High Molecular Weight Glutenin Genes Research Articles

Molecular Characterization and SNP-Based Molecular Marker Development of Two Novel High Molecular Weight Glutenin Genes from Triticum spelta L.

Spelt wheat (Triticum spelta L., 2n=6x=42, AABBDD) is a valuable source of new gene resources for wheat genetic improvement. In the present study, two novel high molecular weight glutenin subunits (HMW-GS) 1Ax2.1* at Glu-A1 and 1By19* at Glu-B1 from German spelt wheat were identified. The encoding genes of both subunits were amplified and cloned by allele-specific PCR (AS-PCR), and the complete sequences of open reading frames (ORF) were obtained. 1Ax2.1* with 2478 bp and 1By19* with 2163 bp encoded 824 and 720 amino acid residues, respectively. Molecular characterization showed that both subunits had a longer repetitive region, and high percentage of α-helices at the N- and C-termini, which are beneficial for forming superior gluten macropolymers. Protein modelling by AlphaFold2 revealed similar three-diamensional (3D) structure features of 1Ax2.1* with two x-type superior quality subunits (1Ax1 and 1Ax2*) and 1By19* with four y-type superior quality subunits (1By16, 1By9, 1By8 and 1By18). Four cysteine residues in the three x-type subunits (1Ax2.1*, 1Ax1 and 1Ax2*) and the cysteine in intermediate repeat region of y-type subunits were not expected to participate in intramolecular disulfide bond formation, but these cysteines might form intermolecular disulfide bonds with other glutenins and gliadins to enhance gluten macropolymer formation. The SNP-based molecular markers for 1Ax2.1* and 1By19* genes were developed, which were verified in different F2 populations and recombination inbred lines (RILs) derived from crossing between spelt wheat and bread wheat cultivars. This study provides data on new glutenin genes and molecular markers for wheat quality improvement.

High molecular weight glutenin gene diversity in Aegilops tauschii demonstrates unique origin of superior wheat quality

Central to the diversity of wheat products was the origin of hexaploid bread wheat, which added the D-genome of Aegilops tauschii to tetraploid wheat giving rise to superior dough properties in leavened breads. The polyploidization, however, imposed a genetic bottleneck, with only limited diversity introduced in the wheat D-subgenome. To understand genetic variants for quality, we sequenced 273 accessions spanning the known diversity of Ae. tauschii. We discovered 45 haplotypes in Glu-D1, a major determinant of quality, relative to the two predominant haplotypes in wheat. The wheat allele 2 + 12 was found in Ae. tauschii Lineage 2, the donor of the wheat D-subgenome. Conversely, the superior quality wheat allele 5 + 10 allele originated in Lineage 3, a recently characterized lineage of Ae. tauschii, showing a unique origin of this important allele. These two wheat alleles were also quite similar relative to the total observed molecular diversity in Ae. tauschii at Glu-D1. Ae. tauschii is thus a reservoir for unique Glu-D1 alleles and provides the genomic resource to begin utilizing new alleles for end-use quality improvement in wheat breeding programs.

Cloning and characterization of high molecular weight glutenin gene from Triticum aestivum cultivar Dacke

<p class="abstract"><strong>Background:</strong> High molecular weight (HMW) glutenin protein plays a crucial role in determining dough viscoelastic properties that determines the quality of wheat flour. The aim of the present study was to isolate, clone and analyze (<em>in silico</em>) the HMW glutenin gene of <em>Triticum aestivum</em> cultivar Dacke.</p><p class="abstract"><strong>Methods:</strong> Primers were designed to amplify a 2445 bp fragment of HMW glutenin gene. Ax type HMW glutenin gene from <em>Triticum aestivum</em> cultivar Dacke was isolated using PCR and it was sequenced by primer walking. </p><p class="abstract"><strong>Results:</strong> Amplified HMW glutenin gene was designated as HMWGAx. Sequence analysis revealed a complete open reading frame encoding 815 amino acid residues with N- and C terminal non-repetitive domain and a central repetitive domain. The calculated molecular weight of the deduced HMW glutenin protein was ~88 kDa and the number of cysteine residues in the HMWGAx was four, in accordance with other x type HMW glutenin proteins. Phylogenetic analysis revealed 100% homology to the previously studied Ax2* type HMW glutenin gene from cultivar Cheyenne. Predicted secondary structure results showed that it was similar to1Ax1 type of common wheat (<em>Triticum aestivum</em>), having superior flour milling quality.</p><p><strong>Conclusions:</strong> Sequence analysis suggests that HMWGAx protein significantly and positively correlates with the properties of elasticity and extensibility of gluten. </p>

Two Novel Y-Type High Molecular Weight Glutenin Genes in Chinese Wheat Landraces of the Yangtze-River Region.

High molecular weight glutenin subunits (HMW-GSs) are key determinants for the end-use quality of wheat. Chinese wheat landraces are an important resource for exploring novel HMW-GS genes to improve the wheat baking quality. Two novel Glu-1Dy HMW-GSs (designated as 1Dy12.6 and 1Dy12.7) were identified and cloned from two Chinese wheat landraces Huazhong830 and Luosimai. The 1Dy12.6 and 1Dy12.7 subunits were deposited as the NCBInr Acc. No KR262518, and KR262519, respectively. The full open reading frames (ORFs) of 1Dy12.6 and 1Dy12.7 were 2022 bp and 1977 bp, encoding for proteins of 673 and 658 amino acid residues, respectively. Each contains four typical primary regions of HMW-GSs (a signal peptide, N- and C-terminal regions, and a central repetitive region). Their deduced molecular masses (70,165 Da and 68,400 Da) were strikingly consistent with those identified by MALDI-TOF-MS (69,985Da and 68,407 Da). The 1Dy12.6 is the largest 1Dy glutenin subunits cloned in common wheat up to date, containing longer repetitive central domains than other 1Dy encoded proteins. In comparison with the most similar active 1Dy alleles previously reported, the newly discovered alleles contained a total of 20 SNPs and 3 indels. The secondary structure prediction indicated that 1Dy12.6 and 1Dy12.7 have similar proportion of α-helix, β-turn, and β-bend to those of 1Dy10 (X12929). The phylogenetic analysis illustrated that the x- and y-type subunits of glutenins were well separated, but both 1Dy12.6 and 1Dy12.7 were clustered with the other Glu-1Dy alleles. Our results revealed that the 1Dy12.6 and 1Dy12.7 subunit have potential to strengthen gluten polymer interactions, and are valuable genetic resources for wheat quality improvement.

Allelic variations of functional markers for high molecular weight glutenin genes in Indian wheat (Triticum aestivum L.) cultivars and their correlation with bread loaf volume

Glutenins, the polymeric proteins are composed of high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits. High molecular weight glutenin subunit (HMW-GS) plays a key role in determining bread making quality. The HMW-GS encoded by Glu-1 loci (GluA1, GluB1 and GluD1) are highly polymorphic. Thus it is very important to identify the alleles at Glu-1 loci for all three genomes for wheat quality improvement. In the present study 53 Indian wheat genotypes were evaluated for HMW-GS composition using PCR-based DNA markers. In total, two alleles (Ax2*, Ax1 or Axnull) at Glu-A1, four allele/allelic pairs (Bx7, Bx7 + By8, Bx7 + By9, Bx17 + By18) at Glu-B1 and two allelic pairs (Dx2 + Dy12 and Dx5 + Dy10) at Glu-D1 were identified using diagnostic DNA markers. Allele or allelic pair Ax2* (62.3 %) at Glu-A1, Bx7 + By8 (64 %) at Glu-B1 and Dx2 + Dy12 (58.5 %) at Glu-D1 were present most frequently in wheat genotypes taken for study. Moreover, the allelic combination Ax2*, Bx17 + By18 and Dx5 + Dy10 exhibited highest bread loaf volume in comparison to other allelic combinations studied. The set of markers used here thus represents a useful alternative for screening of HMW-glutenin allele (s) for improvement of bread making quality in Indian wheat genotypes.

Molecular genetic mapping of QTL associated with flour water absorption and farinograph related traits in bread wheat

Wheat (Triticum aestivum L.) flour functionality during the dough development and baking processes is an important quality attribute considered by the baking industry. A flour with high water absorption during mixing means more water and less flour is needed, compared to a flour with less water absorption. The objective of this study was to identify quantitative trait loci (QTL) influencing water absorption and dough rheological properties of hard red spring wheat. QTL were mapped on a genetic linkage map that comprised 531 simple sequence repeats (SSRs) and diversity array technology (DArT) marker loci. Composite interval mapping with 139 recombinant inbred lines (RILs) was used to identify QTL within and across two field environments. Six QTL on chromosomes 1A, 1B, 4B, 4D, and 5A were detected for farinograph water absorption. These QTL also confirmed earlier studies that flour water absorption is a function of protein content, starch damage, and gluten strength. In this study, dough rheological properties such as dough development time, dough stability, mixing tolerance index, and time to breakdown were influenced by the high-molecular weight glutenin genes Glu-B1 and Glu-D1.

Genetic Mapping Analysis of Bread‐Making Quality Traits in Spring Wheat

ABSTRACTIn this study we assess the genetic architecture of bread‐making quality traits in spring wheat (Triticum aestivum L.). A mapping population derived from BR34 and ‘Grandin’ was used to measure 20 end‐use quality traits including six kernel, seven milling and flour, four dough mixing strength, and three bread‐making traits. A total of 31 quantitative trait loci (QTL) significantly associated with all but two traits were identified. These QTL were clustered in five chromosomal regions, namely 1BS, 1DL, 4BL, 5BL, and 6AS, and explained a large proportion of trait variation with favorable alleles contributed by both parents. The 1DL cluster containing the high molecular weight glutenin gene, Glu‐D1, had a large genetic influence on dough mixing strength and bread‐making performance. Most of the QTL affecting kernel traits were clustered on 6AS. Inconsistency of QTL locations detected from different environments was observed for the flour and milling traits and was likely due to genotype × environment interaction (G × E) effects. Despite high heritabilities estimated for the 20 quality traits evaluated, no QTL were found for flour brightness and bake water absorption, suggesting that these traits may be controlled by QTL with small effects that could not be detected due to the small population size. Because of the complex inheritance of these traits, it will be necessary to validate these QTL in different spring wheat backgrounds evaluated in similar growth conditions as used in this study before the marker information can be used for breeding applications.

DNA analysis in charred grains of naked wheat from several archaeological sites in Spain

In the present work we attempt to recover endogenous ancient DNA from cereal grains preserved under different conditions: charred, partially charred and waterlogged. A total of 126 grains from naked wheat and 18 from barley from different sites on the Eastern Iberian Peninsula ranging from the beginning of agriculture in the region to the turn of the Common Era, were studied. Two different extraction protocols were used, a standard phenol–chloroform method and a silica-based DNA extraction procedure implemented for artificially charred seeds. Amplifications were directed to three markers: the large subunit of ribulose 1,5 biphosphate carboxylase (rbcL) and the microsatellite WCT12 in the chloroplast genome and the x and y subunits of the high molecular weight glutenin gene (Glu-1) in the nucleus. The first two were used to assess the preservation status of the samples, while with the third we tried to identify the wheat grains at species level. It was possible to obtain eleven positive amplifications in 8 partially charred seeds but only two amplifications of the Glu-1 gene from a single sample of the Early Bronze age were genome-specific. Different contamination sources were identified and reported. Cloning and alignment of sequenced clones showed a correspondence of the amplified fragment to modern wheat D genome haplotypes. This result suggests that the sample corresponds to hexaploid wheat (Triticum aestivum L.), thus being the first ancient DNA evidence to date for the cultivation of hexaploid wheat in the prehistoric agriculture of the Iberian Peninsula. Moreover, obtained results highlight contamination problems associated to the study of ancient archaeobotanical charred seeds suggest that the combination of a silica-based extraction method together with the amplification of specific targets is a good strategy for recovering endogenous ancient DNA from this kind of material.

Quantitative trait loci influencing end-use quality traits of hard red spring wheat breeding lines

Wheat bread-making quality is influenced by a complex group of traits including dough visco-elastic characteristics. In this study, quantitative trait locus/loci (QTL) mapping and analysis were conducted for endosperm polymeric proteins together with dough mixing strength and bread-making properties in a population of 139 (MN98550 × MN99394) recombinant inbred lines that was evaluated at three environments in 2006. Eleven chromosome regions were associated with endosperm polymeric proteins, explaining 4.2–31.8% of the phenotypic variation. Most of these polymeric proteins QTL coincided with several QTL for dough-mixing strength and bread-making properties. Major QTL clusters were associated with the low-molecular weight glutenin gene Glu-A3, the two high-molecular weight glutenin genes Glu-B1 and Glu-D1, and two regions on chromosome 6D. Alleles at these QTL clusters have previously been proven useful for wheat quality except one of the 6D QTL clusters.

Quantitative trait loci influencing endosperm texture, dough-mixing strength, and bread-making properties of the hard red spring wheat breeding lines

Wheat end product quality is determined by a complex group of traits including dough viscoelastic characteristics and bread-making properties. Quantitative trait loci (QTL) mapping and analysis were conducted for endosperm texture, dough-mixing strength, and bread-making properties in a population of 139 (MN99394 × MN98550) recombinant inbred lines that were evaluated at three environments in 2006. Based on the genetic map of 534 loci, six QTL were identified for endosperm texture, with the main QTL on chromosomes 1A (R(2) = 6.6%-17.3%), 5A (R(2) = 6.1%-17.1%), and 5D (R(2) = 15.8%-22%). Thirty-four QTL were identified for eight dough-mixing strength and bread-making properties. Major QTL clusters were associated with the low-molecular weight glutenin gene Glu-A3, the two high-molecular weight glutenin genes Glu-B1 and Glu-D1, and two regions on chromosome 6D. Alleles at these QTL clusters have previously been proven useful for wheat quality, except one of the QTL clusters on chromosome 6D. A QTL cluster on chromosome 6D is one of the novel chromosome regions influencing dough-mixing strength and bread-making properties. The QTL for endosperm texture on chromosomes 1A, 5A, and 5B also influenced flour ash content (12.4%-23.3%), flour protein content (10.5%-12.5%), and flour colour (7.7%-13.5%), respectively.

Conserved globulin gene across eight grass genomes identify fundamental units of the loci encoding seed storage proteins

The wheat high molecular weight (HMW) glutenins are important seed storage proteins that determine bread-making quality in hexaploid wheat (Triticum aestivum). In this study, detailed comparative sequence analyses of large orthologous HMW glutenin genomic regions from eight grass species, representing a wide evolutionary history of grass genomes, reveal a number of lineage-specific sequence changes. These lineage-specific changes, which resulted in duplications, insertions, and deletions of genes, are the major forces disrupting gene colinearity among grass genomes. Our results indicate that the presence of the HMW glutenin gene in Triticeae genomes was caused by lineage-specific duplication of a globulin gene. This tandem duplication event is shared by Brachypodium and Triticeae genomes, but is absent in rice, maize, and sorghum, suggesting the duplication occurred after Brachypodium and Triticeae genomes diverged from the other grasses ~35 Ma ago. Aside from their physical location in tandem, the sequence similarity, expression pattern, and conserved cis-acting elements responsible for endosperm-specific expression further support the paralogous relationship between the HMW glutenin and globulin genes. While the duplicated copy in Brachypodium has apparently become nonfunctional, the duplicated copy in wheat has evolved to become the HMW glutenin gene by gaining a central prolamin repetitive domain.

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 F(2) populations segregating for the Glu-A1 or Glu-D1 locus were successfully genotyped with primers UMN19, UMN25 and UMN26 labeled with fluorescent dyes.

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.

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.

Molecular characterization of the HMW glutenin genes Dt x1.5 + Dt y10 from Aegilops tauschii and their PCR-mediated recombinants

The high molecular weight (HMW) glutenin subunits, D t x1.5 + D t y10, are special types of storage proteins found in Aegilops tauschii that are never found in common wheat (Triticum aestivum). This study reports the characterization of the complete open reading frames (ORFs) of the HMW glutenin genes, D t x1.5 and D t y10, using a restrict-enzyme based method named the ‘restricted deletion method’ (RDM). The D t x1.5 and D t y10 were found to have an identical structure compared with the other published HMW glutenin genes. Comparison of the deduced protein sequences also indicated that the D t y10 in Ae. tauschii differed from its counterpart Dy10 in common wheat, by having insertions and deletions in the central repetitive domain. This result confirms the two subunits with same mobility in SDS-PAGE are different types of HMW glutenin subunits. In addition, four PCR-mediated recombinants of the D t x1.5 and D t y10 genes were amplified using a PCR program with shorter extension time. The recombinants had a similar structure to their corresponding natural genes, but a significantly different central repetitive domain. Western blot analysis exhibited a normal expression of the recombinants in E. coli. In addition to its usefulness for studying structure and function of the HMW glutenin subunits, the PCR-mediated recombination may provide an efficient method to generate novel HMW glutenin genes for wheat breeding.

Large-scale chromatin decondensation induced in a developmentally activated transgene locus

The high molecular weight (HMW) glutenin-encoding genes in wheat are developmentally activated in the endosperm at about 8 days after anthesis. We have investigated the physical changes that occur in these genes in two transgenic lines containing about 20 and 50 copies each of the HMW glutenin genes together with their promoters. Using fluorescence in-situ hybridisation (FISH) and confocal imaging, we demonstrate that, in non-expressing tissue, each transgene locus consists of one or two highly condensed sites, which decondense into many foci upon activation of transcription in endosperm nuclei. Initiation of transcription can precede decondensation but not vice versa. We show that, in one of the lines, cytoplasmic transcript levels are high after onset of transcription but disappear by 14 days after anthesis, whereas small interfering RNAs, which indicate post-transcriptional gene silencing (PTGS), are detected at this stage. However, the transcript levels remain high at the transcription sites, most of the transgene copies are transcriptionally active and transcriptional activity in the nucleus ceases only with cell death at the end of endosperm development.

Molecular structure of a novel y-type HMW glutenin subunit gene present in Triticum tauschii

An unusually small y-type high molecular weight (HMW) glutenin subunit gene from Triticum tauschii was sequenced. This gene, encoded at the Glu-D t 1 locus was designated 12.4 t and is the smallest HMW glutenin subunit gene described so far in Triticum species. Oligonucleotide primers based on published sequences of HMW glutenin genes were designed to amplify the encoding region and the central repetitive domain of the gene, which produced fragments of 1.4 and 0.85 kb, respectively. PCR products were cloned and sequenced. The derived amino acid sequence was compared with the amino acid sequences of the HMW glutenin subunits Dy12 t , from T. tauschii, and subunits Dy10 and Dy12 of T. aestivum. The amino acid sequence deduced from the nucleotide sequence demonstrated that deletions of hexapeptides and nonapeptides were responsible for the reduction in the size of this HMW glutenin subunit. The estimated molecular weight of the Dy12.4 t subunit, calculated on the basis of the deduced amino acid sequence, was 45,228 Daltons. There were also single amino acid differences in the N-, C-terminal and central repetitive domains of this gene in comparison to the three other y-type subunits encoded at the Glu-D1 locus. The Dy12.4 t subunit showed the highest similarity to the Dy12 subunit present in the hexaploid wheat Chinese Spring.

Identification of chromosome arms influencing expression of the HMW glutenins in wheat

The high-molecular-weight (HMW) glutenin genes, located on the group 1L chromosome arms, are a major determinant for baking quality in wheat ( Triticum aestivum L.). In addition, the HMW glutenin genes provide a valuable model system for studying the evolution and regulation of orthologous and paralogous genes in polyploid species. The goal of this study was to identify loci that modify the expression of the HMW glutenins, and to map them to specific chromosome arms. Comparisons were made between endosperms with zero versus three (or three versus six) doses for each of the 42 chromosome arms of wheat. SDS-PAGE and scanning densitometry were used to quantify the protein expression levels of the four HMW glutenin genes in cv. Chinese Spring, for each of the dosage comparisons. Fifteen chromosome arms were found to have significant effects on Glu-B1-1, excluding the structural gene dosage effect: eight positive effects on 1AL, 2AS, 2BL, 2DS, 5DS, 6AL, 6DL, and 7AL and seven negative effects on 1BS, 1DS, 1DL, 4DL, 6BS, 6DS, and 7AS. Nineteen chromosome arms had significant effects on Glu-B1-2, excluding the structural gene dosage effect: eight positive effects on 1AL, 2AS, 2BS, 3AL, 4BL, 6DS, 7BL and 7DS and 11 negative effects on 1AS, 1BS, 1DS, 1DL, 2AL, 2BL, 3DS, 4BS, 4DL, 5BL, and 6BS. Twenty chromosome arms had significant effects on Glu-D1-1, excluding the structural gene dosage effect: 11 positive effects on 1AL, 1BL, 2BS, 2DS, 5BS, 5DS, 6AL, 6DS, 6DL, 7AL, and 7BL and nine negative effects on 1AS, 1BS, 1DS, 2BL, 4DL, 5BL, 5DL, 6BL, and 7DS. Twenty-five chromosome arms had significant effects on Glu-D1-2, excluding the structural gene dosage effect: 17 positive effects on 1BL, 2AS, 2BS, 2DS, 2DL, 3AS, 3AL, 3BS, 5AS, 5BS, 5DL, 6AL, 6DL, 7AL, 7BS, 7BL, and 7DL and eight negative effects on 1DS, 4DL, 5AL, 5BL, 6BS, 6BL, 6DS and 7DS. Of the 164 gene-chromosome arm tests performed, about 52% (85/164) showed no significant effects, and 48% (79/164) showed significant effects, excluding the structural gene dosage effects. Of the significant effects, 56% (44/79) were positive effects, and 44% (35/79) were negative effects. Comparisons of dosage effects on orthologous loci (both x-type or both y-type HMW glutenins) showed that orthologous HMW glutenin genes are largely influenced by the same regulatory systems. Less correlation was found for comparisons between paralogous genes, although considerable conservation was observed at this level as well. These observations suggest that after polyploidization, many of the duplicated orthologous regulatory loci were inactivated by mutation, thus consolidating control over the HMW glutenin genes. Possible candidates for orthologous regulatory genes were identified in maize and barley. This study represents the first comprehensive search of the wheat genome for regulators of the HMW glutenins.

Evaluation of baking properties and gluten protein composition of field grown transgenic wheat lines expressing high molecular weight glutenin gene 1Ax1

The unique breadmaking properties of wheat are closely related to the quality and quantity of high molecular weight (HMW) glutenins present in wheat flour. We have produced several transgenic wheat lines expressing the high molecular weight glutenin subunit (HMW-GS) gene 1Ax1. They were analyzed for stability of gene expression and the effect of over-expressed 1Ax1 protein on protein composition, agronomic traits and flour functionality in R4 seeds obtained from plants grown in the field. The expression of 1Ax1 in R4 seeds was similar to that found in R3 seeds harvested from plants reared in a growth chamber, indicating that the high level expression of 1Ax1 under its own promoter was stable under field conditions. Quantitative differences were observed in gliadins, flour yield and single kernel characteristics between 1Ax1 transgenic wheat and the Bobwhite control. No qualitative differences in the gliadin or low molecular weight glutenin subunits were seen between the control and transgenic plants. Two of the transgenic lines showed some very high molecular weight proteins in addition to the 1Ax1 and the native HMW-GS. Purification and N-terminal sequencing of these proteins did not reveal any similarity to HMW-GS. In some of the transgenic lines, mixing time, loaf volume and water absorbance improved relative to the control cultivar. This was the first large scale baking and mixing test of field grown transgenic wheat. Our results show that the integration of a seventh HMW-GS gene (1Ax1), and its expression resulting in the presence of six HMW-GS in the wheat endosperm, neither caused any gene silencing nor any undesirable effect on yield, protein composition or flour functionality.

Evolution of the high molecular weight glutenin loci of the A, B, D, and G genomes of wheat

We used PCR to obtain phylogenetically informative sequences from the high molecular weight glutenin genes of wheat. The validity of partial sequence comparisons as a means of studying glutenin phylogenetics was established by constructing neighbour-joining trees from partial alignments of 12 published glutenin allele sequences. PCR was then used to obtain 20 novel glutenin allele sequences from various Triticum and Aegilops species, including a 3000 year old preserved wheat. A neighbour-joining tree derived from all known glutenin allele sequences had eight clades, representing the eight loci from which the allele sequences were derived, and was split into two halves, one comprising alleles from the Glu-1-1 loci and the other comprising Glu-1-2 alleles. The topology was compatible with the postulated relationships between the A, B, D, and G genomes. The Glu gene duplication event was tentatively dated at 7.2-10.0 million years ago (MYA), the origin of the four genomes at 5.0-6.9 MYA, and the split between the B and G genomes at 2.5-3.5 MYA. The Glu-B1-1 alleles in cultivated wheats fell into two subgroups that diverged 1.4-2.0 MYA, suggesting that emmer was domesticated twice. The D allele sequences were relatively diverse, indicating that the hybridization event that resulted in the hexaploid bread wheats might have occurred more than once.