Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat

Specific amplification of the complete coding region of all six high-molecular-weight (HMW) glutenin genes present in hexaploid wheat was obtained by the polyerase chain reaction (PCR). Primers specific for the N-terminal region of the 1Dx gene and for the repetitive domain of the y-type HMW glutenin genes were also developed. Although the primers were constructed on the basis of the nucleotide sequences of HMW glutenin genes present in T. aestivum L. cv Cheyenne, they were very efficient in amplifying HMW glutenin genes of diploid and tetraploid wheat species. PCR analysis of HMW glutenin genes of T. urartu Tuman., T. longissimum (Schweinf. & Muschl.) Bowden and T. speltoides (Tausch) Gren. ex Richt, showed a high degree of length polymorphism, whereas a low degree of length variation was found in accessions of T. tauschii (Coss.) Schmal. Furthermore, using primers specific for the repetitive regions of HMW genes, we could demonstrate that the size variation observed was due to a different length of the central repetitive domain. The usefulness of the PCR-based approach to analyze the genetic polymorphism of HMW glutenin genes, to isolate new allelic variants, to estimate their molecular size and to verify the number of cysteine residues is discussed.     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Molecular marker-assisted selection of HMW glutenin alleles related to wheat bread quality by PCR-generated DNA markers

While quality in hexaploid wheat (Triticum aestivum L. em Thell.) is a very complex trait, it is known that the water-insoluble gluten proteins are responsible for the elasticity and chohesiveness (strength) of dough and are therefore important determinants of breadmaking quality. High-molecular-weight (HMW) glutenin subunits encoded by genes on the long arm of group 1 chromosomes have been associated with gluten strength, and a portion of the variability between cultivars can be attributed to glutenin subunit composition. Good or poor wheat breadmaking quality is associated with two allelic pairs at the Glu-D1 complex locus, designated 1Dx5–1Dy10 and 1Dx2–1Dy12, respectively. Among the HMW glutenin subunits encoded at Glu-B1, Bx7 is quite common, being associated with either of two subunits, By8 or By9. Both allelic pairs contribute moderately well to good breadmaking quality by increasing dough elasticity. Glutenin subunit screening is accomplished using electrophoresis (SDS-PAGE). In this paper, I report the development of an alternative screening method based on glutenin genes themselves using the polymerase chain reaction (PCR). This easy, quick and non-destructive PCR-based approach is an efficient alternative to standard procedures for selecting bread-wheat genotypes with good breadmaking characteristics. Received: 14 August 1999 / Accepted: 21 March 2000     

Wheat storage proteins: diversity of HMW glutenin subunits in wild emmer from Israel

Summary The diversity of HMW glutenin subunits in the tetraploid wild progenitor of wheat, Triticum turgidum var. dicoccoides was studied electrophoretically in 231 individuals representing 11 populations of wild emmer from Israel. The results show that (a) The two HMW glutenin loci, Glu-A1 and Glu-B1, are rich in variation, having 11 and 15 alleles, respectively, (b) Genetic variation in HMW glutenin subunits is often severely restricted in individual populations, supporting an island population genetic model, (c) Significant correlations were found between glutenin diversity and the frequencies of specific glutenin alleles and physical (climate and soil) and biotic (vegetation) variables. Our results suggest that: (a) at least part of the glutenin polymorphisms in wild emmer can be accounted for by environmental factors and (b) the endosperm of wild emmer contains many allelic variants of glutenin storage proteins that are not present in bread wheat and could be utilized in breeding varieties with improved bread-making qualities.     

Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat (Triticum turgidum ssp. dicoccum Schrank)

Emmer wheat (Triticum turgidum ssp. dicoccum Schrank) is hulled wheat that survives in marginal areas of the Mediterranean Region. The HMW and LMW glutenin subunit composition of 97 accessions of emmer wheat from Spain have been analysed by SDS-PAGE. For the HMW glutenin subunits, four allelic variants were detected for the Glu-A1 locus; one of them has not been previously described. For the Glu-B1 locus, three of the nine alleles detected have not been found before. A high degree of variation was evident for the LMW glutenin subunits, and up to 23 different patterns were detected for the B-LMW glutenin subunits. Considering both types of proteins (HMW and LMW), 30 combinations were found between all the evaluated lines. This wide polymorphism can be used to transfer new quality genes to wheat, and to widen its genetic basis. Received: 13 June 2000 / Accepted: 3 July 2000     

New DNA markers for high molecular weight glutenin subunits in wheat

End-use quality is one of the priorities of modern wheat (Triticum aestivum L.) breeding. Even though quality is a complex trait, high molecular weight (HMW) glutenins play a major role in determining the bread making quality of wheat. DNA markers developed from the sequences of HMW glutenin genes were reported in several previous studies to facilitate marker-assisted selection (MAS). However, most of the previously available markers are dominant and amplify large DNA fragments, and thus are not ideal for high throughput genotyping using modern equipment. The objective of this study was to develop and validate co-dominant markers suitable for high throughput MAS for HMW glutenin subunits encoded at the Glu-A1 and Glu-D1 loci. Indels were identified by sequence alignment of allelic HMW glutenin genes, and were targeted to develop locus-specific co-dominant markers. Marker UMN19 was developed by targeting an 18-bp deletion in the coding sequence of subunit Ax2* of Glu-A1. A single DNA fragment was amplified by marker UMN19, and was placed onto chromosome 1AL. Sixteen wheat cultivars with known HMW glutenin subunits were used to validate marker UMN19. The cultivars with subunit Ax2* amplified the 362-bp fragment as expected, and a 344-bp fragment was observed for cultivars with subunit Ax1 or the Ax-null allele. Two co-dominant markers, UMN25 and UMN26, were developed for Glu-D1 by targeting the fragment size polymorphic sites between subunits Dx2 and Dx5, and between Dy10 and Dy12, respectively. The 16 wheat cultivars with known HMW glutenin subunit composition were genotyped with markers UMN25 and UMN26, and the genotypes perfectly matched their subunit types. Using an Applied Biosystems 3130xl Genetic Analyzer, four F₂ populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.     

Genetic polymorphism in low-molecular-weight glutenin genes from Triticum aestivum, variety Chinese Spring

Low-molecular-weight (LMW) glutenin subunits consist mainly of two domains, one at the N- terminus which contains repeats of short amino-acid motifs, and a non-repetitive one rich in cysteine, at the C- terminal region. In previous reports, polyacrylamide-gel electrophoresis has been used to show that large size variation exists among LMW and HMW glutenin subunits, and it has been suggested that deletions and insertions within the repetitive region are responsible for these variations in length. In this study, PCR-amplification of genomic DNA (Triticum aestivum variety Chinese Spring) was used to isolate three full-length LMW glutenin genes: LMWG-MB1, LMWG-MB2 and LMWG-MB3. The deduced amino-acid sequences show a high similarity between these ORFs, and with those of other LMW glutenin genes. Comparisons indicate that LMWG-MB1 has probably lost a 12-bp fragment through deletion and that LMWG-MB1 and LMWG-MB2 have an insertion of 81 bp within the repetitive domain. The current study has shown direct evidence that insertions and/or deletions provide a mechanistic explanation for the allelic variation, and the resultant evolution, of prolamin genes. Single-base substitutions at identical sites generate stop codons in both LMWG-MB2 and LMWG-MB3 indicating that these clones are pseudogenes. Received: 7 May 1999 / Accepted: 17 June 1999     

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.     

Characterisation of high- and low-molecular weight glutenin subunits associated to the D genome of Aegilops tauschii in a collection of synthetic hexaploid wheats

Synthetic hexaploid wheats (2n=6x=42, AABBDD) involving genomes from Triticum turgidum (2n= 4x=28, AABB) and Aegilops tauschii (2n=2x=14, DD) have been produced as a means for introducing desirable characteristics into bread wheat. In the present work we describe the genetic variability present at the Glu-D ^t 1 and Glu-D ^t 3 loci, encoding high- (HMW) and low-molecular-weight (LMW) glutenin subunits respectively, derived from Ae. tauschii, using electrophoretic and chromatographic methods, in a collection of synthetic hexaploid wheats. A wide variation both in mobility and surface hydrophobicity of HMW glutenin subunits was observed between different accessions of Ae. tauschii used in the production of the synthetic hexaploids. A combination of electrophoretic and chromatographic methods improves the identification of HMW glutenin subunits; in fact subunits with identical apparent mobility were revealed to have a different surface hydrophobicity by reversed-phase high performance liquid chromatography. None of the Dx5^t subunits present in Ae. tauschii showed the presence of the extra cysteine residue found in the HMW glutenin subunit Dx5 of Triticum aestivum, as revealed by selective amplification with polymerase chain reaction (PCR). The wide variability and the high number of subunits encoded by the Glu-D ^t 3 locus suggests that Ae. tauschii may be a rich source for enhancing the genetic variability of glutenin subunits in bread wheat and improving bread-making properties. Received: 3 March 2001 / Accepted: 23 March 2001     

Inheritance of glutenin protein subunits of wheat

Summary The inheritance of the high-molecular-weight (HMW) glutenin protein subunits in hexaploid wheat has been investigated by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis to examine the segregation of these subunits in 496 test-cross seeds. The parents of the f₁ hybrid were chosen so that the test-cross seeds segregated for all the HMW glutenin bands. Two glutenin subunits from one parent, believed to be controlled by genes on chromosome 1D, segregated as alternatives to two glutenin subunits from the other parent, a result that supports the assumption that these subunits are controlled by allelic genes at each of two loci that are very closely linked. Similar results were obtained for glutenin subunits believed to be controlled by chromosome IB, which suggests that these subunits are controlled also by allelic genes at each of two loci that are very closely linked. A single glutenin subunit band, believed to be controlled by chromosome 1A, segregated as an alternative to a single glutenin band from the other parent, except that one seed did not possess either band. It was concluded that these bands are controlled either by allelic genes or by nonallelic genes that are very closely linked.     

Multiplex PCR identification of wheat HMW glutenin subunit genes by allele-specific markers

Wheat bread-making quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular weight (HMW) glutenin subunits encoded by the Glu-1 genes. A multiplex polymerase chain reaction (PCR) method was developed to identify the allele composition of HMW glutenin complex Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) in common wheat genotypes. The study of multiplex PCR to obtain a well-balanced set of amplicons involved examination of various combinations of selected primer sets and/or thermal cycling conditions. One to three simultaneously amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis and differences between Ax1, Ax2* and Axnull genes of Glu-A1 loci, Bx6, Bx7 and Bx17 of Glu-B1, and Dx2, Dx5 and Dy10 genes of Glu-D1 loci were revealed. A complete agreement was found in identification of HMW glutenin subunits by both multiplex PCR analysis and SDS-PAGE for seventy-six Polish cultivars/strains of both spring and winter common wheat. Rapid identification of molecular markers of Glu-1 alleles by multiplex PCR can be an efficient alternative to the standard separation procedure for early selection of useful wheat genotypes with good bread-making quality.     

小伞山羊草高分子量麦谷蛋白亚基及其基因的鉴定

运用SDS_PAGE和分子克隆技术 ,对小伞山羊草 (Aegilopsumbellulata ,UU ,2n =2x =14)的高分子量麦谷蛋白亚基 (1Ux ,1Uy)及其编码基因进行了鉴定。SDS_PAGE分析表明小伞山羊草不同基因型中的 1Ux的电泳迁移率接近或慢于普通小麦 1Dx2 .2亚基的电泳迁移率 ,1Uy亚基的电泳迁移率一般接近或慢于普通小麦的 1Dy类亚基。采用PCR扩增技术获得了 1Ux和 1Uy亚基编码基因的全长编码区 ,并对一个 1Uy基因的全长编码区进行了全序列测定。对推导的氨基酸序列进行比较发现 1Ux和 1Uy亚基具有与来自于其他物种的高分子量麦谷蛋白亚基一致的一级结构 ,聚类分析显示 1Ux和 1Uy亚基与D基因组编码的高分子量麦谷蛋白亚基在起源和进化上具有较高的相似性。     

高冰草中高分子量麦谷蛋白亚基的编码基因

通过SDS-PAGE法分析了高冰草(Agropyron elongatum (Host) Nevski)种子麦谷蛋白亚基,发现高冰草的麦谷蛋白亚基种类比普通小麦更加丰富。通过基因组PCR法用高分子量麦谷蛋白亚基基因的特异引物从高冰草核基因组中分离出了7条麦谷蛋白亚基的全编码序列,分别命名为AgeloG1~AgeloG7。其中的5条已进行全序列测定,对AgeloG1和AgeloG4进行了末端测序。尽管其中的4条基因的编码序列(AgeloG4, AgeloG5, AgeloG6和AgeloG7)小于1.8 kb,但是对从克隆到的序列推导出的氨基酸序列与已经发表的小麦高分子量麦谷蛋白亚基序列进行对比分析发现,这些亚基与来自小麦的高分子量麦谷蛋白亚基具有很高的同源性。并且对信号肽、N-、C-末端的氨基酸序列分析显示,这7条序列编码的亚基皆为y-型亚基。用5条全部测序的编码序列与普通小麦的A、B、D、粗山羊草的D、圆柱山羊草的C、伞穗山羊草的U、黑麦的R染色体的编码高分子量麦谷蛋白的序列进行了聚类分析。表明,AgeloG2与小麦1Dy, AgeloG3与小麦1By, AgeloG5、AgeloG6和AgeloG7与小麦1Ay在起源和进化上有较高的相似性。     

Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica

Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter. Three Ae. cylindrica and two T. timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined. A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae. cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T. timopheevi HMW subunits as 1Gx, 1Ax and 1Ay. It was not clear whether or not a 1Gy HMW subunit was present in T. timopheevi. The PCR products from Ae. cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T. timopheevi were derived from 1Ax and 1Ay genes. The 1Cx, 1Gx and 1Gy sequences were not amplified successfully. The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat (Triticum aestivum). Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high M_r polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species. There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat. The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE. The truncated 1Dx gene from Ae. cylindrica failed to express in E. coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts.     

High-Molecular-Weight Glutenin Subunit Genesin in Decaploid Agropyron elongatum

Seven genes encoding glutenin subunits that present in Agropyron elongatum (Host) Nevski were cloned by PCR analysis and named AgeloG1 to AgeloG7. The complete open reading frames (ORFs) of the seven genes were amplified with primers special for high-molecular-weight (HMW) glutenin subunit genes and subsequently cloned and sequenced. Five of them were completely sequenced, and the other two (AgeloG1 and AgeloG4) were sequenced at the two ends only. Comparison of amino acid sequences suggested that the primary structure of the subunits encoded by the seven genes was very similar to that of y-type HMW glutenin subunits published from wheat, though four of them (AgeloG4, AgeloG5, AgeloG6 and AgeloG7) were shorter than 1.8 kb. Phylogenetic analysis of the five completely sequenced genes and those subunit genes of Triticum aestivum L. (AABBDD), Aegilops tauschii Coss. (DD), Aegilops caudata L. (CC), Secale cereale L. (RR) and Aegilops umbellulata Zhuk. (UU) indicated that the AgeloG2 was most closely related to 1Dy; the AgeloG3 was to 1By; the AgeloG5, AgeloG6 and AgeloG7 were to 1Ay.     

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.     

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2^t and T2, and was considered as part of the same allele at the Glu-D ^t 1 locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2^t and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an ω-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of ω-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F₂ grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii (Glu-D ^t 1 and Gli-D ^t 1) segregated as alleles of the Glu-D1 and Gli-D1 loci of bread wheat. A new locus encoding the ω-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively. Received: 13 October 2000 / Accepted: 18 April 2001     

Isolation and characterization of Glu-1 genes from the St genome of Pseudoroegneria libanotica

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.     

Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat

The visco-elastic properties of bread flour are firmly associated with the presence or absence of certain HMW subunits coded by the Glu-1 genes. Identifying allelic specific molecular markers (AS-PCR) associated with the presence of Glu-1 genes can serve as a valuable tool for the selection of useful genotypes. This paper reports the use of primers designed from nucleotide sequences of the Glu-D1 gene of wheat (AS-PCR for Glu-D1y10) that recognise and amplify homologous sequences of the Glu-R1 gene subunits of rye. The primers amplify the complete coding regions and provided two products of different size in rye, in wheats carrying the substitution 1R(1D) and in rye-wheat aneuploid lines carrying the long arm of chromosome 1R. The location, the molecular characterisation of these sequences and their expression during grain ripening seem to demonstrate that the amplification products correspond to structural genes encoding the high-molecular-weight (HMW) glutenins of rye. The homology of the rye gene to subunits encoding HMW glutenins in wheat was confirmed by Southern blots and sequencing. The amplification-products were cloned, sequenced and characterised, and the sequences compared with the main glutenin subunits of wheat and related species. Further, an RT-PCR experiment was performed using primers designed from the sequence of both amplified products. This assay demonstrated that both sequences are expressed in endosperm during grain ripening. The results of these analyses suggest that both gene subunits correspond to x- and y-type genes of the Glu-R1 locus of rye. Received: 11 December 2000 / Accepted: 17 April 2001