrigley, C. W. 1974. Proteins: Composition, structure, and function. In “Wheat: Production and Utilization” (G. E. Inglett, ed.), pp. 119-145. Avi Publ., Westport, Connecticut. Bushuk, W., Briggs, K. G., and Shebeski, L. H. 1969. Protein quantity and quality as factors in the evaluation of bread wheats. Can. J. Plant Sci. 49, 113-122. Butcher, J. 1975. Effect of aleurone cell damages on wheat fibre values as determined by the neutral detergent technique. J. Sci. Food Agric. 26,345-350. Butcher, J., and Stenvert, N. L. 1973a. Conditioning studies on Australian wheat. I. The effect of conditioning on milling behavior. J. Sci. Food Agric. 24, 10551066. Butcher, J., and Stenvert, N. L. 1973b. Conditioning studies on Australian wheat. 111. The role of the rate of water penetration into the wheat grain. J. Sci: Food Agric. 24, 1077-1084. Buttrose, M. S. 1963. Ultrastructure of the developing wheat endosperm. Aust. J. Biol. Sci. 16, 305-316. Caepoiu, N., Euststiu, N., Ittu, Gh., and Shlescu, N. N. 1975. Wheat breeding for improved nutritional quality in Romania. Proc. Int. Winter Wheat Conf., 2nd, 1976 pp. 362-366. Cain, R. F., Schulta, H. W., and Peters, L. J. 1975. “Nutritional Evaluation of Wheat,” 1st Annu. Rep. National Wheat Institute, Department of Food Science and Technology, Oregon State University, Corvallis. Carbonero, P., and Garcia-Olmedo, F. 1969. Purothionins in Aegilops Triticum spp. Ezperientiu 25, 1110. Cataldo, D. A., Schrader, L. E., Peterson, D. M., and Smith, D. 1975. Factors affecting seed protein concentration in oats. I. Metabolism and distribution of N and carbohydrate in two cultivars that differ in groat protein concentration. Crop Sci. 15, 19-23. Chamberlain, N., Collins, T. H.. and Elton, G. A. H. 1962. The chorleywood bread process. Baker‘s Dig. 36, 52-53. Chan, P. H., Sakano, K., Singh, S., and Wildman, S. G. 1972. Crystalline Fraction I protein : Preparation in large yield. Science 176, 1145-1146. Chapman, V., Mittler, T. E., and Riley, R. 1976. Equivalence of the A genome of bread wheat and that of T . urartu. Genet. Res. 27, 69-76. Charbonnier, L. 1973. Studies of the all-soluble proteins of wheat flour. Biochimie 55, 1217-1225.

GENETIC CONTROL OF PROTEINS IN WHEAT

551

Charbonnier, L. 1974. Isolation and characterization of omega-gliadin fractions. Biochim. Biophys. Acta 359, 142-151. Chen, C. H., and Bushuk, W. 1970a. Nature of proteins in Triticale and its parental species. I. Solubility characteristics and amino acid composition of endosperm proteins. Can. J. Plant Sci. 50, 9-14. Chen, C. H., and Bushuk, W. 1970b. Nature of proteins in TriticaZe and its parental species. 11. Gel filtration and disc electrophoresis results. Can. J. Plant Sci. 50, 15-24. Chen, K., Gray, J. C., and Wildman, S. G. 1975. Fraction I protein and the origin of polyploid wheats. Science 190, 1304-1306. Chmeleva, Z. V., and Medredev, A. M. 1973. Effect of fertilizers on the accumulation of protein and lysine in cultivars of spring wheat (In Russian). Bull. Appl. Bot., Genet. Plant Breed. 52,242-248 (Chem. Abstr. 8 1 2 4 ~ ) . Cole, E. W., Ng, H., and Mecham, D. K. 1973. Further studies on the fractionation and characterization of the gel proteins of wheat flour by column chromatography. Cereal Chem. 50, 336345. Cole, E. W., Ng, H., and Mecham, D. K. 1976. Some chemical and physical properties of the mercuric chloride-solubilized gel protein from different wheat varieties. Cereal Chem. 53, 250-257. Concon, J. M. 1972. Determination of lysine in cereal proteins. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 292-311. Avi Publ., Westport, Connecticut. Crosby, A. R. 1957. Nucleolar activity of lagging chromosomes in wheat. A m . J. Bot. 44, 813-822. Crosby Longwell, A., and Svihla, G. 1960. Specific chromosomal control of the nucleolus and of the cytoplasm in wheat. Exp. Cell Res. 20,294-312. Croy, L. I., and Hageman, R. H. 1970. Relationship of nitrate reductase activity to grain protein production in wheat. Crop Sci. 10, 280-285. Crozet, N., Godon, B., Petit, L., and Guilbot, A. 1974. Submicroscopic structure of wheat flour and gluten lipoprotein components. Cereal Chem. 51, 288-299. Cubadda, R. 1975. Chromosome location of genes controlling the synthesis of some soluble proteins in T. durum and T . aestivum. In “Genetics and Breeding of Durum Wheat” (G. T. Scarascia-Mugnozza, ed.), pp. 653-658. Library of the Faculty of Agriculture, Bari, and National Institute for Nutrition, Rome. Cubadda, R., and Quattrucci, E. 1974. Separation by gel electrofocusing and characterization of wheat esterases. J. Sci. Food Agric. 25, 417-422. Cubadda, R., Quattrucci, E., and Virtuoso, G. 1973. Una nuova tecnica per la separazione de forme multiple di esterasi vegetali mediante gel-focalirzazione ionica (In Italian). Boll. SOC.I t . B i d . Sper. 49, 328-332 (Chem. Abstr. 116382~). Cubadda, R., Bozzini, A,, and Quattrucci, E. 1975. Genetic control of esterases in common wheat. Theor. Appl. Genet. 45,29&293. Dahlberg, A. E., Dingman, C. W., and Peacock, A. C. 1969. Electrophoretic characterization of bacterial polyribosomes on agarose-acrylamide composite gels. J. Mol. Biol. 41, 139-147. Dalby, A., and Tsai, C. Y. 1976. Lysine and tryptophan increases during germination of cereal grains. Cereal Chem. 53, 222-226. Dalling, M. J., Tolbert, N. E., and Hageman, R. H. 1972. Intracellular location of nitrate reductase and nitrite reductase. I. Spinach and tobacco leaves. Biochim. Biophys. Acta 283, 505-512. Dalling, M. J., Halloran, G. M., and Wilson, J. H. 1975. The relation between

552

CALVIN F. KONZAK

nitrate reductase activity and grain nitrogen productivity in wheat. Aust. J. Agric. Res. 26, 1-10. D’Appolonia, B. L., ‘Gilles, K. A., Osman, E. M., and Pomeranz, Y. 1971. Carbohydrates. In ‘LWheat-Chemistry and Technology” (Y. Pomeranz, ed.), pp. 301492. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Davis, W. H., Middleton, G. K., and Hebert, T. T. 1961. Inheritance of protein, texture, and yield in wheat. Crop Sci. 1, 235-238. Deckard, E. L., Williams, N. D., and Hammond, J. J. 1975. Nitrate reductase activity of wheat aneuploids. Agron. Abstr. p. 106 (by permission). Dedio, W., Simmonds, D. H., Hill, R. D., and Shealy, H. 1975. Distribution of aamylase in the triticale kernel during development. Can. J . Plant Sci. 55, 29-36. Deponte, R., Parlamenti, R., Petrucci, T., Silano, V., and Tomasi, M. 1976. Albumin a-amylase inhibitor families from wheat flour. Cereal Chem. 53, 805-820. Dexter, J. E., and Dronzek, B. L. 1975a. Protein synthesis in triticale and its durum wheat and rye parents. Cereal Chem. 52,577-586. Dexter, J. E., and Dronzek, B. L 1975b. Note on the amino acid composition of protein fractions from a developing triticale and its rye and durum wheat parents. Cereal Chem. 52, 587-596. Dhaliwal, H. S. 1976. Cytoplasmic relationship between Triticum boeotrium and Triticum urartu. Wheat In!. Serv. 41-42, 1-2 (by permission). Dhaliwal, H. S., and Johnson, B. L. 1976a. Anther morphology and the origin of the tetraploid wheats. Am. J . Bot. 63, 363368. Dhaliwal, H. S., and Johnson, B. L. 1976b. Cross-compatibility of diploids and tetraploids and the origin of the cytoplasm of the tetraploid wheats. Heredity (in press). Dhaliwal, H. S., and Johnson, B. L. 1976c. Chromosomal rearrangement and the origin of the Timopheevi wheats. Can. J . Genet. Cytol. (in press). Dieckert, J. W., and Dieckert, M. C. 1972. The deposition of vacuolar proteins in oilseeds. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 52-85. Avi Publ., Westport, Connecticut. Doekes, G. J. 1968. Comparison of wheat varieties by starch gel electrophoresis of their grain proteins. J. Sci. Food Agric. 10, 169-176. Doekes, G. J. 1973. Inheritance of gliadin composition in bread wheat, Triticum aestivum L. Euphytica 22, 28-33. Doekes, G. J., and Hack, A. 1971. Electrophoresis of grain proteins as an aid in wheat breeding. EUCARPIA Bull. pp. 329-345. Dohan, D. A., Standing, K. G., and Bushuk, W. 1976. A new method of analysis of protein content in grain by proton activation. Cereal Chem. 53, 91-100. Doll, H. 1972. Variation in protein quantity and quality induced in barely by EMS treatment. In “Induced Mutations and Plant Improvement’’ (Proceedings of a Latin American Study Group meeting on Induced Mutations and Plant Improvement organized by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture held in Buenos Aires, 16-20. November, 19701, STI/ PUB/297. IAEA, Vienna. Doll, H. 1973. Inheritance of the high lysine character of a barley mutant. Hereditas 74, 293-294. Doll, H. 1976. Genetic studies of high lysine barley mutants. In “Barley Genetics 111” (H. Gaul, ed.), pp. 542-546. Karl Thiemig, Munich. Donelson, D. H., and Yamazaki, W. T. 1972. Soft wheat flour particle size analysis by integrated sieve and Coulter counter procedures. Cereal Chem. 49, 641-653. Dronzek, B. L., Kaltsikes, P. J., and Bushuk, W. 1970. Eflect of the D genome on

GENETIC CONTROL OF PROTEINS IN WHEAT

553

the protein of three cultivars of hard red spring wheat. Can. J. Plant Sci. 50, 389400. Duffield, R. D., Croy, L. I., and Smith, E. L. 1972. Inheritance of nitrate reductase activity, grain protein, and straw protein in a hard red winter cross. Agron. J. 64, 249-251. DumanoviE, J., Ehrenberg, L., and Denii., M. 1970. Induced variation of protein content and composition in hexaploid wheat. Improv. P h n t Protein Nucl. Tech., Proc. Symp., 1970 STI/PUB/258, pp. 107-120. Dunstone, R. L., and Evans, L. T. 1974. Role of changes in cell size in the evolution of wheat. Aust. J . Plant Physiol. 1, 157-165. DvoFik, J. 1976. The relationship between the genome of Triticum urartu and the A and B genomes of Triticum aestiuum. Can. J . Genet. Cytol. 18, 371-377. Eastin, J. D., Morris, R., Schmidt, J. W., Mattern, P. J., and Johnson, V. A. 1966. Chromosomal association with gliadin proteins in the wheat variety ‘Cheyenne.’ Crop Sci. 7, 674-676. Edgar, K. F., and Draper, S. R. 1975. Physiological aspects of nitrate utilization in a barley variety exhibiting high seed protein content. J . Sci. Food Agric. 26, 1311-1318. Edwards, I. B. 1973. Physiologic and genetic studies of nitrate reductase activity and nitrogen distribution in spring wheat (Triticum aestivum L.) . Ph.D. Thesis, North Dakota State University, Fargo. Edwards, I. B. 1974. Heritability estimates of nitrate reductase activity in spring wheat and the chromosomal location of genes effecting nitrogen reduction. South A f r . Genet. Congr. 6th, 1974 (Abstr.). Eilrich, G. L., and Hageman, R. H. 1973. Nitrate reductase activity and its relationship to accumulation of vegetative and grain nitrogen in wheat. Crop Sci. 13, 59-66. Elton, G. A. H., and Ewart, J. A. D. 1962. Starch gel electrophoresis of cereal proteins. J. Sci. Food Agric. 13, 62-72 Endo, T. R., and Tsunewaki, K. 1975. Genetic diversity of the cytoplasm in Triticum and Aegilops. I. On the origin of the cytoplasm of Aegilops triuncialis L. Seiken Ziho 25-26, 55-66. Eslick, R . F., and Hockett, E. A. 1976. A second locus for high lysine barley. I n “Barley Genetics 111”(H. Gaul, ed.), p. 634. Karl Thiemig, Munich. Evers, A. D. 1970. Development of the endosperm of wheat. Ann. Bot. (London) CN.S.1 34, 547-555. Everson, E. H., and Seeborg, E. F. 1958. The heritability of milling quality in wheat as measured by the separation of the bran and endosperm. Agron. J. 50, 511513. Ewart, J. A. D. 1966a. Fingerprinting of glutenin and gliadin. J. Sci. Food Agric. 17, 30-33. Ewart, J. A. D. 1966b. Cereal proteins: Immunological studies. J . Sci. Food Agric. 17, 279-284. Ewart, J. A. D. 1967a. Amino acid analyses of glutenins and gliadins. J. Sci. Food Agric. IS, 111-116. Ewart, J. A. D. 196713. Amino acid analyses of cereal flour proteins. J . Sci. Food Agric. 18, 548-552. Ewart, J. A. D. 1968. Fractional extraction of cereal flour proteins. J . Sci. Food Agric. 19, 241-245. Ewart, J. A. D. 1969. Isolation and characterization of a wheat albumin. J. Sci. Food Agric. 20, 730-733. Ewart, J. A. D. 1972a. Recent research and dough viscoelasticity. In “Working

554

CALVIN F. KONZAK

and Discussion Meeting, 7th Meeting of the International Association for Cereal Chemists (ICC), June 8-10, 1972” (ICC Secretariat, ed.), pp. 108-112. Int. Assoc. Cereal Chem., Vienna. Ewart, J. A. D. 1972b. Further studies on SS bonds in cereal glutelins. J . Sci. Food Agric. 23, 567-579. Ewart, J. A. D. 1972c. A modified hypothesis for the structure and rheology of glutelins. J . Sci. Food Agric. 23, 687-699. Ewart, J. A. D. 1973. Sodium dodecyl sulphate electrophoresis of wheat gliadins. J. Sci. Food Agric. 24, 685-689. Ewart, J. A. D. 1975. Isolation of a Cappelle-Desprez gliadin. J . Sci. Food Agric. 26, 1021-1025. Farrand, E. A. 1964. Flour properties in relation to modern bread processes in the United Kingdom, with special reference to alpha-amylase and starch damage. Cereal Chem. 41, 99-111. Farrand, E. A. 1969. Starch damage and alpha-amylase as bases for mathematical model relating to flour water absorption. Cereal Chem. 46, 103-116. Farrand, E. A. 1972. Controlled levels of starch damage in a commerical United Kingdom bread flour and effects on absorption, sedimentation value, and loaf quality. Cereal Chem. 49, 479-488. Farrand, E. A. 1974a. Study of relationships between wheat flour protein contents of two United Kingdom varieties and derived flour protein contents a t varying extraction rates. I. Studies on an experimental commercial mill and a laboratory Buhler mill. Cereal Chem. 51, 56-66. Farrand, E. A. 1974b. Study of relationships between wheat protein contents of two United Kingdom varieties and derived flour protein contents at varying extraction rates. 11. Studies by hand dissection of individual grains. Cereal Chem. 51, 66-74. Favret, E. A., Solari, R., Manghers, L., and Avila, A. 1969. Genetic control of the qualitative and quantitative production of endosperm proteins in wheat and barley. New Approaches Breed. Improved Plant Protein, Proc. Panel Meet., 1968 pp. 87-107. Favret, E. A., Manghers, L., Solari, R., A d a , A., and Monesiglio, J. C. 1970. Gene control of protein production in cereal seeds. Improv. Plant Nucl. Techn., Proc. Sump., 1970. pp. 87-97. Feillet, P., and Bourdet, A . 1967. Composition protkique et caractkristiques gbnktique des blks (In French, Engl. Summary). Bull. SOC.Chim. Biol. 49, 1273-1283. Feillet, P., and Nimmo, C. C. 1970. Soluble proteins in wheat. 111. Isolation and characterization of two albumins, ALB 13A and ALB 13B, from flour. Cereal Chem. 47, 447464, Fellers, D. A., Sinkey, V., Shepherd, A. D., and Pence, J. W. 1966. Solubilization and recovery of protein from wheat millfeeds. Cereal Chem. 43, 1-13. Fernandez de Caleya, R., Gonzalez-Pascual, B., Garcia-Olmedo, F., and Carbonero, P. 1972. Susceptibility of phytopathogenic bacteria to wheat purothionins in vitro Appl. Microbial. 23, 998-1000. Fernandez de Caleya, R., Hernandez-Lucas, C., Carbonero, P., and GarciaOlmedo, F. 1976. Gene expression in allopolyploids. Genetic control of lipopurothionins of wheat. Genetics 83, 687-699. Fincher, G. B., Sawyer, W. H., and Stone, B. A. 1974. Chemical and physical properties of an arahinogalactan-peptide from wheat endosperm. Biochem. J . 139, 535-545.

GENETIC CONTROL OF PROTEINS I N WHEAT

555

Rnney, K. F. 1943. Fractionating and reconstituting techniques as tools in wheat flour research. Cereal Chem. 20, 381-396. Finney, K. F. 1971. Fractionating and reconstituting techniques to relate functional (breadmaking) to biochemical properties of wheat flour components. Cereal Sci. Today 16, 342-356. Finnry, K. F. 1975. A sugar-free formula for regular and high protein breads. Baker’s Dig. 49, 18-22. Finney, K. F., and Fryer, H. C. 1958. Effect on loaf volume of high-temperature during the fruiting period of wheat. Agron. J . 50, 28-34. Finney, K. F., Shogren, M. D., Pomeranz, Y., and Bolte, L. C. 1972. Cereal malts in breadmaking. Baker’s Dig. 46, 36-38 and 55. Finney, P. L., Magoffin, C. D., Hoseney, R. C., and Finney, K. F. 1976. Short-time baking systems. I. Interdependence of yeast concentration, fermentation time, proof time, and oxidation requirement. Cereal Chem. 53, 12b134. Finney, P. L., Rubenthaler, G. L., Jeffers, H. C., and Anderson, P. D. 1977. Shorttime baking systems. 111. Malt independence in a sugar-free formula. Cereal Chem. (in press). Fish, W. W., and Abbott, D. C. 1969. Isolation and characterization of a watersoluble wheat protein. J. Sci. Food Agric. 20, 723-730. Fisher, N., Redman, D. G., and Elton, G. A. H. 1968. Fractionation and characterization of purothionin. Cereal Chem. 45, 48. Flavell, R. B., and Smith, D. B. 1974a. Variation in nucleolar organizer rRNA gene multiplicity in wheat and rye. Chromosoma 47,327334. Flavell, R. B., and Smith D. B. 197413. The role of homoeologous group 1 chromosomes in the control of rRNA genes in wheat. Biochem. Genet. 12, 271-279. Fortini, S., Giorgi, B., Giacomelli, M., Mannino, P., and Cordischi, M. 1973. Modification of RuDP and PEP carboxylase activities, protein content and leaf weight in ditelocentric lines of bread wheat in Chinese Spring. Ann. Ist. Sper. Cerealicolt. 4, 133-144. Gale, M. D., and Spencer, D. 1974. Location of genes controlling enzyme production in endosperm. E.WA.C. Newsl. 4, 16-17 (by permission). Garcia-Olmedo, F., and Carbonero, P. 1970. Homoeologous protein synthesis controlled by homoeologous chromosomes in wheat. Phytochemktry 9, 1495-1497. Garcia-Olmedo, F., Sotelo, I., and Garcia-Faure, R. 1968. Identification of Triticum aestivum products in alimentary paste. IV. Petroleum ether soluble lipoproteins. A n . Inst. Nac. Invest. Agron. (Spain) 17, 433-443. Garcia-Olmedo, F., Carbonero, P., Aragoncillo, C., Fernandez de Caleya, R., and Torres, J. V. 1975. Expression of homoeologous molecular systems in wheat alloploids. In “Heterosis in Plant Breeding. Section I, Principles of Heterosis” Proc. V I I Eucarpia Congress, Budapest, 1974 (in press). Garen, A. 1968. Sense and nonsense in the genetic code. Science 160, 149-159. Gavrilyuk, I. P., Gubareva, N. K., and Konarev, V. G. 1973. Methods for extraction, seperation into fractions and identification of proteins used for genome analysis of cultivated plants. (In Russian, Engl. Summary) Bull. Appl. Bat., Genet. Plant Breed. 52, 249-281 (Chem. Abstr. 40116m). Gibson, R. A., and Paleg, L. G. 1975. Further experiments on the a-amylase containing lysosomes of wheat aleurone cells. Aust. J. Physiol. 2, 4149. Glasziou, K . T. 1969. Control of enzyme formation and inactivation in plants. Annu. R e v . Plant Physiol. 20, 63-88.

556

CALVIN F. KONZAK

Goering, K. J., Fritts, D. H., and Allen, K. G. D. 1974. A comparison of the 10sa of birefrigence with the percent gelatinization and viscosity on potato, wheat, rice, corn, cow cockle, and several barley starches. Cereal Chem. 51, 764-771. Goering, K. J., Jackson, L. L., and Dehaas, B. W. 1975. Effect of some nonstarch components in corn and barley starch granules on the viscosity of heated starch-water suspensions. Cereal Chem. 52, 493-500. Graham, J. S. D., Jennings, A. C., Morton, R. K., Palk, B. A., and Raison, J. K. 1962. Protein bodies and protein synthesis in developing wheat endosperm. Nature (London) 196, 967-969. Graham, J. S. D., Morton, R. K., and Raison, J. K. 1963. Isolation and characterization of protein bodies from developing wheat endosperm. Aust. J. Biol. Sci. 16, 375-384. Grant, D.R. 1974. Studies of the role of ascorbic acid in chemical dough development. I. Reaction of ascorbic acid with flour-water suspensions. Cereal Chem. 51, 684-692. Green, M. R.,and Pastewka, J. V. 1974s. Simultaneous differential staining by a cationic carbocyanine dye of nucleic acids, proteins, and conjugated proteins. I. Phosphoproteins. J . Histochem. Cytochem. 22,767-773. Green, M. R., and Pastewka, J. V. 1974b. Simultaneous differential staining by a cationic carbocyanine dye of nucleic acids, proteins, and conjugated proteins. 11. Carbohydrate and sulfated carbohydrate-containing proteins. J. Histochem. Cytochem. 22, 774-781. Green, M. R.,Pastewka, J. V., and Goerttler, K. L. 1973. Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal. Biochem. 56, 43. Greenaway, W. T., and Watson, C. A. 1975. The gluto-matic for semiautomatic determination of wet and dry gluten content of wheat flour. Cereal Chem. 52, 367473. Greenwood, C. T. 1976. Starch. I n “Advances in Cereal Science and Technology” (Y. Pomeranz, ed.), pp. 119-157. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Greenwood, C. T., and Ewart, J. A. D. 1975. Hypothesis for the structure of glutenin in relation to rheological properties of gluten and dough. Cereal Chern. 52(3, 11) 146r-153r. Grimes, G. W., Mahler, H. R., and Perlman, P. S. 1974. Nuclear gene dosage effects on mitochondria1 mass and DNA. J . Cell Biol. 61, 565-574. Gunthardt, H.,and McGinnis, J. 1957. Effect of nitrogen fertilization on amino acids in whole wheat. J. Nutr. 61,167-170. Haag, D., Tschahargane, C., and Goerttler, K. L. 1971. Simultaneous differential staining of nucleic acids and proteins in histological tissues by means of J-band effect. Histochemie 26, 190-193. Hall, O., Johnson, B. L., and Olered, R. 1966. Evaluation of genome relationships in wheat from their protein homologies, Proc. Int. Wheat Genet. Symp., Srd, 1963 pp. 47-54. Halloran, G. M. 1975.Genetic analysis of grain protein percentage in wheat. Theor. Appl. Genet. 46,74-86. Hamauzu, Z., Toyomasu, T., and Yonezawa, I?. 1974. Molecular weight determination of gliadin fractions in gel filtration by SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium. Agric. Biol. Chem. 38, 2445-2450. Hammes, G. G., and Wu,C. W. 1971. Regulation of enzyme activity. Science 172, 1205-1211.

GENETIC CONTROL OF PROTEINS IN WHEAT

557

Hanif, M., and Langer, R. H. M. 1972. The vascular system of the spikelet in wheat (Triticum aestivum). Ann. Bat. 36, 721-728. Harlan, J. R. 1970. Evolution of cultivated plants. I n “Genetic Resources in Plants: Their Exploration and Conservation” (0.H. Frankel and E. Bennett, eds.), Int. Biol. Program, pp. 19-32. Davis, Philadelphia, Pennsylvania. Harlan, J. R. 1975. “Crops and Man.” American Society of Agronomy, Crop Science Society of America, Madison, Wisconsin. Harlan, J. R., and Zohary, D. 1966. Distribution of wild wheats and barley. Science 153, 1074-1080.

Harris, D. R. 1967. New light on plant domestication and the origins of agriculture. A review. Geogr. Rev. 57, 90-107. Hart, G. E. 1970. Evidence for triplicate genes for alcohol dehydrogenase in hexaploid wheat. Proc. Natl. Acad. Sci. U S . A . 66, 1136-1141. Hart, G. E. 1971. Alcohol dehydrogenase isozymes of Triticum: Dissociation and recombination of subunits. Mol. Gen. Genet. 111,61-65. Hart, G. E. 1973a. Homoeologous gene evolution in hexaploid wheat. Proc. Int. Wheat Genet. Symp., 4th, f973 pp. 805410. Hart, G. E. 1973b. Acid phosphatase of hexaploid wheat: linkage to homoeologous chromosomes of structural genes for six isozymes. Proc. Int. Cong. Genet. fSth Genet. 74 Suppl. 2, s110. Hart, G. E. 1975. Glutamate oxaloacetate transaminase isozymes of Triticum: Evidence for multiple systems of triplicate structural genes in hexaploid wheat. In “Isozymes, 111. Developmental Biology” ( C . L. Markert, ed.), pp. 637-657. Academic Press, New York. Hart, G . E., and Langston, P. 1975. Evidence for triplicate endopeptidase and lipoxygenase structural gene sets in hexaploid wheat. Isozyme Bull. 8, 12. Hart, G. E., McMillin, D. E., and Sears, E. R. 1976. Determination of the chromosomal location of a glutamate oxaloacetate transaminase structural gene using Triticum-Agropyron translocations. Genetics 83, 49-61. Haunold, A., Johnson, V. A., and Schmidt, J. W. 1962. Genetic measurements of protein in the grain of Triticum aestivum L. Agron. J . 54, 203-206. Hehn, E. R., and Barmore, M. A. 1965. Breeding wheat for quality. Adv. Agron. 17, 85-114.

Henningsen, K. W., Baynton, J. E., von Wettstein, D., and Boardman, N. K. 1973. Nuclear genes controlling chloroplast development in barley. Biochem. Gene Expression Higher Org., Proc. Symp., 1972 pp, 457-478. Hernandez, H. H., Walsh, D. E., and Bauer, A. 1974. Nitrate reductase of wheat: Its relation to nitrogen fertilization. Cereal Chem. 51, 330-336. Hernandez-Lucas, C., Fernandez de Caleya, R., and Carbonero, P. 1974. Inhibition of brewer’s yeasts by wheat purothionins. Appl. Microbial. 28, 165-168. Hertel, W. 1974. Biosynthesis of wheat protcins (In German). Getreide, Mehl, Brat 28, 10-12 (Chem. Ahstr. 47444K). Hickey, C. A., Murphy, E. L., and Calloway, D. H. 1972. Intestinal-gas production following ingestion of commercial wheat cereals and milling fractions. Cereal Chem. 49, 276-282. Hillel, J., and Simchen, G. 1973. Mating systems, population structure and chromosome behavior in Triticum speltoides and T . longissimum. Proc. Int. Wheat Genet. Symp., 4th, 1973 pp. 521-526. Hochstrasser, K., Werle, E., Schwarz, S., and Siegelmann, R. 1969. Uber pflnnzliclie Proteaseinhibitoren, 111. Reindarstellung der Trypsininhibitoren aus Keimen

558

CALVIN F. KONZAK

von Weiaen- und Roggensamen, Lokalisierung der aktiven Zentren (In German, Engl. Summary.) Hoppe-Seyler’s Z. Physiol. Chem. 350, 249-254 (Chem. Abstr.

.

74681G)

Hoseney, R. C., and Finney, K. F. 1971. Functional (breadmaking) and biochemical properties of wheat flour components. XI. Review. Baker’s Dig.45, 30-36, 39-40, and 64-67. Hoseney, R. C., Finney, K. F., Bhorgren, M. D., and Pomeranz, Y . 1969a. Functional (breadmaking) and biochemical properties of wheat flour components. 11. Role of water-solubles. Cereal Chem. 46, 117-125. Hoseney, R. C., Finney, K. F., Shogren, M. D., and Pomeranz, Y . 1969b. Functional (breadmaking) and biochemical properties of wheat flour comopnents. 111. Characterization of gluten protein fractions obtained by ultracentrifugation. Cereal Chem. 48, 126135. Hoseney, R. C., Finney, K. F., Shogren, M. D., and Pomeranz, Y . 1969c. Functional (breadmaking) and biochemical properties of wheat flour components. IV. Gluten protein fractionation by solubilizing in 70% ethyl alcohol and in dilute lactic acid. Cereal Chem. 46, 495-502. Hoseney, R. C., Finney, K. F., Pomeranz, Y., and Shogren, M. D. 1969d. Functional (breadmaking) and biochemical properties of wheat flour components. V. Role of total extractable lipids. Cereal Chem. 46, 606-613. Hoseney, R. C., Finney, K. F., and Pomeranz, Y . 1970a. Functional (breadmaking) and biochemical properties of wheat flour components. VI. Gliadin-lipid- glutenin interaction in wheat gluten. Cereal Chem. 47, 135-140. Hoseney, R. C., Pomeranz, Y., and Finney, K. F. 1970b. Functional (breadmaking) and biochemical properties of wheat flour components. VII. Petroleum ethersoluble lipoproteins of wheat flour. Cereal Chem. 47, 153-160. Hoseney, R. C., Finney, K. F., and Shogren M. D. 1972. Functional (breadmaking) and biochemical properties of wheat flour components. IX. Replacing total free lipid with synthetic lipid. Cereal Chem. 49,366371. Hsieh, S. C., and Hsieh, J. S. 1976. Studies on protein and amino acid improvemenls by chromosome manipulation in wheat (In Chinese, Engl. Summary). Nat. Sci. Council Monthly (Taiwan) 10, 44-52. Huebner, F. R. 1970. Comparative studies on glutenins from different classes of wheat. J . Agric. Food Chem. 18,256-259. Huebner, F. R., and Wall, J. S. 1974. Wheat glutenin subunits. I. preparative separation by gel filtration and ion-exchange chromatography. Cereal Chem. 51, 228-240. Huebner, F. R., and Wall, J. S. 1975. Fractionation and quantitative differences of glutenin from wheat varieties varying in baking quality. Cereal Chem. (in press). Huebner, F. R., and Wall J. S. 1976. Fractionation and quantitative differences of glutenin from wheat varieties varying in baking quality. Cereal Chem. 53, 258469. Huebner, F. R., Donaldson, G. L., and Wall, J. S. 1974. Wheat glutenin subunits. 11. Compositional differences. Cereal Chem. 51, 240-249. Huffaker, R. C., and Peterson, L. W. 1974. Protein turnover in plants and possible means of its regulation. Annu. Rev. Plant Physiol. 25,363392. Inamine, E. S., Noble, E. G., and Mecham, D. K. 1967. Solubilization and fractionation of wheat flour proteins insoluble in dilute acetic acid. Cereal Chem. 44, 143-151. Inglett, G. E., and Anderson, R. A. 1974. Flour milling. In “Wheat: Production

GENETIC CONTROL OF PROTEINS IN WHEAT

559

and Utilization” (G. E. Inglett, ed.), pp. 186-198. Avi Publ., Westport, Connecticut. Ingversen, J. 1975. Structure and composition of protein bodies from wild-type and high-lysine barley endosperm. Hereditas 81, 69-76. Ingversen, J. 1976. Ultrastructural and biochemical differences between the protein bodies from wild type and high lysine barley seeds. In “Barley Genetics 111” (H. Gaul, ed.), p. 547. Karl Thiemig, Munich. Ingversen, J., and KGie, B. 1973. Lysine-rich proteins in high lysine Hordeum uulgare grain. Phytochemistry 12, 1107-1111. Ingversen, J., KZie, B., and Doll, H. 1973. Induced seed protein mutant of barley. Ezperientiu 29, 1151-1152. Irani, B. N., and Bhatia, C. R. 1972. Chromosomal location of alcohol dehydrogenase gene(s) in rye, using wheat-rye addition lines. Genetica 43, 195-200. Irvine, G. N. 1971. Durum wheat and paste products. In “Wheat: Chemistry and Technology” ( Y . Pomeranz, ed.), pp. 777-798. Am. Assoc. Cereal Chem. St. Paul, Minnesota. Islam, A. K. M. R., Shepherd, K. W., and Sparrow, D. H. B. 1976. Addition of individual barley chromosomes to wheat. In “Barley Genetics 111” (H. Gaul, ed.), pp. 260-270. Karl Thiemig, Munich. Jaaska, V. 1974. The origin of tetraploid wheats based on electrophoretic studies of enzymes. Eesti NSV Tead. Akad. Toim., Biol. 23,201-220. Jacob, F., and Monod, J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 31tL356. Jagannathan, D. R., and Bhatia, C. R. 1972. Effect of rye chromosome 2 substitution on endosperm proteins of wheat. Theor. Appl. Genet. 42,89-92. Jahn-Deesbach, W., and Jurgens, U. 1973. Der Einfluss variierter Stickstoffgahen auf die Ertragsmorphologie und die Stickstoffeinlagerung bei Sommerweizen in einem Gef&sversuch. IV. Stickstoffeinlagerung in die verschiedenen Proteinfraktionen des Ganzkornes und der einzelnen Mahlfraktionen. (In German, Engl. Summary) 2. Acker- PfEanzenbau 138, 276-286 (Chem. Abstr. 12366e). Jain, H. K., Singh, M. P., and Raut, R. N. 1968a. Genomic differentiation and RNA synthesis in wheat. Proc. Znt. Wheat Genet. Symp., 3rd, 1968 pp. 99-104. Jain, H. K., Raut, R. N., and Nerwal, S. K. 196813. Nuclear organizer as hyperactive locus for RNA synthesis. Heredity 24, 59-67. Jain, H. K., Singhal, N. C., Singh, M. P., and Austin, A. 1975. An approach to breeding for higher protein content in bread wheat. In “Breeding For Seed Protein Improvement Using Nuclear Techniques,” pp. 39-46. Proc. IAEA Symp., Vienna. Jain, H. K., Singhal, N. C., and Austin, A. 1976. Breeding for higher protein yields in bread wheat: Experimental approach and a phenotypic marker. Z. Pflanzenzuecht. 77, 100-111. Jambunathan, R., Mertz, E. T., and Axtell, J. D. 1975. Fractionation of soluble proteins of high lysine and normal sorghum grain. Cereal Chem. 52, 119-121. Jaworski, A., and Key, J. L. 1972. Distribution of ribosomal deoxyribonucleic acid in subcellular fractions of higher plants. Plant Physiol. 53, 366-369. Jenkins, L. D., Loney, D. P., and Meredith, P. 1974. Periodicity of growth and starch deposition in the developing wheat grain. Cereal Chem. 51, 718-733. Jha, M. P., Kaul, A. K., Raghaviah, P., and Swaminathan, M. S. 1971. Identification of chromosomes carrying factors for seed storage proteins. Wheat In!. S e w . 32, 9-13.

Jimenez, J. R. 1968. The effect of the opaque-2 and floury-2 genes on the produc-

560

CALVIN F. KONZAR

tion of protein in maize endosperm. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Johnson, B. L. 1968. Electrophoretic evidence on the origin of Triticum zhukovskyi. Proc. Znt. Wheat Genet. Symp., Srd, 196s pp. 105-110. Johnson, B. L. 1972a. Seed protein profiles and the origin of the hexaploid wheats. Am. J . Bot. 59, 952-960. Johnson, B. L. 1972b. Protein electrophoretic profiles and the origin of the B genome of wheat. Proc. Natl. Acad. Sci. USA. 89, 1398-1402. Johnson, B. L. 1975. Identification of the apparent B genome donor of wheat. Can. J . Genet. Cytol. 17, 21-39. Johnson, B. L. 1976. Polyploid wheats and fraction 1 protein. Science 192, 1252. Johnson, B. L., and Dhaliwal, H. S. 1976. Reproductive isolation of Triticum boeoticum and Triticum urartu and the origin of tetraploid wheats. A m . J . Bot. 83, 1088-1094. Johnson, B. L., and Hall. 0. 1966. Electrophoretic studies of species relationships in Trilicum. Acta Agric. Scand., Suppl. 18, 222224. Johnson, B. L., Barnhart, D., and Hall, 0. 1967. Analysis of genome and species relationships in the polyploid wheats by protein electrophoresis. Am. J . Bot. 54, 1089-1098. Johnson, J. A., and Sanchez, R. S. 1972. New no-fermentation process controls bread flour and costs. Baker’s Dig. 48, 30-32. Johnson, J. A., Khan, M. N. A., and Sanchez, C. R. S. 1972. Wheat cultivars, environment and bread baking quality. Cereal Sci. Today 17, 323-326. Johnson, V. A., and Lay, C. L. 1974. Genetic improvement of plant protein. J . Agric. Food Chem. 22, 558-566. Johnson, V. A., and Mattern, P. J. 1972. “Improvement of the Nutritional Quality of Wheat through Increased Protein Content and Improved Amino Acid Balance,” Summary report of research findings July 1, 1966-Dec. 31, 1972, Contract AID/Csd-1208. Agency for International Development, Department of State, Washington, D.C. Johnson, V. A., and Mattern, P. J. 1975. “Improvement of the Nutritional Quality of Wheat Protein through Increased Protein Content and Improved Amino Acid Balance,” Report of research findings Jan. 1, 1973-March 31, 1975, Contracts AID/Csd-1208 and AID/ta-C-1093. Agency for International Development, Department of State, Washington, D.C. Johnson, V. A., Schmidt, J. W., Mattern, P. J., and Haunold, A. 1963. Agronomic and quality characteristics of high protein Fz-derived families from a soft red winter-hard wheat cross. Crop Sci. 3, 7-10. Johnson, V. A., Mattern, P. J., and Schmidt, J. W. 1967. Nitrogen relations during spring growth in varieties of Triticum aestivum L. differing in grain protein content. Crop. Sci. 7 , 664-667. Johnson, V. A., Whited, D. A., Mattern, P. J., and Schmidt, J. W. 1968a. Nutritional improvement of wheat by breeding. Proc. Znt. Wheat Genet. Sym?., Srd, 1968 pp. 457-461. Johnson, V. A., Schmidt, J. W., and Mattern, P. J. 1968b. Cereal breeding for better protein impact. Econ. Bot. 22, 16-25. Johnson, V. A., Mattern, P. J., and Schmidt, J. W. 1972a. Wheat protein improvement. Int. Rice Res. Inst., Rice Breed. pp. 407418. Johnson, V. A., Mattern, P. J., and Schmidt, J. W. 197213. Genetic studies of wheat protein. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 126-136. Avi Publ. Go., Westport, Connecticut.

GENETIC CONTROL OF PROTEINS I N WHEAT

561

Johnson, V. A., Dreier, A. F., and Grabouski, P. H. 1973. Yield and protein responses to nitrogen fertilizer of two winter wheat varieties differing in inherent protein content of their grain. Agron. J. 65, 259-263. Johnson, V. A., Mattern, P. J., Stroike, J. E., and Wilhelmi, K. D. 1975a. Breeding for improved nutritional quality in wheat. Proc. Int. Winter Wheat Conf. 2nd, 1975 pp. 316-333. Johnson, V. A., Mattern, P. J., and Vogel, K. P. 1975b. Cultural genetic, and other factors affecting quality of wheat. In “Bread: Social, Nutritional, and Agricultural Aspects of Wheat Bread” (A. Spencer, ed.), pp. 127-140. Applied Science Publ., London. Jones, I. K., Phillips, J. W., and Hird, F. J. R. 1974. The estimation of rheologically important thiol and disulphide groups in dough. J . Sci. Food Agric. 25, 1-10. Jones, R. W., and Cluskey, J. E. 1963. Preparation of aluminum lactate. (Communication to the Editor.) Cereal Chem. 40, 589-591. Jones, R. W., Babcock, G. E., Taylor, N. W., and Senti, F. R. 1961. Molecular weight of wheat gluten fractions. Arch. Biochem. Biophys. 94, 483-488. Jones, R. W., Babcock, G. E., Taylor, N. W., and Dimler, R. J. 1963. Fractionation of wheat gluten by gel filtration. Cereal Chem. 40, 409414. Joppa, L. R. 1973. Development of disomic substitution lines of durum wheat (Tn‘ticum turgidum L.) Proc. Znt. Wheat Genet. Symp., 4th, 1973 pp. 685-690. Joppa, L. R., Bietz, J. A., and McDonald, C. 1975. Development and characteristics of a disomic-1D addition line of durum wheat. Can. J. Genet. Cytol. 17, 355463. Joudrier, M. P., and Cauderon, Y. 1976. Localisation chromosomique de genes contr8lant la synth6se de certains constituants p amylasique du grain de B16 tendre (In French, Engl. Summary). Note. C. R . Hebd. Seances Acad. Sci., Ser. D . 282, 115-118 (Chem.Abstr. 161857j). Juliano, B. 0. 1972. Studies on protein quality and quantity of rice. In “Symposium: Seed Proteins” (G. E. Inglett, ed.), pp. 114-125. Avi Publ., Westport, Connecticut. Kakade, M. L. 1974. Biochemical basis for the differences in plant protein utilization. J. Agric. Food Chem. 22, 550-555. Kaltsikes, P. J., Evans, L. E., and Bushuk, W. 1968a. Durum type wheat with high breadmaking quality. Science 159, 211-213. Kaltsikes, P. J., Evans, L. E., and Larter, E. N. 196813. Identification of a chromosome segment controlling breadmaking quality in common wheat. Can. J. Genet. Cytol. 10, 763. Kamagata, T., and Nishikawa, K. 1973. Genetic variation of peroxidase isozymes in wheat (In Japanese) Jpn. J . Genel. 48, 422423 (abstr.). Kanazana, H., and Yonezawa, D. 1973. Studies on polypeptide composition of “low molecular weight glutenin” (In Japanese, English Summary). Nippon Nogei Kagaku Kaishi 47, 17-22 (Chem. Abstr. 11550g). Kanazana, H., and Yonezawa, D. 1974a. Aggregative polypeptides as component polypeptides of wheat glutenin (In Japanese, English Summary). Nippon Nogei Kngaku Kaishi 48, 113-117 (Chem. Abstr. 2242ln). Kanaaawa, H., and Yonezawa, D. 1974b. Conversions of intra- to interchain disulfide bonds in gluten polypeptides by S H S S exchange reaction (In Japanese, English Summary). Nippon Nogei Kagaku Kaishi 48, 245-253 (Chem. Abstr. f47137j). Kannangara, C. G . 1969. The formation of ribulose diphosphate carboxylase proteins during the chloroplast development in barley. Plant Physiol. 44, 1533-1537.

562

CALVIN F. KONZAK

Kasarda, D. D. 1970. The conformational structure of wheat proteins. Baker’s Dig. 44,20-26, Kasarda, D. D. 1975. Celiac disease: Malabsorption of nutrients induced by a toxic factor in gluten, In “Protein Nutritional Quality of Foods and Feeds” (M. Friedman, ed.), Vol. 1, Part 2,pp. 565-593. Dekker, New York. Kasarda, D. D., Bernardin, J. E., and Thomas, R. S. 1967. Reversible aggregation of alpha-gliadin to fibrils. Science 155, 203-205. Kasarda, D. D., Nimmo, C. C., and Kohler, G. 0. 1971.Proteins and the amino acid composition of wheat fractions. Zn “Wheat-Chemistry and Technology” (Y. Pomerans, ed.), pp. 227-299. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Kasarda, D. D., Qualset, C. O., and Platt, 8. G. 1973. Varietal relationships of the alpha-gliadin proteins in wheat. Proc. Int. Wheat Genet. Symp., 4th, 19Y3 pp. 811-815. Kasarda, D. D., Da Rosa, D. A., and Ohms, J. I. 1974a. N-terminal sequence of cun-gliadin.Biochim. Biophys. Acla 351, 290-294. Kasarda, D. D., Nimmo, C. C., and Bernardin, J. E. 1974b. Structural aspects and genetic relationships of gliadins. In “Coeliac Disease” J. M. Hekkens and A. S. Pena, eds.), pp. 25-36. H. E. Stenfert Kroese, Leiden, Netherlands. Kasarda, D. D., Bernardin, J. E., and Qualset, C. 0. 1974c. Relationship of gliadin protein components to chromosomes through the use of substitution lines. Cereal Sci. Today 19,403. Kasarda, D. D., Bernardin, J. E., and Nimmo, C. C. 1976a. Wheat proteins. In “Advances in Cereal Science and Technology” (Y. Pomeranz, ed.), pp. 158-236. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Kasarda, D. D., Bernardin, J. E., and Qualset, C. 0. 1976b. Relationship of gliadin protein components to chromosomes in hexaploid wheats (Trilicum aestivum L.).Proc. Natl. Acad. Sci. USA. 73, 3646-3650. Kato, M., Hiromi, K., and Morita, Y.1974. Purification and kinetic studies of wheat bran @-amylase. Evaluation of subsite affinities. J . Biochem. (Tokyo) 75, 563576. Kaul, A. K., and Sosulski, F. W. 1965. Inheritance of flour protein content in a Selkirk x Gab0 cross. Can. J . Genet. Cytol. 7, 12-17. Kawashima, N., and Wildman, S. G. 1971. Studies of Fraction I protein. I. Effect of crystallization of Fraction I protein from tobacco leaves on ribulose diphosphate carboxylase activity. Biochim. Biophys. Acta 299,240-249. Kawashima, N., and Wildman, S. G. 1972. Studies on Fraction I protein. IV. Mode of inheritance of primary structure in relation to whether chloroplast or nuclear DNA contains the code for a chloroplast protein. Biochirn. Biophys. Acta 282, 42-49. Kendall, M. J., Schneider, R., Cox, P. S., and Hawkins, C. F. 1972. Gluten subfractions in coeliac disease. Lancet 2, 1065-1067. Kent, N. L. 1966. Subaleurone cells of high protein content. Cereal Chem. 43, 585601. Kent, N. L., and Evers, A. D. 1969. Variation in protein composition within the endosperm of hard wheat. Cereal Chem. 46,293-300. Kerber, E. R. 1964. Wheat: Reconstitution of the tetraploid component (AABB) of hexaploids. Science 143, 253-255. Kerber, E. R., and Tipples, K. H. 1969. Effects of the D genome on milling and baking properties of wheat. Can. J . Plant Sci. 49,225-263.

GENETIC CONTROL OF PROTEINS IN WHEAT

563

Khan, A. H.1972. Improvement of quality and yield of wheat by mutation breeding. Wheat hj. S e w . 36, 7-8. Khrabrova, M. A,, Galai, V. S., and Maystrenko, 0. 1. 1973. Electrophoretic patterns of gluten proteins of some common wheat varieties and of a set of Chinese Spring ditelosomics. In “Cytogenetic Studies on Aneuploids of Common Wheat” (0. I. Maystrenko and V. V. Xhvostova, eds.) (In Russian, English Summary), pp. 218-232. Acad. Sci. U.S.S.R., Siberian Branch, Inst. Cytol. Genet. Khush, G. S. 1973. “Cytogenetics of Aneuploids.” Academic Press, New York. Kies, C., and Fox, H.M. 1970a. Determination of the first-limiting amino acid of wheat and Triticale grain for humans. Cereal Chem. 47, 615-625. Kies, C., and Fox, H.M.1970b. Protein nutritive value of wheat and Triticale grain for humans, studied at two levels of protein intake. Cereal Chem. 47, 671-678. Kihara, H. 1944. Discovery of the DD analyzer, one of the ancestors of Triticum vulgare. Agric. Hort. 19, 889-890. Kihara, H. 1954.Consideration on the evolution and distribution of Aegilops species based on the analyser-method. Cytologk 19, 336357. Kihara, H.1959. Fertility and morphological variation in the substitution and reetoration backcrosses of the hybrids, Triticum vulgare x Aegilops caudata. Proc. Int. Congr. Genet., loth, 1968 Vol. 1, pp. 142-171. Kihara, H. 1966. Factors effecting the evolution of common wheat. Indian J. Genet. Plant Breed. 26a, 14-28. Kihara, H.1968. Cytoplasmic relationships in the Triticinae. Proc. Znt. Wheat Genet. Symp., Srd, 1968 pp. 125-134. Kihara, H. 1974. The history of all organisms is inscribed in the chromosomes (in Japanese). Kromosomo 96-97, 2995-3008. Kihara, H. 1975. Origin of cultivated plants with special reference to wheat. Seiken Ziho 25-26, 1-24. Kihara, H.,and Lilienfeld, F. A. 1949.A new synthesized 6 x wheat. Proc. Znt. Congr. Genet., Sth, 1948 Hereditas, Suppl. 1, 307-319. Kihara, H.,and Tsunewaki, K. 1968. Some fundamental problems underlying the program for hybrid wheat breeding. Seiken Ziho 16,l-14. Kilborn, R. H., and Tipples, K. H.1972a. Factors affecting mechanical dough development. I. Effect of mixing intensity and work input. Cereal Chem. 49, 3447. Kilborn, R. H.,and Tipples, K. H.1972b. Factors affecting mechanical dough development. 11. Implications of mixing a t a constant rate of energy input. Cereal Chem. 49, 48-53. Kimber, G. 1966. Estimate of the number of genes involved in the genetic suppression of the cytological diploidization of wheat Nature (London) 212, 317-318. Kimber, G. 1973. The relationships of the S-genome diploids to polyploid wheats. Proc. Int. Wheat Genet. Symp., 4th. 1973 pp. 81-85. Kimber, G., and Athwal, R. S. 1972. A reassessment of the course of evolution in wheat. Proc. Natl. Acad. Sci. U S A . 69, 912915. Klepper, L. A. 1975. Nitrate assimilation enzymes and seed protein in wheat. Proc. Int. Winter Wheat Conf.,grid. 1976 pp. 334-340. Kneen, E., and Sandstedt, R. M. 1943.An amylase inhibitor from certain cereals. J. A m . Chem. SOC.65, 1247. Kobrehel, K., and Feillet, P. 1975. Identification of genomes and chromosomes involved in peroxidase synthesis of wheat seeds. Can. J. Bot. 53, 2336-2344.

564

CALVIN F. KONZAK

Kobrehel, K., and Gautier, M. F. 1974. Variability in peroxidase isozymes in wheat and related species. Can. J . Bot. 52, 755-759. Koenig, V. L., Ogrins, A., Trimbo, H. B., and Miller, B. S. 1974. The electrophoretic analysis of flour from several varieties of hard red winter wheat grown a t several locations. J . Sci. Food. Agric. 15,492497. Kolderup, F. 1975. Effects of temperature, photoperiod and light quantity on protein production in wheat grains. J . Sci. Food. Agric. 26, 583-592. Konarev, V. G., Gubareva, N. K., Gavrilyuk, I. P., and Bushuk, U'. 1972. Identification of genome D in wheats with respect to gliadin (In Russian, English Summary). Vestn. Skh. Nauki (Moscow) 17,108-114 (Chem. Abstr. 111649~). Kruger, J. E. 1973. Changes in the levels of proteolytic enzymes from hard red spring wheat during growth and maturation. Cerea2 Chem. 50, 122-131. Kruger, J. E. 1976. Changes in the polyphenol oxidases of wheat during kernel growth and maturation. Cereal Chem. 53, 201-213. Kruger, J. E., and LaBerge, D. E. 1974. Changes in peroxidase activity and peroxidase isoayme patterns of wheat during kernel growth and maturation. Cereal Chem. 51, 345-354. Ku, H. S. 1973. Stimulation of nitrate reductase by a protein fraction. Agron. Abstr. p. 35 (by permission). Ku, H., Omata, S., and Tolbert, N. E. 1973. Nitrate reductase inhibitors from leaf tissue. Agron. Abstr. p. 36 (by permission). Kulp, K. 1972. Physicochemical properties of starches of wheats and flours. Cereal Chem. 49, 697-706. Kung, S. D. 1976. Tobacco fraction I protein: A unique genetic marker. Science 191, 429-434.

Kung, S. D., Sakano, K., and Wildman, S. G. 1974a. Multiple peptide composition of the large and small subunits of Nicotiana tabaccum Fraction I protein ascertained by fingerprinting and electrofocusing. Biochem. Biophys. Acta 365, 138-147. Kung, S. D., Sakano, K., and Wildman, S. G. 1974b. Evolution of Fraction I protein. Phnt Physiol. 53, (Suppl.) 63 (Abstr. No. 356). Kuspira, J., and Unrau, J. 1957. Genetic analysis of certain characters in common wheat using whole chromosome substitution lines. Can. J . Plant Sci. 37, 300-326. Kuspira, J., and Unrau, J. 1958. Determination of the number and dominance of genes on substituted chromosomes in common wheat. Can. J . Phnt Sci. 38, 199-205.

Kwanyuen, P., and Wildman, S. G. 1975. Nuclear DNA codes for Nicotiana ferredoxin. Biochim. Biophys. Acta 405, 167-174. La Clerc, J. A,, Bailey, L. H., and Wessling, H. L. 1918. Milling and baking tests of Einkorn, Emmer, Spelt, and Polish wheat. Agron. J. 10, 215-217. Landry, J., and Moureaux. T. 1970. HBtBrogBnBi'tB des glutelines du grain de Mays: Extraction selertive et composition en acides amines des trois fractiones isoldes (In French). Bull. Soc. Chim. Biol. 52, 1021-1037 (Chem. Abstr. 7877Sh). Larkin, R. A., MacMasters, M. M., Wolf, M. J., and Rist, C. E. 1951. Studies on the relation of bran thickness to millability of some Pacific Northwest wheats. Cereal Chem. 28, 247-258. Larkin, R. A., MacMaster, M. M., and Rist, C. E. 1952. Relation of endosperm cell wall thickness t o the milling quality of seven Pacific Northwest wheats. Cereal Chem. 29, 407-413. Larsen, J. 1974. The localization of phenol reaction genes in hexaploid wheat. E.W.A.C. Newslett. No. 4,80 (by permission).

GENETIC CONTROL OF PROTEINS IN WHEAT

565

Larsen, J., and Kimber, G. 1973. The effect of the genotype of Triticum speltoides on the pairing of homologous chromosomes. Cnnnd. J . Genet. Cytol. 15, 233-236. Law, C. N. 1968. Genetic analysis using inter-varietal substitution lines. Proc. Int. Wheat Genet. Symp., Srd, 196s pp. 331-342. Law, C. N., and Worland, A. J. 1972. “Aneuploidy in Wheat and its Uses in Genetic Analysis.” Annu. Rep., 1972, pp. 25-65. Plant Breed. Inst., Cambridge, England. Law, C. N., and Worland, A. J. 1973. Chromosome substitutions and their use in the analysis and prediction of wheat varietal performance. Proc. Int. Wheat Genet. Symp., 4th, 1975 pp. 41-49. Lawrence, J. M., Day, K . M., Huey, E., and Lee, B. 1958. Lysine content of wheat varieties, species, and related genera. Cereal Chem. 35, 169-178. Lawrence, J. M., Lin, S.-C. and Grant, D. R. 1970. Dye-binding capacity and amino acid content of wheat-protein-gel-electrophoresis-bands. Cereal Chem. 47, 110117.

Leaback, D. H., and Wrigley, C. W. 1976. The isoelectric focusing of proteins. I n “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.), 4th ed., Vol. 2. Heinemann, London (in press). Lebsock, K. L., Fifield, C . C., Gurney, G. M., and Greenaway, W. T. 1964. Variation and evaluation of mixing tolerance, protein content, and sedimentation value in early generations of spring wheat, Triticum aestivum L. Crop Sci. 4, 111-174. Lee, J. W., and Wrigley, C. w. 1963, The protein composition of gluten extracted from different wheats. Aust. J. E x p . Agric. Anim. Husb. 3, 85-88. Lein, I< Aegilops species and their 2>( relatives. Seiken Ziho 25-26, 67-78. Munck, L. 1976. Developing the concept, of quality in barley breeding programs for malt and feed. I n “Barley Genetics 111” (H. Gaul, ed.), pp. 526535. Karl Thiemig, Munich. Munck, L., Karlsson, K. E., Hagberg, A,, and Eggum, B. 0. 1970. Gene for improved nutritional value in barley seed protein. Science 168, 985-987. Mylotte, M., Egan-Mitchell, B., McCarthy, C. F., and McNicholl, B. 1973. Incidence of coeliac disease in the west of Ireland. Br. M e d . J . 1, 703-705. Nagabhushan, N., Gulyas, A., and Zalik, S. 1974. Comparison of plant cytoplasmic

570

CALVIN F. KONZAK

ribosomal proteins by two dimensional polyacrylamide gel electrophoresis. PEant Physiol. 53, 516518. Nagayoshi, T. 1973. Genetical studies on amylase isozymes of D genome chromosomes in common wheat (in Japanese). Jpn. J . Genet. 48, 433 (abstr.). Nagayoshi, T., Kakuda, T., and Yasuhara, A. 1974. Genetical studies on esterase isorymes of D genome chromosomes in common wheat (in Japanese). Jpn. J . Genet. 49, 312 (Abstr.). Nakai, Y. 1973. Isozymes variations in Aegilops and Triticum. 11. Esterase and acid phosphatase isozymes studied by the gel isoelectrofocusing method. Seiken Ziho 24, 45-73. Nakai, Y., and Tsunewaki, K. 1974. Genetic studies on the developmental changes of esterase isozymes in aneuploids of common wheat (in Japanese). Jpn. J . Genet. 49, 313. (Abstr.) National Research Council. Board on Agriculture and Renewable Resources. 1976. “Genetic Improvement of Seed Proteins,” pp. 1-394. Washington D.C. Neyra, C. A., and Hageman, R. H. 1974. Dependence of nitrite reduction on electron transport in chloroplasts. Plant Physiol. 54, 480-483. Nielsen, H. C., Beckwith, A. C., and Wall, J. S. 1968. Effect of disulphide-bond cleavage on wheat gliadin fractions obtained by gel filtration. Cereal Chem. 45, 3747.

Nilan, R. A. 1964. “The Cytology and Genetics of Barley, 1951-1962.” Washington State Univ. Press, Pullman. Nilan, R. A., Sideris, E. G., Kleinhofs, A., Sander, C., and Konzak, C. F. 1973. Azide-a potent mutagen. Mutat. Res. 17, 142-144. Nilan, R. A., Kleinhofs, A., and Konzak, C. F. 1976a. The role of induced mutation in supplementing natural genetic variability. Proc. N . Y . Acad. Sci. (in press). Nilan, R. A., Kleinhofs, A., and Sander, C. 1976b. Azide mutagenesis in barley. In “Barley Genetics 111” (H. Gaul, ed.), pp. 113-122. Karl Thiemig, Munich. Nimmo, C. C., O’Sullivan, M. T., and Bernardin, J. E. 1968. The relation of a globulin of wheat flour to purothionin. Cereal Chem. 45, 28. Nimmo, C. C., Kasarda, D. D., and Lew, E. J.-L. 1974. Physical characterization of the wheat protein purothionin. J . Sci. Food Agric. 25,607-617. Nishikawa, K. 1973. Alpha-amylase isozymes and phylogeny of hexaploid wheat. Proc. Int. Wheat Genet. Symp., 4th, i9Y3 pp. 851-855. Nishikawa, K., and Kamagata, T. 1974. Organ-specific peroxidase isorymes in aneuploids of common wheat (in Japanese). Jpn. J . Genet. 49, 314-315 (abstr.). Nishikawa, K., and Nobuhara, M. 1971. Genetic studies of m-amylase isozymes in wheat. I. Location of genes and variation in tetra- and hexaploid wheat. Jpn. J . Genet. 46, 345-353. Nishikawa, K., Hina, Y., Oonishi, S., and Furuta, Y. 1976. The D genome dependent isozymes of a-amylase in wheat. Wheat Znf. Serv. Nos. 41-42, March, 1976, p. 8 (by permission). Nittler, L. W., and Jensen, H. A. 1974. Cultivar differences among barley plants grown with different nutrient treatments. Agron. J. 66, 397-399. Noda, I