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.     

High-molecular-weight glutenin subunits in the Cylindropyrum and Vertebrata section of the Aegilops genus and identification of subunits related to those encoded by the Dx alleles of common wheat

The high-molecular-weight (HMW) glute-nin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1D^tx subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1D^tx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae. ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat. Received: 5 November 1999 / Accepted: 12 February 2000     

Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits

Wheat HMW glutenin subunit genes 1Ax1 and 1Dx5 were introduced, and either expressed or overexpressed, into a commercial wheat cultivar that already expresses five subunits. Six independent transgenic events were obtained and characterized by SDS-PAGE and Southern analysis. The 1Dx5 gene was overexpressed in two events without changes in the other endosperm proteins. Overexpression of 1Dx5 increased the contribution of HMW glutenin subunits to total protein up to 22%. Two events express the 1Ax1 subunit transgene with associated silencing of the 1Ax2* endogenous subunit. In the SDS-PAGE one of them shows a new HMW glutenin band of an apparent M_r lower than that of the 1Dx5 subunit. Southern analysis of the four events confirmed transformation and suggest that the transgenes are present in a low copy number. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic wheat expressing the 1Ax1 subunit transgene and overexpressing the Dx5 gene. Transgenes and expression patterns were stably transmitted to the progenies in all the events except one where in some of the segregating T₂ seeds the silencing of all HMW glutenin subunits was reverted associated with a drastic lost of transgenes from a high to a low copy number. The revertant T₂ seeds expressed the five endogenous subunits plus the 1Ax1 transgene. Received: 16 June 1999 / Accepted: 29 July 1999     

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species

Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.     

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.     

Comparative analysis of the D genome-encoded high-molecular weight subunits of glutenin

Four genes encoding novel 1Dx-type high-molecular weight (HMW) subunits were amplified by polymerase chain reaction, two each from Aegilops tauschii and bread wheat Triticum aestivum. The two subunits from Ae. tauschii (1Dx2.1^t and 1Dx2^t) were both very similar in sequence to subunit 1Dx2 from bread wheat. In contrast, the two novel bread wheat subunits (1Dx2.2 and 1Dx2.2*) differed from subunit 1Dx2 in having different internally duplicated regions (of 132 and 186 amino acid, respectively) within their repetitive domains. These duplicated sequences were located adjacent to the regions from which they had been duplicated and had complete intact repeat motifs at each end. The implications of these results for HMW subunit evolution and wheat quality improvement are discussed.     

Characterization of HMW prolamines and their coding sequences from Crithopsis delileana

The high-molecular-weight (HMW) prolamines subunits and their coding sequences from wheat-related diploid species Crithopsis delileana were investigated. Only one HMW prolamine subunit with the similar electrophoresis mobility to the y-type HMW glutenin subunit of hexaploid wheat was observed in two accessions of C. delileana by SDS-PAGE analyses of the total storage protein fractions. It was confirmed by sequencing and expression analysis that this prolamine subunit was an x-type subunit. The amino acid sequence of this subunit had the similar typical structure to those of x-type HMW glutenin genes previously described in wheat. An in-frame stop codon was found in the coding sequences of y-type prolamine subunits. It was found by specifically extraction of HMW prolamines and sequence analysis that the coding regions of Ky prolamine subunit gene is very likely to be not expressed as a full-length protein. Phylogenetic analysis indicated that the Kx subunit could be clustered together with 1Ax1 subunit by an interior paralleled branch, and Ky subunit (inactive) was most closely related to the 1Ay subunit. The coding sequences of Kx subunit could successfully be expressed in bacterial expression system, and the expressed protein had the same electrophoresis mobility as the Kx subunit from the seed of C. delileana. It was the first time that the HMW prolamines subunits encoded by K genome of C. delileana were characterized.     

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.     

Cloning and molecular characterization of a novel x-type glutenin gene Glu-D(t)1 from Aegilops tauschii

A novel gene encoding an x-type high molecular weight glutenin subunit (HMW-GS), designated 1Dx1.1 ^t , was isolated from Aegilops tauschii. It is the largest HMW-GS gene reported so far in this species and its product has a slower mobility than that of subunit 1Ax1 in SDS-PAGE. The open reading frame (ORF) of the gene was 2,628 bp, encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1^t had high similarity with other 1Dx subunits but also had two unique characteristics. Firstly, a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits was absent from subunit Dx1.1^t. Secondly, three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence (GQL) were present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1^t clustered with other known 1Dx subunits.     

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     

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。     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定(英文)

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列。表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似。在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区。但是,本研究所克隆的沉默型1Ay基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白。讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及1Ay基因沉默的机制。     

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality

Summary A gene encoding the high-molecular-weight (HMW) subunit of glutenin 1Ax1 was isolated from bread wheat cv Hope. Comparison of the deduced amino acid sequence with that previously reported for an allelic subunit, 1Ax2^*, showed only minor differences, which were consistent with both subunits being associated with good bread-making quality. Quantitative analyses of total protein extracts from 22 cultivars of bread wheat showed that the presence of either subunit 1Ax1 or 1Ax2^*, when compared with a null allele, resulted in an increase in the proportion of HMW subunit protein from ca. 8 to 10% of the total. It is suggested that this quantitative increase in HMW subunit protein may account for the association of 1Ax subunits with good quality.EMBL Data Library. Accession number: X61009     

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S^s genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S^sx49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S^sy49077) from the same accession. Consequently, 1S^sx49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S^sx49077 subunit is presented.     

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T₂ plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列.表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似.在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区.但是,本研究所克隆的沉默型1Av基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白.讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及lAy基因沉默的机制.     

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.     

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