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.     

Identification of new low-molecular-weight glutenin subunit genes in wheat

To clarify the composition of low-molecular-weight glutenin subunits (LMW-GSs) in a soft wheat cultivar, we cloned and characterized LMW-GS genes from a cDNA library and genomic DNA in Norin 61. Based on alignment of the conserved N- and C- terminal domains of the deduced amino-acid sequences, these genes are classified into 12 groups. One of these groups (group 5), the corresponding gene of which has not been reported previously, contains two additional hydrophobic amino-acid clusters interrupting the N-terminal repetitive domain. Other groups (groups 11 and 12), which were not identified in other cultivars as a protein product, showed all eight cysteines in the C-terminal conserved domain. With specific primer sets for these groups it was revealed that Glu-D3 and Glu-A3 encoded the former and the latter, respectively. Both groups of genes were expressed in immature seeds. The presence of these groups of LMW-GSs may affect the dough strength of soft wheat. Received: 26 March 2001 / Accepted: 16 July 2001     

Evidence of intralocus recombination at the <Emphasis Type="Italic">Glu-3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

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.

Abstract

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.

     

Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci

Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.     

Phylogenetic relationship of a new class of LMW-GS genes in the M genome of Aegilops comosa

A new class of low molecular weight glutenin subunit (LMW-GS) genes was isolated and characterized from Aegilops comosa (2n?=?2x?=?14, MM). Although their DNA structure displayed high similarity to LMW-i type genes, there are some key differences. The deduced amino acid sequences of their mature proteins showed that the first amino acid residue of each gene was leucine and therefore they were designated as LMW-l type subunits. An extra cysteine residue was present in the signal peptide and the first cysteine residue of mature proteins located at the end of repetitive domain. Additionally, a long insertion of 10?C22 residues (LGQQPQ_5?C17) occurred in the end of the C-terminal II. Comparative analysis demonstrated that LMW-l type glutenin genes possessed a great number of single-nucleotide polymorphisms and insertions/deletions. A new classification system was proposed according to the gene structure and phylogenetic analysis. In this new system, LMW-GS is classified into two major classes, LMW-M and LMW-I, with each including two subclasses. The former included LMW-m and LMW-s types while the latter contained LMW-l and LMW-i types. Analysis of their evolutionary origin showed that the LMW-l genes diverged from the group 2 of LMW-m type genes at about 12?C14?million years ago (MYA) while LMW-i type evolved from LMW-l type at approximately 8?C12 MYA. The LMW-s type was a variant form of group 1 of LMW-m type and their divergence occurred about 4?C6 MYA. In addition to homologous recombination, non-homologous illegitimate recombination could be an important molecular mechanism for the origin and evolution of LMW-GS gene family. The secondary structure prediction suggested that the novel LMW-l type subunits, such as AcLMW-L1 and AcLMW-L2, may have positive effects on dough properties.     

Comparison of low- and high molecular-weight wheat glutenin allele effects on flour quality

Five crosses were made, using a set of New Zealand wheat cultivars, to measure the effect of glutenin allele differences on baking quality parameters. The alleles involved were: Glu-A1 (2*, 1 and n), Glu-D1 (5+10, 2+12), Glu-A3 (c, d and e), Glu-B3 (Sec-12, Sec-13, b and g), Glu-D3 (a and b). The allelic variation of F₃ individual plants was identified by SDS-PAGE, and plants with the same HMW-GS and LMW-GS patterns were grouped. Quality parameters were then measured on the grouped F₄ bulks. Quality parameters measured for this study were wholemeal flour protein content (WFP), grain hardness (HAR), SDS sedimentation volume (SED), Pelshenke time (PEL), mid-line peak value (MPV) and the mid-line peak time (MPT) of a mixograph. The results showed there were significant quality differences within most populations associated with the possession of a particular allele, reaching magnitudes of up to 42% for the range between populations. Most glutenin allelic comparisons showed significant differences for at least one of the resultant measured quality parameters. Allelic differences of Glu-A1 significantly influenced all characters except MPT, with the null allele apparently inferior; possession of 5+10 at Glu-D1 significantly increased Pelshenke time and SED volumes relative to allele 2+12; WFP, SED and MPV were significantly affected by the Glu-A3 alleles tested. Glu-B3 alleles significantly affected all characters except hardness and the Glu-D3 alleles tested significantly affected all characters other than hardness and SDS sedimentation volume. Received: 8 June 1999 / Accepted: 25 July 2000     

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.     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Allelic variations in <Emphasis Type="Italic">Glu-1</Emphasis> and <Emphasis Type="Italic">Glu-3</Emphasis> loci of historical and modern Iranian bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars

Proline and glutamine-rich wheat seed endosperm proteins are collectively referred to as prolamins. They are comprised of HMW-GSs, LMW-GSs and gliadins. HMW-GSs are major determinants of gluten elasticity and LMW-GSs considerably affect dough extensibility and maximum dough resistance. The inheritance of glutenin subunits follows Mendelian genetics with multiple alleles in each locus. Identification of the banding patterns of glutenin subunits could be used as an estimate for screening high quality wheat germplasm. Here, by means of a two-step 1D-SDS-PAGE procedure, we identified the allelic variations in high and low-molecular-weight glutenin subunits in 65 hexaploid wheat (Triticum aestivum L.) cultivars representing a historical trend in the cultivars introduced or released in Iran from the years 1940 to 1990. Distinct alleles 17 and 19 were detected for Glu-1 and Glu-3 loci, respectively. The allelic frequencies at the Glu-1 loci demonstrated unimodal distributions. At Glu-A1, Glu-B1 and Glu-D1, we found that the most frequent alleles were the null, 7 + 8, 2 + 12 alleles, respectively, in Iranian wheat cultivars. In contrast, Glu-3 loci showed bimodal or trimodal distributions. At Glu-A3, themost frequent alleles were c and e. At Glu-B3 the most frequent alleles were a, b and c. At Glu-D3 locus, the alleles b and a, were the most and the second most frequent alleles in Iranian wheat cultivars. This led to a significantly higher Nei coefficient of genetic variations in Glu-3 loci (0.756) as compared to Glu-1 loci (0.547). At Glu-3 loci, we observed relatively high quality alleles in Glu-A3 and Glu-D3 loci and low quality alleles at Glu-B3 locus.     

Genetic characterization of storage proteins in a set of F1-derived haploid lines in bread wheat

Wheat storage proteins were evaluated by SDS-PAGE in a population of 206 doubled haploid (DH) lines, produced from a cross between bread wheat cvs Chinese Spring (CS) and Courtot (CT). The analysis of gliadins and high- and low-molecular-weight glutenins gave rise to 11 protein markers between parental varieties. Among these, one each was encoded at the Glu-A1, Gli-A1, Gli-A2, Gli-A5, Glu-B3, Gli-B1 and Gli-D1 loci and four were encoded at the Glu-D3 locus. Only the Gli-A2 marker showed a distorted segregation. A distance of 1.94 cM was evaluated between the Gli-A1 locus and the recently found Gli-A5 locus. Among the DH lines, only nine exhibited an unexpected pattern. The chromosome allocation was determined for almost all the LMW-GS and gliadin bands of CS using nullitetrasomic and ditelosomic lines. Two C LMW-GS were found to be coded by 6DS. Similarly, substitution lines into CT allowed the allelic determination of numerous LMW-GS and gliadin bands. A correspondence between gliadin markers separated in SDS-PAGE and in A-PAGE revealed that the common allele Gli-Aa between CS and CT determined in A-PAGE was able to be separated into two alleles when SDS-PAGE was used.     

Identification of LMW glutenin-like genes from Secale sylvestre host

Three low-molecular-weight (LMW) glutenin-like genes (designated as Ssy1, Ssy2 and Ssy3) from Secale sylvestre Host were isolated and characterized. The three genes consist of a predicted highly conservative signal peptide with 20 amino acids, a short N-terminal region with 13 amino acids, a highly variable repetitive domain and a less variable C-terminal domain. The deduced amino acid sequences of the three genes were the LMW-m type due to a methionine residue at the N-terminus. The phylogenic analysis indicated that the prolamin genes could be perfectly clustered into five groups, including HMW-GS, LMW-GS, alpha/beta-, gamma- and omega-prolamin. The LMW glutenin-like genes of S. sylvestre were more orthologous with the LMW-GS genes of wheat and B hordein genes of barley, which also had been confirmed by the homology analysis with the LMW-GS of wheat at Glu-A3, Glu-B3 and Glu-D3 loci. These results indicated that a chromosome locus (designated as Glu-R3) might be located on the R genome of S. sylvestre with the functions similar to the Glu-3 locus in wheat and its related species.     

LMW-GS genes in Agropyron elongatum and their potential value in wheat breeding

To study the usefulness of low-molecular-weight glutenin subunits (LMW-GS) of Agropyron elongatum (Host) Nevski to wheat (Triticum aestivum L.) quality improvement, we characterized LMW-GS genes of A. elongatum. Nine LMW-GS genes of A. elongatum, which were named AeL1 to AeL9, were cloned by genomic PCR. After sequencing, we obtained complete open reading frames from AeL2 to AeL8 and partial genes of AeL1 and AeL9. All nine sequences are homoeologous to those of wheat and related grasses. Comparison of the deduced amino acid sequences with those of published LMW-GS suggests that the basic structures of all the subunits are very similar. However, except for AeL4 and AeL5, which contain the identical N-terminal sequence with LMW-m, other LMW-GS sequences separated from A. elongatum cannot be classified according to previous criteria for the three types: LMW-m (methionine), LMW-s (serine), and LMW-i (isoleucine), and then 12 groups. In addition, there are some characters in the LMW-GS sequences of A. elongatum: AeL2, AeL3, and AeL6 involve a Cys residue in the signal peptide respectively, which is absent in most of LMW-GS; AeL3, AeL6, AeL8, and AeL9 start their first Cys residues in the N-terminal repetitive domains, respectively; both AeL2 and AeL5 have nine Cys residues, with an extra Cys residue in the N-terminal repetitive domain and the repetitive and glutamine-rich domain; AeL2, AeL3, AeL6, and AeL9 comprise long repetitive domains. Phylogenetic analysis indicates that there is a relatively weak sequence identity between the LMW-GS genes from A. elongatum cloned in this study and those reported from other plants. Three LMW-GS sequences, AeL2, AeL3, and AeL6, are clustered to Glu-A3 from wheat than to those from other plants. The possible use of these genes in relation to the high quality of hybrid wheat is discussed.     

High Molecular Weight Glutenin Subunit Composition of Indian Wheats and Their Relationships with Dough Strength

One hundred and seventy two wheat varieties including twenty-five durum wheat cultivars were evaluated for high molecular weight glutenin subunit (HMW-GS) composition using SDS-PAGE. The relationship between HMW-GS and sedimentation tests for dough strength was studied. Three alleles were present at the Glu-A1 locus, eight at Glu-B1 and two at Glu-D1 in bread wheat. The data indicated the prevalence of the Glu-A1b allele (63.5%) at the Glu-A1 and Glu-D1a (71.4%) at Glu-D1 loci. Three alleles, namely Glu-B1b (30.61%), Glu-B1c (25.85%) and Glu-B1i (34.00%) represented about 90% of the alleles at Glu-B1 locus. The combination of Glu-A1b, Glu-B1i and Glu-D1d alleles exhibited highest dough strength as measured by sedimentation value in comparison to other combinations (p<0.001). However, this combination was present only in 7% of the samples evaluated. In durum wheat, the null allele (Glu-A1c) was observed more frequently (76%) than the Glu-A1b allele (24%). Glu-B1f and Glu-B1e alleles represented equally (32% each). Protein subunits 13+16 and 6+8 were found correlated positively (p<0.05) with improved dough strength as compared to subunit 20 in durum wheat. This information can be a valuable reference for designing breeding programme for the improvement of bread and pasta making quality of bread and durum wheats, respectively in India.     

Analysis of diversity of russian and Ukrainian bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (HMWGS) in Russian and Ukrainian bread wheat cultivars was analyzed. The diversity of spring wheat cultivars for alleles of the Glu-1 loci is characterized by medium values of the polymorphism polymorphism information content (PIC), and in winter wheats it varies from high at the Glu-A1 locus to low at the Glu-D1 locus. The spring and winter cultivars differ significantly in the frequencies of alleles of the glutenin loci. The combination of the Glu-A1b, Glu-B1c, and Glu-D1a alleles prevails among the spring cultivars, and the combination of the Glu-A1a, Glu-B1c, and Glu-D1d alleles prevails among the winter cultivars. The distribution of the Glu-1 alleles significantly depends on the moisture and heat supply in the region of origin of the cultivars. Drought resistance is associated with the Glu-D1a allele in the spring wheat and with the Glu-B1b allele in the winter wheat. The sources of the Glu-1 alleles were identified in the spring and wheat cultivars. The analysis of independence of the distribution of the spring and winter cultivars by the market classes and by the alleles of the HMWGS loci showed a highly significant association of the alleles of three Glu-1 loci with the market classes in foreign cultivars and independence or a weak association in the Russian and Ukrainian cultivars. This seems to be due to the absence of a statistically substantiated system of classification of the domestic cultivars on the basis of their quality.     

Allelic variation of high-molecular-weight glutenin genes in bread wheat

The composition and quantity of high-molecular-weight glutenin subunits plays an important role in determining the bread-making quality of wheat. Molecular-genetic analysis of allelic composition of high-molecular-weight glutenin genes in 102 bread wheat cultivars and lines from different geographical regions was conducted. Three alleles at the Glu-A1 locus, nine alleles at the Glu-B1 locus, and two alleles at the Glu-D1 locus were identified. Among the investigated cultivars and lines, 21 were characterized by intracultivar polymorphism. High allelic variation of high-molecular-weight glutenin subunit genes was shown for the collection: 21 and 9 combinations were defined in monomorphic and polymorphic cultivars and lines, respectively. However, the major part of the collection (66.7%) contained four allelic combinations: Glu-A1b Glu-B1c Glu-D1d, Glu-A1b Glu-B1c Glu-D1-2a, Glu-A1a Glu-B1c Glu-D1d, and Glu-A1b Glu-B1c Glu-D1d/Glu-D1-2a. Fourteen cultivars of bread wheat were selected, and they were characterized by a favorable allelic composition of Glu-1 loci.     

Use of recombinant inbred lines of wheat for study of associations of high-molecular-weight glutenin subunit alleles to quantitative traits

Summary The high-molecular-weight glutenin subunits (HMW glutenin), encoded by alleles at homoeologous lociGlu-A1,Glu-B1, andGlu-D1 on the long arms of chromosomes1A,1B, and1D of a set of F₈ random recombinant inbred lines (RIL) derived from the bread wheat cross Anza × Cajeme 71, were classified by SDS-PAGE. Anza has poor breadmaking quality and HMW-glutenin subunits (Payne numbers) null (Glu-A1c), 7+8 (Glu-B1b), and 2+12 (Glu-D1a); Cajeme 71 has good quality and 1 (Glu-A1a), 17+18 (Glu-B1i), and 5+10 (Glu-D1d). The combinations of these alleles in the RIL were examined for associations with grain yield and four indicators of grain quality — protein content, yellowberry, pearling index, and SDS sedimentation volume. Data were obtained from a field experiment with three nitrogen fertilization treatments on 48 RIL and the parents. Orthogonal partitioning of the genetic variance associated with the three HMW glutenin subunit loci into additive and epistatic (digenic and trigenic) effects showed strong associations of these loci with grain yield and the indicators of quality; however, the associations accounted for no more than 25% of the differences between the parents. Genetic variance was detected among the RIL, which had the same HMW glutenin genotype for all traits. Epistatic effects were absent for grain yield and yellowberry, but were substantial for grain protein content, pearling index, and SDS sedimentation volume. All three loci had large single-locus additive effects for grain yield, protein, and SDS sedimentation volume. Yellowberry was largely influenced byGlu-B1 andGlu-D1, whereas pearling index was associated withGlu-A1 andGlu-B1. Even though the observed associations-of effects of HMW glutenin loci with the quantitative characters were small relative to the total genetic variability, they are of considerable importance in understanding the genetics of wheat quality, and are useful in the development of new wheat varieties with specific desired characteristics.     

Identification of LMW Glutenin-Like Genes from <Emphasis Type="Italic">Secale sylvestre</Emphasis> Host

Three low-molecular-weight (LMW) glutenin-like genes (designated as Ssy1, Ssy2, and Ssy3) from Secale sylvestre Host were isolated and characterized. The three genes consist of a predicted highly conservative signal peptide with 20 amino acids, a short N-terminal region with 13 amino acids, a highly variable repetitive domain and a less variable C-terminal domain. The deduced amino acid sequences of the three genes were the LMW-m type due to a methionine residue at the N-terminus. The phylogenetic analysis indicated that the prolamin genes could be perfectly clustered into five groups, including HMW-GS, LMW-GS, α/β-, γ-, and κ-prolamin. The LMW glutenin-like genes of S. sylvestre were more orthologous with the LMW-GS genes of wheat and B hordein genes of barley, which also had been confirmed by the homology analysis with the LMW-GS of wheat at Glu-A3, Glu-B3, and Glu-D3 loci. These results indicated that a chromosome locus (designated as Glu-R3) might be located on the R genome of S. sylvestre with the functions similar to the Glu-3 locus in wheat and its related species.     

Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar

Some allelic forms of low-molecular-weight glutenin subunit (LMW-GS) can greatly influence the end-use of wheat flours, understanding the function of each allele of LMW-GS is important to wheat quality breeding. A LMW-GS gene XYGluD3-LMWGS 1(AY263369) has been cloned from bread wheat cultivar Xiaoyan 6. The deduced protein contained nine cystine residues, one more than that in all other LMW-GSs reported previously, indicating that it is either a new gene or a new allele of a known LMW-GS gene. In this study, the gene was expressed in E. coil in large scale for the testing of its functional property. Reactive Red 120-Agarose resin was used efficiently to purify the expressed LMW-GS proteins from bacteria, with the lactic acid–sodium lactate buffer (pH 4.5) which contained low concentration SDS as elution solution. The purified protein (belonging to the LMW-m family, MW about 35 KDa) was supplemented into a base flour, the results of 10 g dough mixing test indicated that incorporation of the LMW-GS increased the strength of the dough, with significant increases in mixing time (MT) and peak width (PW), and decrease in breakdown in resistance (RBD) compared with the control. In addition, the dough with incorporation of the LMW-GS had more glutenin macropolyeric protein than the control, suggesting that the LMW-GS participated in forming larger glutenin polymers, and greatly contributed to dough strength. The changes in mixing parameters and the amount of glutenin macropolyeric protein were related to the quantity of incorporating subunits.     

Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.)

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A^mA^m, 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T–C transition, namely arginine → cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A^m and A genomes was estimated at 3.98 MYA.Q. Zhang had the same contribution to this work as the first author.     

Molecular characterization of a LMW-GS gene located on chromosome 1B and the development of primers specific for the Glu-B3 complex locus in durum wheat

 Low-molecular-weight glutenin subunits (LMW-GS) represent a specific class of wheat storage proteins encoded at the Glu-3 loci. Particularly interesting are the LMW-GS encoded at the Glu-B3 locus because they have been shown to play an important role in determining the pasta-making properties of durum wheat. Genes encoding LMW-GS have been characterized but only a few of them have been assigned to specific loci. Notably, no complete LMW-GS gene encoded at the Glu-B3 locus has yet been described. The present paper reports the isolation and characterization of a lmw-gs gene located at the Glu-B3 locus. The clone involved, designated pLDNLMW1B, contains the entire coding region and 524 bp of the 5′ upstream region. A nucleotide comparison between the pLDNLMW1B clone and other LMW-GS genes showed the presence of some peculiar structural characteristics, such as short insertions in the promoter region, the presence of a cysteine codon in the repetitive domain, and a more regular structure of this region, which could be important for its tissue-specific expression and for the functional properties of the encoded protein, respectively. Received : 30 May 1997 / Accepted : 29 July 1997