Low-molecular-weight Glutenin Subunits Genes Research Articles

Characterization of Glutenin Genes in Bread Wheat by Third-Generation RNA Sequencing and the Development of a Glu-1Dx5 Marker Specific for the Extra Cysteine Residue.

High-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) in a mature grain play important roles in the formation of a glutenin macropolymer and gluten quality. To characterize the expressed glutenin genes of the bread wheat variety Xinmai 26 during seed development, a total of 18 full-length transcripts were obtained by the newly emerged third-generation RNA sequencing of the PacBio Sequel II platform, including 5 transcripts of HMW-GS genes and 13 transcripts of LMW-GS genes (8 intact genes and 5 pseudogenes). Combined with the patterns of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), allelic types of the obtained glutenin genes were, respectively, determined, wherein molecular characterization deduced by transcript1528 (1Dx5) and transcript907 (Glu-A3c) indicated their great influence on dough quality. In addition, a specific functional marker dCAPS5 was developed for the single-nucleotide substitution at position 353 of the 1Dx5 subunit, which was further intensively compared with the other proposed markers to efficiently utilize the 1Dx5 subunit with the extra cysteine residue. This study provides an efficient method to accurately identify and utilize glutenin genes in bread wheat, which is helpful in understanding the contributions of glutenin genes to wheat quality.

Enriching LMW-GS alleles and strengthening gluten properties of common wheat through wide hybridization with wild emmer.

Two advanced lines (BAd7-209 and BAd7-213) with identical high-molecular-weight glutenin subunit composition were obtained via wide hybridization between low-gluten cultivar chuannong16 (CN16) and wild emmer D97 (D97). BAd7-209 was better than BAd7-213, and both of them were much better than CN16 in a dough quality test. We found that BAd7-209 had more abundant and higher expression levels of low-molecular-weight glutenin subunit (LMW-GS) proteins than those of BAd7-213. Twenty-nine novel LMW-GS genes at Glu-A3 locus were isolated from BAd7-209, BAd7-213 and their parents. We found that all 29 LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Twenty-seven genes encode LMW-m-type subunits, and two encode LMW-i-type subunits. BAd7-209 had a higher number of LMW-GS genes than BAd7-213, CN16, and D97. Two wild emmer genes MG574329 and MG574330 were present in the two advanced lines. Most of the LMW-m-type genes showed minor nucleotide variations between wide hybrids and their parents that could be induced through the wide hybridization process. Our results demonstrated that the wild emmer LMW-GS alleles could be feasibly transferred and integrated into common wheat background via wide hybridization and the potential value of the wild emmer LMW-GS alleles in breeding programs designed to improve wheat flour quality.

Low molecular weight glutenin subunit gene composition at Glu-D3 loci of Aegilops tauschii and common wheat and a further view of wheat evolution.

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat. Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01-AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

Characterization of novel LMW glutenin subunit genes at the Glu-M3 locus from Aegilops comosa.

We report the characterization of seven novel low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops comosa (2n = 2x = 14, MM). We found that all seven LMW-GS genes possessed the same primary structure shared by other known LMW-GSs. Three genes (comosa-M1, comosa-M2, and comosa-M3) encode LMW-m-type subunits, two (comosa-I1 and comosa-I2) encode LWM-i-type subunits, and two (comosa-L1 and comosa-L2) encode LWM-l-type subunits. The comosa-M1 possessed seven cysteine residues, which resulted from a single-nucleotide polymorphism (SNP) of the G-A transition in the fifth position of the N-terminal sequence. Two l-type subunits, comosa-L1 and comosa-L2, contained nine cysteine residues with an extra cysteine residue located in the signal peptide and repetitive domain. In addition, a long insertion of 13 residues (LGQQPQ8/LVQQPQ8) was present in the end of the C-terminal II. Phylogenetic analysis implied that the comosa-L2 may be the intermediate type during the evolution of LMW-l and LMW-i-type genes. Our results demonstrated that the novel LMW-GSs, such as comosa-M1, comosa-L1, and comosa-L2, may have positive effects on dough properties.

Development of haplotype-specific molecular markers for the low-molecular-weight glutenin subunits

Low-molecular-weight glutenin subunits (LMW-GSs) are one of the major components of gluten, and their allelic variation has been widely associated with different wheat end-use quality parameters. These proteins are encoded by multigene families located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3); the genes at each locus are divided by large intergenic and highly recombinogenic regions. Among the methods used for the LMW-GS allele identification, polymerase chain reaction (PCR)-based molecular markers have the advantages of being simple, accurate, and independent from the plant stage of development. However, the available LMW-GS molecular markers are either incapable of capturing the complexity of the LMW-GS gene family or difficult to interpret. In the present study, we report the development of a set of PCR-based molecular markers specific for the LMW-GS haplotypes present at each Glu-3 locus. Based on the LMW-GS gene sequences available in GenBank, single nucleotide polymorphisms (SNPs) specific for each Glu-3 haplotype were identified and the relevant PCR primers were designed. In total, we developed three molecular markers for the Glu-A3 and Glu-B3 loci, respectively, and five molecular markers for the Glu-D3 locus. The markers were tested on 44 bread wheat varieties previously characterized for their LMW-GS genic profile and found to be equally or more efficient than previously developed LMW-GS PCR-based markers. This set of markers allows an easier and less ambiguous identification of specific LMW-GS haplotypes associated with gluten strength and can facilitate marker-assisted breeding for wheat quality.

Allelic variation at high-molecular weight and low-molecular weight glutenin subunit genes in Moroccan bread wheat and durum wheat cultivars

Glutenin is a major protein fraction contributing to the functional properties of gluten and dough. The glutenin constitutes 30–40% of the protein in wheat flour and about half of that in gluten. It is essential to identify correct glutenin alleles and to improve wheat quality by selecting alleles that exert favorable effects. Moroccan wheat cultivars are unique in West Asia and North Africa region, since many of them possess resistance to Hessian fly, a pest, which is becoming important in other countries in the region. Hence, these cultivars are being used as donor for the resistance in the breeding program. Here, we determine the allelic variation in high-molecular weight glutenin subunits (HMW-GS) and low-molecular weight glutenin subunits (LMW-GS) in Moroccan cultivars of bread and durum wheat using the gene-specific PCR markers. In 20 cultivars of bread wheat, 9 different allele variants were detected at HMW-GS and 13 different allele variants were detected at LMW-GS, in which the alleles Glu-A1b (2*), Glu-B1i (17 + 18), Glu-B1c (7*/7 + 9), Glu-D1d (5 + 10), Glu-A3c, Glu-B3 h, and Glu-D3b were the most frequents. In 26 cultivars of durum wheat, less allelic variation was found: seven different allele variants at HMW-GS and six different allele variants at LMW-GS were identified, in which the major alleles were Glu-A1c (null), Glu-B1b (7 + 8), Glu-B1e (20), Glu-A3c, and Glu-B3d. The mean value of the genetic diversity for the glutenin loci was 0.502 in bread wheat and 0.449 in durum wheat. Most of the glutenin alleles carried by Moroccan bread wheat cultivars impart good bread-making quality. Most of the durum wheat glutenin alleles were related to low strength dough or poor quality and need to be improved. To improve quality of Moroccan durum wheat, essentially, Glu-A1c and Glu-B3d alleles of the genes should be replaced with the better alleles through breeding.

Evidence of intralocus recombination at the Glu-3 loci in bread wheat (Triticum aestivum L.).

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci. The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

Definition of the low molecular weight glutenin subunit gene family members in a set of standard bread wheat (Triticum aestivum L.) varieties

Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the viscoelastic properties of wheat dough. The LMW-GSs are encoded by multi-gene families at the Glu-A3, Glu-B3 and Glu-D3 loci, with more than 15 genes present in most bread wheat varieties. However, the genic profile associated with different alleles has not been clearly defined. Here, the LMW-GSs in a set of standard varieties were analyzed using molecular markers. In most cases, each Glu-3 allele was represented by a specific haplotype; however, some alleles were undistinguishable. The Glu-A3e and Glu-A3g alleles showed an identical marker haplotype, as did the alleles Glu-B3c and Glu-B3d, and Glu-B3f and Glu-B3ab. In contrast, two haplotypes among varieties designated Glu-D3c were differentiated. The marker profiles present at the Glu-D3 locus exhibited less variation compared to the genes at the Glu-A3 and Glu-B3 loci. Results show the potential of the LMW-GS gene marker system in the characterization of the LMW-GS alleles present in specific bread wheat varieties, and its reconciliation with protein-based nomenclature. This approach will advance the understanding of the contribution of each of the LMW-GS gene alleles in the control of the end-use quality.

Comprehensive identification of LMW-GS genes and their protein products in a common wheat variety.

Although it is well known that low-molecular-weight glutenin subunits (LMW-GS) from wheat affect bread and noodle processing quality, the function of specific LMW-GS proteins remains unclear. It is important to find the genes that correspond to individual LMW-GS proteins in order to understand the functions of specific proteins. The objective of this study was to link LMW-GS genes and haplotypes characterized using well known Glu-A3, Glu-B3, and Glu-D3 gene-specific primers to their protein products in a single wheat variety. A total of 36 LMW-GS genes and pseudogenes were amplified from the Korean cultivar Keumkang. These include 11 Glu-3 gene haplotypes, two from the Glu-A3 locus, two from the Glu-B3 locus, and seven from the Glu-D3 locus. To establish relationships between gene haplotypes and their protein products, a glutenin protein fraction was separated by two-dimensional gel electrophoresis (2-DGE) and 17 protein spots were analyzed by N-terminal amino acid sequencing and tandem mass spectrometry (MS/MS). LMW-GS proteins were identified that corresponded to all Glu-3 gene haplotypes except the pseudogenes. This is the first report of the comprehensive characterization of LMW-GS genes and their corresponding proteins in a single wheat cultivar. Our approach will be useful to understand the contributions of individual LMW-GS to the end-use quality of flour.

Composition, variation, expression and evolution of low-molecular-weight glutenin subunit genes in Triticum urartu.

BackgroundWheat (AABBDD, 2n = 6x = 42) is a major dietary component for many populations across the world. Bread-making quality of wheat is mainly determined by glutenin subunits, but it remains challenging to elucidate the composition and variation of low-molecular-weight glutenin subunits (LMW-GS) genes, the major components for glutenin subunits in hexaploid wheat. This problem, however, can be greatly simplified by characterizing the LMW-GS genes in Triticum urartu, the A-genome donor of hexaploid wheat. In the present study, we exploited the high-throughput molecular marker system, gene cloning, proteomic methods and molecular evolutionary genetic analysis to reveal the composition, variation, expression and evolution of LMW-GS genes in a T. urartu population from the Fertile Crescent region.ResultsEight LMW-GS genes, including four m-type, one s-type and three i-type, were characterized in the T. urartu population. Six or seven genes, the highest number at the Glu-A3 locus, were detected in each accession. Three i-type genes, each containing more than six allelic variants, were tightly linked because of their co-segregation in every accession. Only 2-3 allelic variants were detected for each m- and s-type gene. The m-type gene, TuA3-385, for which homologs were previously characterized only at Glu-D3 locus in common wheat and Aegilops tauschii, was detected at Glu-A3 locus in T. urartu. TuA3-460 was the first s-type gene identified at Glu-A3 locus. Proteomic analysis showed 1-4 genes, mainly i-type, expressed in individual accessions. About 62% accessions had three active i-type genes, rather than one or two in common wheat. Southeastern Turkey might be the center of origin and diversity for T. urartu due to its abundance of LMW-GS genes/genotypes. Phylogenetic reconstruction demonstrated that the characterized T. urartu might be the direct donor of the Glu-A3 locus in common wheat varieties.ConclusionsCompared with the Glu-A3 locus in common wheat, a large number of highly diverse LMW-GS genes and active genes were characterized in T. urartu, demonstrating that this progenitor might provide valuable genetic resources for LMW-GS genes to improve the quality of common wheat. The phylogenetic analysis provided molecular evidence and confirmed that T. urartu was the A-genome donor of hexaploid wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0322-3) contains supplementary material, which is available to authorized users.

Novel LMW glutenin subunit genes from wild emmer wheat (Triticum turgidum ssp. dicoccoides) in relation to Glu-3 evolution.

Four low-molecular-weight-isoleucine (LMW-i)-type and one novel chimeric (between LMW-i and LMW-methionine (m) types) low-molecular-weight glutenin subunit (LMW-GS) genes were characterized from wild emmer wheat (Triticum dicoccoides), designated as emmer-1 to emmer-5. All five LMW-GS genes possessed the same primary structure shared by other published LMW-GSs. The three genes emmer-1, emmer-3, and emmer-5 are similar, with the exception that emmer-3 and emmer-5 lost a few repeat motifs compared to emmer-1. Gene duplication and insertions/deletions of repeat motifs mediated through unequal crossing over may be responsible for the generation of these three Glu-3 alleles. Although the first residue of mature peptide of emmer-4 is isoleucine, it is not typical LMW-i-type LMW-GS. Phylogenetic analysis indicated that emmer-4 is located in the LMW-m subgroup, suggesting a closer relationship with LMW-m-type gene Y14104 of T. durum. Sequence alignment indicated that the emmer-4 is likely a chimeric gene generated by illegitimate recombination between LMW-i and LMW-m type. Unequal crossing over and illegitimate recombination are effective mechanisms for enriching both copy numbers and variations of LMW-GSs.

High-level expression of LMW-GS and α-gliadin genes promoted by the expressed tag sequence of 5′ end in Escherichia coli

Wheat storage protein genes, especially low molecular weight glutenin subunit (LMW-GS) and gliadin genes are difficult to be expressed in Escherichiacoli, mainly due to the presence of highly repetitive sequences. In order to establish a high efficiency expression system for these genes, five different expression plasmids combining with 9 genes, viz. 6 LMW-GS and 3 α-gliadin genes isolated from common wheat and related species, were studied for heterologous expression in E. coli. In this study, when an expressed tag sequence encoding signal peptide, His-S or GST-tag was fused to the 5′ end of LMW-GS or gliadin gene as the leading sequence, all recombination genes could be stably expressed at a high level. On the contrast, as expected, the inserted genes encoding mature protein failed without an expressed tag sequence. This result indicated that using expressed tag sequences as leading sequences could promote LMW-GS and gliadin genes to be well expressed in E. coli. Further transcriptional analysis by quantitative real-time PCR (qRT-PCR) showed transcription levels of recombination genes (e.g. GST-Glutenin, His-S-Glutenin and SP∗-His-Glutenin) were 4-fold to 33-fold higher than those of the LMW-GS genes, which suggested these expressed tag sequences might play an important role in stimulating transcription. The possible molecular mechanism under this phenomenon was discussed.

Characterisation of low molecular weight glutenin subunit genes from Pseudoroegneria spicata and Pd. strigosa.

We report the characterisation of nine novel low molecular weight glutenin subunit (LMW-GS) genes from two Pseudoroegneria species, Pd. spicata and Pd. strigosa. We found that all nine LMW-GS genes possess the same primary structure shared by other published LMW-GS. Five genes encode LMW-i type subunits, three encode LMW-m type subunits and one encodes a peptide similar to B-3 hordeins of Hordeum chilense. No LMW-s type subunit genes were found in Pseudoroegneria. One subunit, PSt24-LMW-2, contains six conserved cysteine residues, and the other eight subunits all contain eight cysteine residues. We show that one cysteine residue is located in the signal peptide of PSt24-LMW-1, suggesting a mature peptide containing only seven cysteine residues. Phylogenetic analysis indicates that the LMW-GS genes from the St genome cluster together and suggests a distant relationship with LMW-GS of the A and B genomes of wheat. Slippage/unequal crossing over and illegitimate recombination are effective mechanisms for enriching variations of seed storage proteins.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Cloning and characterization of low-molecular-weight glutenin subunit alleles from Chinese wheat landraces (Triticum aestivum L.).

Low-molecular-weight glutenin subunits (LMW-GS) are of great importance in processing quality and participate in the formation of polymers in wheat. In this study, eight new LMW-GS alleles were isolated from Chinese wheat landraces (Triticum aestivum L.) and designated as Glu-A3-1a, Glu-A3-1b, Glu-B3-1a, Glu-B3-1b, Glu-B3-1c, Glu-D3-1a, Glu-D3-1b, and Glu-D3-1c, which were located at the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. Based on the proteins encoded, the number of deduced amino acids of Glu-B3 alleles was approximately 50 more than those of Glu-A3 and Glu-D3 alleles. The first cysteine of Glu-A3 and Glu-D3 alleles was located at the N-terminal domain, while that of Glu-B3 alleles was found in the repetitive domain, which may lead to the different functioning in forming disulfide bonds. All the eight genes were LMW-m types and the new allele of Glu-B3-1a which had nine cysteine residues may be the desirable LMW-GS gene for improving bread-making quality.

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 2554bp. 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 (> −700bp) 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.

Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum, Aegilops crassa, Ae. cylindrica and Ae. tauschii

Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n ¼ 4x ¼ 28), Ae. cylindrica (2n ¼ 4x ¼ 28), Ae. tauschii (2n ¼ 2x ¼ 14) and Triticum aestivum (2n ¼ 6x ¼ 42) were obtained using five sets of specific allele primer pairs. Only the sequences of the first primer pair were complete coding sequences (cds) of LMW-GS, and had 305, 304, 306 and 305 LMW-m amino acid residues in Ae. crassa, Ae. cylindrica, Ae. tauschii and T. aestivum, respectively. The repetitive domain and repeat motif numbers of all LMW glutenin subunits showed eight conserved cysteine residues that lead to the same functional activity in different genome. Based on DNA and predicted protein sequences, phylogenetic trees for all sets of sequences were drawn. At the DNA level, the species closest to T. aestivum for the second, third, fourth and fifth set of sequences were Ae. cylindrica, Ae. tauschii and Ae. crassa, respectively. At the protein level, the species closest to T. aestivum based on the first, second and fifth set of sequences were Ae. cylindrica, Ae. crassa and Ae. crassa, respectively. For other sets of sequences, bread wheat proved to be a distinct species. The LMW-GS gene sequences have been recorded in the GenBank with accession numbers JQ726549–JQ726568.

Novel insights into the composition, variation, organization, and expression of the low-molecular-weight glutenin subunit gene family in common wheat

Low-molecular-weight glutenin subunits (LMW-GS), encoded by a complex multigene family, play an important role in the processing quality of wheat flour. Although members of this gene family have been identified in several wheat varieties, the allelic variation and composition of LMW-GS genes in common wheat are not well understood. In the present study, using the LMW-GS gene molecular marker system and the full-length gene cloning method, a comprehensive molecular analysis of LMW-GS genes was conducted in a representative population, the micro-core collections (MCC) of Chinese wheat germplasm. Generally, >15 LMW-GS genes were identified from individual MCC accessions, of which 4–6 were located at the Glu-A3 locus, 3–5 at the Glu-B3 locus, and eight at the Glu-D3 locus. LMW-GS genes at the Glu-A3 locus showed the highest allelic diversity, followed by the Glu-B3 genes, while the Glu-D3 genes were extremely conserved among MCC accessions. Expression and sequence analysis showed that 9–13 active LMW-GS genes were present in each accession. Sequence identity analysis showed that all i-type genes present at the Glu-A3 locus formed a single group, the s-type genes located at Glu-B3 and Glu-D3 loci comprised a unique group, while high-diversity m-type genes were classified into four groups and detected in all Glu-3 loci. These results contribute to the functional analysis of LMW-GS genes and facilitate improvement of bread-making quality by wheat molecular breeding programmes.

Molecular characterization of LMW-GS genes in Brachypodium distachyon L. reveals highly conserved Glu-3 loci in Triticum and relatedspecies

BackgroundBrachypodium distachyon L. is a newly emerging model plant system for temperate cereal crop species. However, its grain protein compositions are still not clear. In the current study, we carried out a detailed proteomics and molecular genetics study on grain glutenin proteins in B. distachyon.ResultsSDS-PAGE and RP-HPLC analysis of grain proteins showed that Brachypodium has few gliadins and high molecular weight glutenin subunits. In contrast the electrophoretic patterns for the albumin, globulin and low molecular weight glutenin subunit (LMW-GS) fractions of the grain protein were similar to those in wheat. In particular, the LMW-C type subunits in Brachypodium were more abundant than the equivalent proteins in common wheat. Southern blotting analysis confirmed that Brachypodium has 4–5 copies of LMW-GS genes. A total of 18 LMW-GS genes were cloned from Brachypodium by allele specific PCR. LMW-GS and 4 deduced amino acid sequences were further confirmed by using Western-blotting and MALDI-TOF-MS. Phylogenetic analysis indicated that Brachypodium was closer to Ae. markgrafii and Ae. umbellulata than to T. aestivum.ConclusionsBrachypodium possessed a highly conserved Glu-3 locus that is closely related to Triticum and related species. The presence of LMW-GS in B. distachyon grains indicates that B. distachyon may be used as a model system for studying wheat quality attributes.

Molecular cloning and characterization of five novel low molecular weight glutenin subunit genes from Tibetan wheat landraces (Triticum aestivum L.)

The low molecular weight glutenin subunits (LMW-GS) are the major components of glutenins and are important for the end-use quality of wheat. Five novel LMW-GS genes (designated as LMW-Jiachazharen, LMW-Bangdadongmai-3, LMW-Jiachaaigan, LMW-Maoyintumai and LMW-Rikezehongmai) were isolated from Tibetan wheat landraces. The coding regions of LMW-Jiachazharen, LMW-Bangdadongmai-3, LMW-Jiachaaigan, LMW-Maoyintumai and LMW-Rikezehongmai were 912, 897, 915, 927 and 906 bp in length, which encoded 302, 297, 303, 307 and 300 amino acid residues, respectively. Analysis of the deduced amino acid sequences showed that the five novel genes were classified as LMW-m type, with the predicted molecular weights of 32,013.97, 31,622.89, 32,107.07, 32,939.41 and 31,731.64 Da, respectively. The LMW-Jiachazharen, LMW-Jiachaaigan, LMW-Maoyintumai possessed seven cysteine residues, which resulted from a single-nucleotide polymorphism (SNP) of the G–A transition. However, except for eight conserved cysteine residues, LMW-Bangdadongmai-3 contained an extra one, as the result of a SNP of the T–C transition. In addition, the corresponding five LMW-GS were identified and confirmed by sodium SDS-PAGE and MALDI-TOF-MS, respectively. Phylogenetic analysis indicated that the five novel genes were glutenin-like proteins and designated as LMW-m type genes. The five novel genes may be new candidate LMW-GS genes with potential value for wheat quality improvement.