Biochemical Genetics, Vol. 25, Nos. 1/2, 1987

Chromosomal Location of Genes Coding for Endosperm Proteins of Hordeum chilense, Determined by Two-Dimensional Electrophoresis of Wheat- H. chilense Chromosome Addition Lines

P. I. Payne, l L. M. Holt, 1 S. M. Reader, 1 and T. E. Miller 1

Received 10 Apr. 1986~Final 30 July 1986

The proteins of H o r d e u m chilense grain were resolved into 25 major components by two-dimensional electrophoresis. Their solubilities in aque- ous alcohol solutions were determined to distinguish prolamin storage proteins from metabolic and structural proteins. The prolamins were divided into two groups, based on the presence or absence of intermolecular disulfide bonds determined by gel-filtration chromatography. Using an incomplete set of Chinese Spring wheat-H, chilense disomic addition lines, the structural genes of 21 of the 26 most dominant seed proteins were assigned to chromosomes. The great majority of the prolamin genes, including those coding for a high molecular weight (HMW) prolamin subunit, was present on chromosome 1n ch. However, a small number of prolamin genes also occurred

vh eh on chromosomes 5H and 7H . A minor protein, probably belonging to the nonstorage group of proteins, is coded by genes on 5H oh. Various ditelosomie addition lines and ditelosomic and disomic substitution lines for chromo- some 7H ch were also analyzed by electrophoresis. This technique revealed that the genes for three major prolamins occur on the/3 arm of chromosome 7H ch and that a gene for a minor protein, also thought to be a prolamin, occurs on the o~ arm. These results are discussed in relation to the evolution of prolamin genes in the Tri t iceae.

KEY WORDS: Hordeum chilense; endosperm protein genes; two-dimensional electrophoresis.

1 Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ, England.

53 0006-2928/87/0200-0053505.00/0 © 1987 Plenum Publishing Corporation

54 Payne, Holt, Reader, and Miller

INTRODUCTION

In recent years advances in immature embryo culture have made possible wider crosses within the Triticeae. Crosses between wheat and its relatives have led to the production of sets of addition lines of individual alien chromosome pairs added to the wheat genome. These lines are being exploited in various studies, one being to determine the chromosomal distribution of endosperm storage-protein genes in different members of the Triticeae (Soliman et al., 1980; Lawrence and Shepherd, 1981; Shewry et al.,1985a). Alien chromosome addition and substitution lines are also potentially of great use in studying the genetics and chromosomal location of genes in wheat controlling proteins which are more conserved than storage proteins and show little or no allelic variation between varieties.

Hybrids between bread wheat and the wild barley, H o r d e u m chilense, were first described by Martin and Chapman (1977). The resulting amphi- ploid (Chapman and Miller, 1978) was used by Miller et al. (1981) to develop several disomic addition lines. These and more recently developed lines, including chromosome substitutions (Miller et al., 1985), were used in this study to determine the chromosomal location of the genes coding for the major proteins in the grain. Since the endosperm makes up the great bulk of the grain, it will be assumed that all these proteins occur in this tissue.

MATERIALS AND METHODS

Plant Material

The following genotypes were studied. 1. Tr i t icum aes t ivum cv. Chinese Spring (2n = 6x = 42; AABBDD). 2. Hordeum chilense (2n = 14; HOhHCh). 3. T. aes t ivum Chinese Spring-H. chilense disomic addition lines

A(7HCb), C(5HCh), F(4HCh), and G(1H ch) and two 5HCh/6H ch translo- cated addition lines (2n = 6x = 44).

4. Monosomic addition line B (2H oh) (2n = 6x = 43). 5. Ditelocentric addition lines 7HCho~ and 7HCh/3 and substitution lines

(7A)7H oh, (7B)7H ~h, (7D)7H ~h, (7A)7H~hc~, (7D)7H~%~, and (TB)TH~hB.

6. Ditelocentric addition line 1HOBS.

Two-Dimensional Electrophoresis

Endosperm proteins, extracted with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, were fraetionated by the methods of O'Farrell (1975) and O'Farrell et al., (1977) as modified previously for wheat proteins (Holt et al.,

Hordeum chilense Proteins 55

1981; Jackson et al. , 1983). The methods employ two first dir,~rsicms, isoelectric focusing (IEF) and nonequilibrium pH-gradient electrophoresb (NEPHGE) . The former separates the neutral and acidic proteins~ m~d th,-~ latter the basic proteins. The common second dimension was sodium dod.ecy] sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

RESULTS

Characterization of the Proteins of H. chilense

A two-dimensional fractionation of the total endosperm grain protein~, of ,J. c h i l e n s e is shown in Fig. 1. The most strongly stained components on the~ two-dimensional map have been numbered 1 to 25. Polypeptides of ~omk~a?! molecular weight, less than 20,000, have not been studied. The +.wo--

NEPHGE IEF

4 15

Fig. 1. Two-dimensional fractionation of the endosperm proteins of Hordeurn chilense. The most strongly stained components have been numbered. The overlapping regions of the NEPHGE and IEF separations have been eliminated, as described by Jackson et al. (1983). Because one protein component had markedly different migration properties on NEPHGE and IEF, the two photographs had exceptionally to be cut diagonally.

56 Payne, Holt, Reader, and Miller

dimensional map is considerably simpler than the corresponding map of endosperm proteins from Chinese Spring wheat (Payne et al., 1984a) as might be expected, for the former species is diploid and the latter is hexaploid.

In Fig. 2, the two-dimensional map of endosperm proteins for Chinese Spring has been drawn diagrammatically. The chromosomal location of the genes which code for nearly all of the components is known (Fig. 4 of Payne et al., 1985). When protein extracts of Chinese Spring and H. chilense are mixed prior to electrophoresis, most of the latter are well resolved and are shown in Fig. 2 as open circles. Unfortunately components 20 to 25 are obscured by Chinese Spring proteins so that the chromosomal location of their structural genes could not be deduced from electrophoretic analyses of the addition lines.

In order to distinguish prolamin storage proteins from other grain proteins which have other functions, flour samples were separately extracted in 70% (v/v) aqueous ethanol and 50% (v/v) aqueous isopropanol at room

NEPHGE IEF

0 , , , .

0

41B,°

O , 5 o

4" °.3 - "6 t o

° d l b ~ , g "

o " ° = : o -, ,7 15

1 ~ 17. ~ B 12 11 <:~ 18 , r~ " 14 1 3 ~ 0 j,

10

e o

o

1 I/J O

I if) 12

Fig. 2. Diagrammatic two-dimensional map of a mixture of Chinese Spring and H. ehilense seed proteins. The components from H. chilense which are distinguishable from Chinese Spring proteins are represented by open circles and are highlighted by single-headed arrows. The numbers correspond to those in Fig. 1 and Table I. The Chinese Spring components are drawn as filled circles. The component marked by a double-headed arrow was shown, as described in the text, to be controlled by genes on chromosome 7D.

Hordeum chilense Proteins 57

temperature, prior to two-dimensional electrophoresis. Prolamins have an unusual amino acid composition and they tend to dissolve specifically in these solvents (Shewry and Miflin, 1985). Component 1 is only partially soluble in ethanol but it is more soluble in isopropanol and is considered to be a prolamin. It has a distinctive two-dimensional map position (Fig. 1) and clearly belongs to the group of proteins called high molecular weight (HMW) subunits of glutenin in wheat (Payne et al., 1985) or, more generally, HMW prolamin subunits in cereals.

The other prolamin components of H. chilense, numbered 2-4, 6-14, 16-20, and 22-24 in Fig. 2, are freely soluble in ethanol, except for 6 and 16, which are freely soluble in isopropanol. These components have a two- dimensional map position similar to that of the prolamins of cultivated barley, Hordeum vulgare (Fig. 2 of Payne, 1986) but, on average, have a slightly faster mobility in the second dimension (SDS-PAGE) than the prolamins of wheat (Fig. 2). The prolamins of wheat are called gliadins and glutenin subunits, according to whether they form intermolecular disulfide bonds (Payne et al., 1984a). The prolamins of cultivated barley are called hordeins.

To characterize these prolamin proteins on the basis of aggregation states, ethanol-soluble proteins were separated through a Sephadex G-100 column into void-volume (aggregated proteins) and sieved fractions (mono- merie proteins) as described by Jackson et al. (1983). Components 2-4, 9-14, 19, and 20 occurred essentially entirely as nonaggregated monomeric pro- teins, whereas components 6 and 16-18 were primarily aggregated (results not shown). Components 7 and 8 occurred equally in aggregated and nonaggregated states. Some prolamins in H. vulgare apparently also have similar dual properties (Field et al., 1983), although none do in wheat (Payne et al., 1984a). Components 22-24 were present in amounts too small to characterize by gel-filtration chromatography.

Three protein components of H. chilense which did not dissolve in 70% aqueous ethanol solutions were 5, 15, and 21. The two-dimensional map position of component 15 (Fig. 2) indicates that it is homologous to the nonstorage proteins of wheat. These, like component 15, are insoluble in both ethanol and isopropanol and they also share the property of staining meta- chromatically with Coomassie blue R to give a pink color.

Component 21 occurs as a horizontal streak in Fig. 1, through its anomalous behavior during isoelectric focusing. Although it is partially soluble in aqueous isopropanol, it stains pink, not blue, and is considered to be homologous to the HMW albumins of wheat. Unfortunately it is completely obscured by one of them in fractionated mixtures of Chinese Spring and H. chilense (Fig. 2).

Component 5 does not appear to be soluble in either of the aqueous alcohol solutions, although partial solubility cannot be ruled out because the

~% Payne, Holt, Reader, and Miller

:=,J~: ~ponent is p resen t only in ve ry sma l l a m o u n t s in t he endospe rm. I ts s t a te of

~ggrega t ion could not be d e t e r m i n e d . T h e solubi l i t ies and a g g r e g a t i o n s ta tes

ot ~ H. chilense gra in pro te ins a re s u m m a r i z e d in T a b l e I.

Two-Dimensional Electrophoresis of Chinese Spring-H. chilense Addition Lines

W h e n ana lyzed by t w o - d i m e n s i o n a l e lec t rophores i s , all of t he ava i l ab le

add i t ion l ines were shown to con ta in all the e n d o s p e r m pro te ins of Ch ine se

Spr ing , and several l ines had add i t iona l componen t s , al l of wh ich cor re -

sponded to t he prev ious ly c h a r a c t e r i z e d pro te ins o f H. chilense shown in Fig.

1. T h e c h r o m o s o m e G add i t ion l ine (Fig. 3) c o n t a i n e d 13 of the 24

c o m p o n e n t s l abe led in Fig. 1. O n e of t h e m is c o m p o n e n t 1, cons ide red to be a

H M W p r o l a m i n subun i t f r o m its so lubi l i ty proper t ies , a l r e a d y discussed, and

Table I. Characterization of the Grain Proteins of Hordeum chilense and the Chromosomal Location of Their Structural Genes

Alcohol Protein Aggregation Chromosome Component solubility a group state location

1 Isopropanol HMW prolamin Aggregated G (1H oh) 2 Ethanol Prolarnin Monomeric " 3 " " " " 4 " " " " 5 Insoluble Not known Aggregated " 6 Isopropanol Prolamin . . . . 7 Ethanol " Aggregated/monomeric " 8 re t r ¢J ip

9a " " Monomeric " 9b ~ ? ? A (7H c~) 10 Ethanol Prolamin Monomeric G ( 1H c~) 11 . . . . . . . . 12 . . . . . . A (7H oh) 13 . . . . . . . . 14 . . . . . . . . 15 Insoluble Nonstorage - - C (5H ~h) 16 Isopropanol Prolamin Aggregated G ( 1H oh) 17 Ethanol . . . . C (5H oh) 18 . . . . . . . . 19 . . . . Monomeric A (7H Ch) 20 . . . . . . ? 21 Isopropanol HMW albumin - - ? 22 Ethanol Prolamin ? G (1H ~h) 23 - - " ? ? 24 - - " ? ? 25 Insoluble Not known ? ?

aAll components stated to be soluble in ethanol are also soluble in isopropanol.

Hordeum chilense Proteins 59

from its two-dimensional map position (Payne et al., 1985). Genes coding for this protein group occur on homoeologous chromosomes in the Triticeae, namely, chromosomes 1A, 1B, and 1D in wheat (Payne et al., 1982), 1R in rye, and 5(1H) in cultivated barley (Shewry et al., 1984b). Thus, it is likely that chromosome G of H. chilense is also a homoeologue.

The remaining proteins coded by chromosome G are 2-11, 16, and 22 (Fig. 3) and all except possibly component 5 are thought to be prolamins. Thus, as in wheat, rye, and cultivated barley (H. vulgare), the chromosome which contains HMW prolamin subunit genes also contains the structural genes for most of the prolamins. It is concluded therefore that chromosome G is homoeologous to chromosomes 1A, 1B, and 1D of bread wheat and accordingly is designated 1H oh. The ditelocentric addition line containing the short arm only of chromosome 1H ~h was also analyzed. All the endosperm proteins coded by chromosome 1H ch are present in this line except for component 1, showing that the genes for the former occur on 1H¢hS and suggesting that the gene for the latter occurs on 7H~hL.

The two-dimensional map of the H. chilense addition line A is shown in Fig. 4. Four extra protein components are present, three major, which correspond to 12, 13, and 14 in Fig. 1, and two minor, 9 and 19. However,

NEPHGE IEF

1 iii

<

I if)

Fig. 3. Two-dimensional map of the seed proteins of Chinese Spring-H. chilense addition G. The protein components of the wild barley are arrowed and numbered.

60

NEPHGE IEF

1

I

r~

Fig. 4. Two-dimensional map of the seed proteins of Chinese Spring-H. chilense addition A.

protein 9 had previously been assigned to chromosome 1H eh and was a minor component in the Chinese Spring-H. ehilense A addition line. Evidently, the minor protein coded by genes on chromosome A must have the same two-dimensional map position as the major 1HOb-encoded protein, and so to distinguish them they are named 9B and 9A, respectively. It is not possible to characterize protein 9B but the other endosperm proteins coded by chromo- some A are all monomeric prolamins (Table I). Recent work by Miller et al.

(1985) has shown that chromosome A can substitute in turn for chromosomes 7A, 7B, and 7D of the Chinese Spring genome. The chromosome was therefore designated 7H oh. This is the first report of prolamin genes occurring on chromosomes of this homoeologous group.

The remaining addition line which was shown to code for H. chilense grain proteins contained chromosome C. Three of the proteins detected in Fig. 1 were present (Fig. 5). Components 17 and 18 are probably aggregated prolamins and component 15 belongs to the nonstorage group of proteins (Table I). Recently, chromosome C was shown to be able to substitute for chromosome 5D of Chinese Spring (Miller and Reader, unpublished) and so should be designated 5H ~h. This is consistent with recent findings of Payne et al. (1985), who showed that five of six abundant, high molecular weight proteins in bread wheat, which they called nonstorage proteins, are controlled by the group 5 chromosomes. However, no prolamin genes have previously

NEPHGE IEF

1 iii

I o0

Fig. 5. Two-dimensionalmapoftheseed proteins of Chinese Spring-H. chilenseaddition C.

been reported to occur on this homoeologous set of chromosomes. Several other chromosome addition lines were analyzed. The additional chromosome in line B (thought to be homoeologous to group 2) and the pair in line F (homoeologous to group 4) (Miller and Reader, unpublished) had two- dimensional maps identical to that of Chinese Spring wheat, showing that no prominent endosperm proteins are coded by genes on these chromosomes. No complete addition line was available for the homoeologous group 6 chromo- some. However, two partially characterized translocation addition lines were available, one thought to consist of 5HChL/6HChS and the other, 5H°hS/6H~hL. The former but not the latter contained components 15, 17, and 18, shown previously to be coded by 5H oh. No grain protein genes could be assigned to 6H oh.

The chromosomal locations of the structural genes for the major endo- sperm proteins are summarized in Table I.

Two-Dimensional Eiectrophoresis of Chinese Spring-/ / . chilense 7H ch Substitution Lines and 7H ch Ditelocentric Addition Lines

The location of genes coding for proteins 12-14 and 19 on chromosome 7H ch was confirmed from the analysis of the disomic substitution line, Chinese Spring (7D)7H °h. As well as these proteins being present in the line (results

62 Payne, Holt, Reader, and Miller

not shown), one component from Chinese Spring was missing (see Fig. 2, double-headed arrow). The gene for this component had previously been assigned to chromosome 7D (Payne et al., 1985) from two-dimensional analyses of nullisomic-tetrasomic lines of Chinese Spring.

Several other lines were available which enabled the endosperm protein genes on 7H ch to be located to chromosome arms. In lines (7A)7Heha and (7D)7HCha, the a arm only of 7H ch had substituted for chromosomes 7A and 7D, respectively. Both lines contained protein 19 but lacked 9B and 12-14. In contrast, another diteloeentric substitution line, (7B)7H~ht3, produced compo- nents 9B and 12-14 but not 19. Analysis of the Chinese Spring ditelocentric 7H~hfl addition line confirmed the findings that components 9B and 12-14 must be coded by genes on the ~ arm of 7H oh, and component 19 on the arm.

DISCUSSION

There are probably only two major groups of storage proteins in flowering plants, the prolamins and the globulins (Shewry and Miflin, 1985). The former appear to be restricted to the grass family, the Gramineae. Within the Triticeae, a tribe containing the economically important cereals, wheat, rye, and barley, the genomic distribution of the prolamin encoding genes has been studied extensively in recent years. The genes coding for the HMW prolamin subunits appear to have maintained the same chromosome position during the evolution of this tribe. These genes lie close to the centromere on the long arms of chromosomes 1A, 1B, and 1D in bread wheat (Payne et al., 1982), chromosome 5(1H) in barley (Shewry et al., 1983), and chromosome 1R in rye (Singh and Shepherd, 1984). These chromosomes all belong to the same homoeologous group.

The same chromosomes also carry either all or the majority of the remaining prolamin genes in these three cereals. The genes occur toward the end of the short arms and so are only weakly linked to the HMW prolamin genes [wheat (Payne et al., 1982); barley (Shewry et al., 1983); rye (Shewry et al., 1984a)]. In wheat there is only one major prolamin (gliadin) locus on the short arm of each group 1 chromosome (Payne et al., 1984b) and there is some evidence for one locus only on the short arm of the homoeologous chromosome in rye (Shewry et al., 1984a). In barley, however, there are two tightly linked major loci, one coding for B-hordeins and the other for C-hordeins (Doll and Brown, 1979). Recent nucleotide sequencing of cDNA clones derived from these two protein groups has shown that they contain significant regions of sequence homology (Forde et al., 1985) and the authors concluded that they had a common evolutionary origin.

Although no recombination mapping experiments have yet been under-

Hordeum chilense Proteins 63

taken for H. chilense, it was shown in this study that the H M W prolamin genes occur on the long arm of chromosome 1H ch and the genes for the majority of the smaller prolamins, on the short arm. A distribution of prolamin genes along this chromosome similar to those of homoeologues in wheat, rye, and barley is therefore probable. In cultivated barley all the prolamin genes occur on chromosome 5(1H), but in the remaining cereals some have become scattered among different chromosomes in the genomes.

In bread wheat, approximately one-third of the prolamin genes, those coding for ~- and t3-gliadins, occur at one locus, toward the end of the short arm of each group 6 chromosome (Dvof~ik and Chert, 1983; Payne and Holt, unpublished results). Nucleotide sequencing studies have shown that these proteins (Kasarda et al., 1984) possess significant homology to 3,-gliadins/ L M W glutenin subunits (Bartels and Thompson, 1983), whose genes occur on the group 1 chromosomes. Since the latter must be derived from the ancestral chromosome carrying the prolamin genes, it is probable that the ~- and ¢/-gliadin genes were transferred to the group 6 chromosomes by a reciprocal translocation, as suggested previously (Payne et al., 1982). :In cultivated rye, Secale cereale, no prolamin genes are found on the homoeologous chromo- some 6R, but genes coding for 75K 3,-secalins are present on 2R (Shewry et al., 1984a), presumably also as a result of a translocation. However, a related species in the same genus, S. montanum, contains the 75K 3,-secalin genes on 6R r~ (homoeologous to group 6 chromosomes of wheat) rather than on the homoeologous group 2 chromosome, 2R m (Shewry et al., 1985a,b). As the 75K 3,-secalins are related to o~- and/3-gliadins, the authors speculated that a translocation occurred in a common ancestor of wheat and rye, transferring prolamin genes from chromosome 1 to chromosome 6. Prior to the divergence ofS. cereale from S. montanum, a further translocation between chromosome 6R and chromosome 2R occurred.

In H. chilense a further variation is seen, for while no detectable prolamin genes occur on chromosome 2H ch or 6H ~h, genes for two prominent proteins, thought to be prolamins from their solubility properties, were found on the/3 arm of chromosome 7H ~h. Presumably another different translocation was responsible. A further complication is that genes for minor proteins, also thought to be prolamins, occur on the o~ arm of 7H ~h and on 5H °h. Such a diversity in genomic distribution of storage protein genes was not shown for wheat (Jackson et al., 1983) using analytical techniques similar to those studied here. More detailed biochemical work, such as amino acid sequencing, is required to determine whether these proteins encoded on chromosomes 5 and 7 are indeed prolamins.

The two-dimensional electrophoretic procedure used in this study has the advantage over other two-dimensional procedures often used to analyze cereal grain proteins (Wrigley and Shepherd, 1973; Lafiandra et al., 1984) of being

64 Payne, Holt, Reader, and Miller

able to fractionate total proteins rather than just prolamin storage proteins. Analysis by this procedure clearly resolves proteins that are coded by three of the seven chromosome pairs in H. chilense and in rye and 11 of the 21 chromosome pairs in bread wheat (Payne et al., 1985). This technique is being exploited at this institute to assist in the development and characterization of wheat-alien chromosome addition lines involving other members of the Triticeae.

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Doll, H., and Brown, A. H. D. (1979). Hordein variation in wild (Hordeum spontaneum) and cultivated (H. vulgare) barley. Can. J. Genet. Cytol. 21:391.

Dvorak, J., and Chen, K.-C. (1983). Distribution of non-structural variation between wheat cultivars along chromosome arm 6Bp: Evidence from the linkage map and physical map of the arm. Genetics 106:325.

Field, J. M., Shewry, P. R., and Miflin, B. J. (1983). Aggregation states of alcohol-soluble storage proteins of barley, rye, wheat and maize. J. Sci. Fd. Agr. 34:362.

Forde, B. G., Kreis, M., Williamson, M. S., Fry, R. P., Pywell, J., Shewry, P. R., Bunce, N., and Miflin, B. J. (1985). Short tandem repeats shared by B- and C-hordein cDNAs suggest a common evolutionary origin for two groups of cereal storage protein genes. EMBO J. 4:9.

Holt, L. M., Astin, R., and Payne, P. I. (1981). Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin. 2. Relative isoelectric points determined by two-dimensional fractionation in polyacrylamide gels. Theor. Appl. Genet. 60:237.

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Kasarda, D. D., Okita, T. W., Bernardin, J. E., Baeker, P. A., Nimmo, C. C., Lew, E. J.-L., Dietler, M. D., and Greene, F. C. (1984). Nucleic acid (cDNA) and amino acid sequences of a-type gliadins from wheat (Triticum aestivum). Proc. Natl. Acad. Sci. USA 81:4712.

Lafiandra, D., Kasarda, D. D., and Morris, R. (1984). Chromosomal assignment of genes coding for the wheat gliadin protein components of the cultivars "Cheyenne" and "Chinese Spring" by two-dimensional (two-pH) electrophoresis. Theor. Appl. Genet. 68:531.

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O'Farrell, P. Z., Goodman, H. M,, and O'Farrell, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133.

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Hordeum chilense Proteins 65

studies on the high-molecular-weight subunits of wheat glutenin. Part 3. Telocentric mapping of the subunit genes on the long arms of the homoeologous group 1 chromosomes. Theor. Appl. Genet. 63:129.

Payne, P. I., Holt, L. M., Jackson, E. A., and Law, C. N. (1984a). Wheat storage proteins: Their genetics and their potential for manipulation by plant breeding. Phil. Trans. R. Soc. Lond B 304:359.

Payne, P. I., Jackson, E. A., Holt, L. M., and Law, C. N. (1984b). Genetic linkage between endosperm storage protein genes on each of the short arms of chromosomes 1A and 1B in wheat. Theor. Appl. Genet. 67:235.

Payne, P. I., Holt, L. M., Jarvis, M. G., and Jackson, E. A. (1985). Two-dimensional fractionation of the endosperm proteins of bread wheat (Triticum aestivum): Biochemical and genetical studies. Cereal Chem. 62:319.

Shewry, P. R., and Miflin, B. J. (1985). Seed storage proteins of economically important cereals. Adv. Cer. Sci. Technol. 7:1.

Shewry, P. R., Finch, R. A., Parmar, S., Franklin, J., and Miflin, B. J. (1983). Chromosomal location of Hor 3, a new locus governing storage proteins in barley. Heredity 50:179.

Shewry, P. R., Bradberry, D., Franklin, J., and White, R. P. (1984a). The chromosomal locations and linkage relationships of the structural genes for the prolamin storage protein (secalins) of rye. Theor. Appl. Genet. 69:63.

Shewry, P. R., Miflin, B. J., and Kasarda, D. D. (1984b). The structural and evolutionary relationships of the prolamin storage proteins of barley, rye and wheat. Phil. Trans. R. Soc. Lond. B 304:297.

Shewry, P. R., Parmar, S., and Miller, T. E. (1985a). Chromosomal location of the structural genes for the Mr 75,000 7-secalins in Secale montanum Guss: Evidence for a translocation involving chromosomes 2R and 6R in cultivated rye (Secale cereale). Heredity 54:381.

Shewry, P. R., Parmar, S., Fulrath, N., Kasarda, D. D., and Miller, T. E. (1985b). Chromosomal locations of the structural genes for secalins in wild perennial rye (Secale montanum) and cultivated rye (S. cereale) determined by two-dimensional electrophoresis. Can. J. Genet. Cytol. 28:76.

Singh, N. K., and Shepherd, K. W. (1984). Mapping of the genes controlling high-molecular- weight glutenin subunits of rye on the long arm of chromosome 1R. Genet. Res. Cambr. 44:117.

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