High frequency of HMW-GS sequence variation through somatic hybridization between Agropyron elongatum and common wheat

A symmetric somatic hybridization was performed to combine the protoplasts of tall wheatgrass (Agropyron elongatum) and bread wheat (Triticum aestivum). Fertile regenerants were obtained which were morphologically similar to tall wheatgrass, but which contained some introgression segments from wheat. An SDS-PAGE analysis showed that a number of non-parental high-molecular weight glutenin subunits (HMW-GS) were present in the symmetric somatic hybridization derivatives. These sequences were amplified, cloned and sequenced, to deliver 14 distinct HMW-GS coding sequences, eight of which were of the y-type (Hy1–Hy8) and six x-type (Hx1–Hx6). Five of the cloned HMW-GS sequences were successfully expressed in E. coli. The analysis of their deduced peptide sequences showed that they all possessed the typical HMW-GS primary structure. Sequence alignments indicated that Hx5 and Hy1 were probably derived from the tall wheatgrass genes Aex5 and Aey6, while Hy2, Hy3, Hx1 and Hy6 may have resulted from slippage in the replication of a related biparental gene. We found that both symmetric and asymmetric somatic hybridization could promote the emergence of novel alleles. We discussed the origination of allelic variation of HMW-GS genes in somatic hybridization, which might be the result from the response to genomic shock triggered by the merger and interaction of biparent genomes.     

Generation of novel high quality HMW-GS genes in two introgression lines of <Emphasis Type="Italic">Triticum aestivum</Emphasis>/<Emphasis Type="Italic">Agropyron elongatum</Emphasis>

Background  

High molecular weight glutenin subunits (HMW-GS) have been proved to be mostly correlated with the processing quality of common wheat (Triticum aestivum). But wheat cultivars have limited number of high quality HMW-GS. However, novel HMW-GS were found to be present in many wheat asymmetric somatic hybrid introgression lines of common wheat/Agropyron elongatum.     

Characterization of high molecular weight glutenin subunit genes from the Ns genome of Psathyrostachys juncea

The Ns genome of the genus Psathyrostachys possesses superior traits useful for wheat improvement. However, very little is known about the high molecular weight (HMW) subunits of glutenin encoded by the Ns genome. In this paper, we report the isolation of four alleles of HMW glutenin subunit gene from Psathyrostachys juncea. Sequence alignment data shows the four alleles have similar primary structure with those in wheat and other wheat-related grasses, with some unique modifications. All four sequences more closely resemble y-type, rather than x-type, glutenins. However, our results show three of the subunits (1Ns2-4) contain an extra glutamine residue in the N-terminal region not found on typical y-type subunits, as well as the x-type subunit specific sequence LAAQLPAMCRL. These three subunits likely represent an intermediate state in the divergence between x- and y-type subunits. Results also indicate that the Ns genome is more closely related to the St genome of Pseudoroegneria than any other Triticeae genomes.     

A novel high molecular weight glutenin subunit from Australopyrum retrofractum

We describe the sequence of a gene encoding a high molecular weight glutenin subunit (HMW-GS) expressed in the endosperm of the wheat relative Australopyrum retrofractum. Although the subunit has a similar primary structure to that HMW-GS genes present in other Triticeae species, its N-terminal domain is shorter, its central repetitive domain includes a unique dodecameric motif, and its C-terminal domain contain an extra cysteine residue. A phylogenetic analysis showed that the Glu-W1 gene is neither a true x- nor a true y-type subunit, although it is more closely related to the y-type genes present in the K and E genomes than to any other published HMW-GS gene. All these results indicated that this novel subunit may undergo a special evolutionary process different from other Triticeae species. A flour supplementation experiment showed that the Glu-W1 subunit has a negative effect on dough quality, which might be the result of interaction between the two closely placed cysteine residues in the C-terminal region.     

Structure,variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence

In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> − 700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5′-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.     

Molecular cloning and comparative analysis of a y-type inactive HMW glutenin subunit gene from cultivated emmer wheat (Triticum dicoccum L.)

Cultivated emmer (Triticum dicoccum, 2n = 4x = 28, AABB) is closely related to bread wheat and possesses extensive allelic variations in high molecular weight glutenin subunit (HMW-GS) composition. These alleles may be an important genetic resource for wheat quality improvement. To isolate and clone HMW-GS genes from cultivated emmer, two pairs of allele-specific (AS) PCR primers were designed to amplify the coding sequence of y-type HMW-GS genes and their upstream sequences, respectively. The results showed that single bands of strong amplification were obtained through AS-PCR of genomic DNA from emmer. After cloning and sequencing the complete sequence of coding and 5'-flanking regions of a y-type subunit gene at Glu-A1 locus was obtained. Nucleotide and deduced amino acid sequences analysis showed that this gene possessed a similar structure as the previously reported Ay gene from common wheat, and is hence designated as Ay1d. The distinct feature of the Ay1d gene is that its coding region contains four stop codons and its upstream region has a 85-bp deletion in the same position of the Ay gene, which are probably responsible for the silencing of y-type subunit genes at Glu-A1 locus. Phylogenetic analysis of HMW glutenin subunit genes from different Triticum species and genomes were also carried out.     

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.     

PCR analysis of x- and y-type genes present at the complex Glu-A1 locus in durum and bread wheat

Genes (x-type) corresponding to different high-molecular-weight glutenin subunits encoded at the Glu-A1 locus present in bread- and durum-wheat cultivars have been selectively amplified by the polymerase chain reaction (PCR). DNA fragments corresponding to an unexpressed x-type gene were also amplified. As unexpressed y-type genes may or may not contain an 8-kb transposon-like insertion, two different sets of primers were designed to obtain amplification of DNA fragments corresponding to these genes. Amplified DNA fragments were also digested with restriction enzymes. The digestion patterns of amplified fragments corresponding to unusual x-type subunits showed similarities with genes encoding the most common subunits 2^* and 1. The unexpressed amplified x-type gene showed a restriction pattern similar to the one obtained with the allelic gene encoding high-molecular-weight glutenin subunit 1; homologies were also found within the repetitive region of the linked y-type genes. On the basis of these observations it is postulated that an ancestral active x-type gene, most likely corresponding to subunit 1, was silenced following the insertion of the 8-kb transposon-like fragment into the linked y-type gene. Received: 8 April 1996 / Accepted: 30 August 1996     

ChAy/Bx, a novel chimeric high-molecular-weight glutenin subunit gene apparently created by homoeologous recombination in Triticum turgidum ssp. dicoccoides

High-molecular-weight glutenin subunits (HMW-GSs) are of considerable interest, because they play a crucial role in determining dough viscoelastic properties and end-use quality of wheat flour. In this paper, ChAy/Bx, a novel chimeric HMW-GS gene from Triticum turgidum ssp. dicoccoides (AABB, 2n = 4x = 28) accession D129, was isolated and characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the electrophoretic mobility of the glutenin subunit encoded by ChAy/Bx was slightly faster than that of 1Dy12. The complete ORF of ChAy/Bx contained 1671 bp encoding a deduced polypeptide of 555 amino acid residues (or 534 amino acid residues for the mature protein), making it the smallest HMW-GS gene known from Triticum species. Sequence analysis showed that ChAy/Bx was neither a conventional x-type nor a conventional y-type subunit gene, but a novel chimeric gene. Its first 1305 nt sequence was highly homologous with the corresponding sequence of 1Ay type genes, while its final 366 nt sequence was highly homologous with the corresponding sequence of 1Bx type genes. The mature ChAy/Bx protein consisted of the N-terminus of 1Ay type subunit (the first 414 amino acid residues) and the C-terminus of 1Bx type subunit (the final 120 amino acid residues). Secondary structure prediction showed that ChAy/Bx contained some domains of 1Ay subunit and some domains of 1Bx subunit. The special structure of this HMW glutenin chimera ChAy/Bx subunit might have unique effects on the end-use quality of wheat flour. Here we propose that homoeologous recombination might be a novel pathway for allelic variation or molecular evolution of HMW-GSs.     

Generation of high frequency of novel alleles of the high molecular weight glutenin in somatic hybridization between bread wheat and tall wheatgrass

Somatic hybridization between bread wheat and tall wheatgrass (Agropyron elongatum) has generated fertile introgression progenies with novel combinations of high molecular weight glutenin subunits (HMW-GS). Most of these novel HMW-GS alleles were stably inherited. Sixteen HMW-GS sequences were PCR amplified from three introgression progeny lines and sequenced. The alignment of these sequences indicated that five, probably derived from point mutations of the parental genes, whereas eight likely represent the product of replication slippage. Three Glu-1Ay sequences appear to have lost the transposon presented in the parental gene. Two subunits carry an additional cysteine residue, which may be favorable to the quality of end-use product. We demonstrate that novel HMW-GS alleles can be rapidly generated via asymmetric somatic hybridization. Heng Liu and Shuwei Liu have contributed equally to the work.     

Molecular characterization and phylogenetic analysis of a novel glutenin gene (Dy10.1t) from Aegilops tauschii.

A novel y-type high molecular mass glutenin subunit (HMM-GS) possessing a mobility that is slightly slower than that of the subunit Dy10 obtained by SDS-PAGE, named Dy10.1t, in the wild wheat Aegilops tauschii was identified by 1- and 2-dimensional gel electrophoresis, capillary electrophoresis, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The gene encoding the HMM subunit Dy10.1t was amplified with allele-specific PCR primers, and the amplified products were cloned and sequenced. The coding domain of the Dy10.1t subunit gene consisted of 1980 bp encoding a protein of 658 residues with an M rs of 68 611 Da, which was similar to the M rs determined by MALDI-TOF-MS. The deduced amino acid sequence indicated that Dy10.1t subunit displayed a greater similarity to the Dy12 subunit, differing by only 8 amino acid substitutions. Six coding region single-nucleotide polymorphisms were discovered in the Dy10.1t gene by multiple alignments (1 per 330 bp), 1 in the N-terminal domain and the others in the central repeats. Five of them resulted in residue substitutions, whereas 3 created enzyme site changes. The homology and neighbour-joining trees constructed from code domain sequences of 20 x- and y-type glutenin genes from different Triticum species separated into 2 halves, which corresponded to the x-type and y-type HMM glutenin alleles. Phylogenetic analysis revealed that the Glu-1 gene duplication event probably occurred at about 16.83 million years ago, whereas the divergence times of A, B, and D genomes within x-type and y-type halves were before 7.047 and 10.54 million years ago, respectively.     

Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing of x-type Gene on Glu-B1

Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evidence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nucleotide sequence of 3 442 bp including upstream sequence of 1 070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contribution of the unique silenced Glu-B1 alleles to quality improvement was also discussed.     

Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale

This work reports the molecular characterisation of new alleles of the previously reported Glu-R1 locus. Wheat lines carrying the chromosome substitution 1R(1D), rye cultivars and related wild species were analysed. Five new x-type and four y-type Glu-R1 glutenin subunits were isolated and characterised. The coding region of the sequences shows the typical structure of the HMW glutenin genes previously described in wheat, with the N and C-terminal domains flanking the central repetitive region. Tri-, hexa- and nona-peptides found in the central repetitive region of wheat glutenin genes were also present in the rye genes. Duplications and deletions of these motifs are responsible for allelic variation at the Glu-R1 locus. Orthologous genes (from different genomes) were more closely related than paralogous genes (x- and y-type), supporting the hypothesis of gene duplication before Triticeae speciation. Differences in the number and position of cysteine residues identified alleles which in wheat are associated with good dough quality. SDS proteins encoded by some characterised alleles were presumptively identified.     

Intermolecular disulfide bonds link specific high-molecular-weight glutenin subunits in wheat endosperm.

A detergent wash extracted soluble proteins from wheat flour, leaving a residue enriched with insoluble glutenin aggregates. Digestion of this residue with endoproteinase Lys-C, which showed a limited specificity for glutenin subunits, produced several peptides with apparent molecular weights close to those of intact high-molecular-weight glutenin subunits. N-terminal sequencing indicated that the isolated peptides were composed of high-molecular-weight glutenin subunit fragments joined by an intermolecular disulfide bond. In two of these peptides, only two components were found, one from an x-type subunit and the other from a y-type subunit. The isolated peptides all contained at least one x-type C-terminal region and one y-type N-terminal region, suggesting a specific orientation to the intermolecular disulfide linkage.     

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.     

小麦高分子量谷蛋白亚基Glu-B1位点沉默基因的克隆与序列分析

二粒小麦（Triticum turgidum L．var．dicoccoides）具有极其丰富的遗传多样性,是栽培小麦品种改良的巨大基因库。在高分子量谷蛋白基因的组成上,它具有许多栽培小麦不存在的变异类型,在Glu—B1位点上的变异更大。我们利用种子贮藏蛋白的SDS—PAGE方法从原产于伊朗的二粒小麦材料PI94640中观察到缺失Glu—B1区的高分子量谷蛋白亚基。利用Glu-1Bx基因保守序列设计PCR引物,对该材料的总DNA扩增,获得了X型亚基编码基因（Glu-1Bxm）的全序列,其全长为3442bp含1070bp的启动子区。序列比较发现,Glu-1Bxm在启动子区序列与Glu—1Bx7的最为相似。而在基因编码区,我们发现Glu—1Bxm仅编码212个氨基酸,由于开放阅读框中起始密码子后第637位核苷酸发生了点突变,即编码谷酰胺的CAA突变为终止密码TAA,可能直接导致了该高分子量谷蛋白亚基的失活,这是我们在小麦Glu—B1位点基因沉默分子证据的首次报道。将Glu—1Bxm全序列与Glu—B1位点其他等位基因进行了系统树分析,发现Glu—1Bxm是较为古老的类型。本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。     

Molecular Mechanisms of HMW Glutenin Subunits from 1Sl Genome of Aegilops longissima Positively Affecting Wheat Breadmaking Quality

A wheat cultivar “Chinese Spring” chromosome substitution line CS-1S^l(1B), in which the 1B chromosome was substituted by 1S^l from Aegilops longissima, was developed and found to possess superior dough and breadmaking quality. The molecular mechanism of its super quality conformation is studied in the aspects of high molecular glutenin genes, protein accumulation patterns, glutenin polymeric proteins, protein bodies, starch granules, and protein disulfide isomerase (PDI) and PDI-like protein expressions. Results showed that the introduced HMW-GS 1S^l×2.3* and 1S^ly16* in the substitution line possesses long repetitive domain, making both be larger than any known x- and y-type subunits from B genome. The introduced subunit genes were also found to have a higher level of mRNA expressions during grain development, resulting in more HMW-GS accumulation in the mature grains. A higher abundance of PDI and PDI-like proteins was observed which possess a known function of assisting disulfide bond formation. Larger HMW-GS deposited in protein bodies were also found in the substitution line. The CS substitution line is expected to be highly valuable in wheat quality improvement since the novel HMW-GS are located on chromosome 1S^l, making it possible to combine with the known superior D×5+Dy10 subunits encoded by Glu-D1 for developing high quality bread wheat.     

A second ‘overexpression’ allele at the <Emphasis Type="Italic">Glu-B1</Emphasis> high-molecular-weight glutenin locus of wheat: sequence characterisation and functional effects

Bread is one of the major constituents of the human diet and wheat (Triticum aestivum L.) is the most important cereal for bread making. The gluten proteins (glutenins and gliadins) are recognised as important components affecting the processing quality of wheat flour. In this research, we investigated a particular glutenin subunit allele in an Australian cultivar, H45. Based on protein and DNA assays, the Glu-B1 allele of H45 seems to be Glu-B1al, an allele that includes a functional duplication of a gene encoding an x-type high-molecular-weight glutenin subunit, and is thought to increase dough strength through overexpression of that subunit. Yet H45 does not have the dough properties that would be expected if it carries the Glu-B1al allele. After confirming that H45 overexpresses Bx subunits and that it has relatively low un-extractable polymeric protein (an indicator of weak dough), we cloned and sequenced two Bx genes from H45. The sequences of the two genes differ from each other, and they each differ by four single-nucleotide polymorphisms (SNPs) from the sequence that has been reported for the Glu-B1al x-type glutenin genes of the Canadian wheat cultivar Glenlea. One of the SNPs leads to an extra cysteine residue in one of the subunits. The presence of this additional cysteine may explain the dough properties of H45 through effects on cross-linkage within or between glutenin subunits. We propose that the Glu-B1 allele of H45 be designated Glu-B1br, and we present evidence that Glu-B1br is co-inherited with low un-extractable polymeric protein.     

Isolation and molecular characterization of high molecular weight glutenin subunit genes 1Bx13 and 1By16 from hexaploid wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.     

The nucleotide and deduced amino acid sequences of an HMW glutenin subunit gene from chromosome 1B of bread wheat (Triticum aestivum L.) and comparison with those of genes from chromosomes 1A and 1D

Summary The nucleotide and deduced amino acid sequences of a high molecular weight glutenin subunit gene derived from chromosome 1B of bread wheat (Triticum aestivum L.) are reported. The encoded protein corresponds to the y-type subunit 1B9. Comparison of the 5 upstream untranslated regions of this gene and a previously reported silent y-type gene derived from chromosome 1A showed a deletion of 85 bp in the latter. A sequence present in this region of the 1By 9 gene shows homology with part of the -300 element which is conserved in the 5 upstream regions of other prolamin genes from barley, wheat and maize (Forde BG et al. 1985). It is suggested that the absence of this element is responsible for the lack of expression of the 1Ay gene. Comparison of the derived amino acid sequence with those reported previously for the silent 1Ay gene and the expressed x-type (1Dx2) and y-type (1Dy12) genes derived from chromosome 1D showed that the three y-type proteins are closely related. In contrast the x-type subunit (1Dx2) shows clear differences in the N-terminal region and in the number, type and organisation of repeats in the central repetitive domain.