Allelic diversity of HMW and LMW glutenin subunits and omega-gliadins in French bread wheat (Triticum aestivum L.)

Wheat endosperm storage proteins, namely gliadins and glutenins, are the major components of gluten. They play an important role in dough properties and in bread making quality in various wheat varieties. In the present study, the different alleles encoded at the 6 glutenin loci and at 3 -gliadin loci were identified from a set of 200 hexaploid wheat cultivars grown primarily in France using SDS PAGE. At Glu-A1, Glu-B1 and Glu-D1, encoding high molecular weight glutenin subunits (HMW-GS), 3, 8 and 5 alleles were observed respectively. Low molecular weight glutenin subunits (LMW-GS) displayed similar polymorphism, as 5 and 11 alleles were identified at loci Glu-A3 and Glu-B3 respectively. Four alleles were observed at Glu-D3 loci. Omega-gliadin diversity was also very high, as 7, 13 and 9 alleles were found at Gli-A1, Gli-B1 and Gli-D1, respectively. A total of 147 (or 149) patterns resulted from the genetic combination of the alleles encoding at the six glutenin loci (or Glu-1 and Gli-1 loci). Although Glu-1 and Glu-3 loci were located on different chromosome arms and were theoretically independent, some associations were revealed due to pedigree relatedness between some French wheat cultivars. The usefulness of allelic identification of LMW-GS together with HMW-GS and gliadins for future genetic and technological wheat improvement is discussed.     

高分子量麦谷蛋白亚基基因Glu-1Ay 在小麦中的表达

小麦是世界上重要的粮食作物.小麦的品质一直是发达国家小麦生产的重要指标.中国在解决了产量需求的今天,品质已经成为小麦育种的主要目标之一.小麦加工品质主要取决于其高分子量麦谷蛋白亚基(HMW-GS)数量及亚基组合.一般来说,高分子量麦谷蛋白亚基对加工品质的影响具有加性效应,因此增加普通小麦 HWM-GS 数量可以改良面团品质[1]..     

Evaluation of geographic distribution of high-molecular-weight glutenin subunits (HMW-GS) in wild and cultivated Triticum species

Variation of high-molecular-weight glutenin subunit (HMW-GS) in 632 wild and cultivated Triticum accessions was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A total of 11 alleles of HMW-GS in diploid species, 22 in tetraploid species, and 15 in hexaploid species were detected. Diploid species on Glu-1A locus and tetraploid species Glu-1B locus showed the highest diversity, respectively. Tetraploid species had the highest level of diversity on three Glu-1 loci, followed by hexaploid and diploid, based on Shannon’s information index, Nei’s genetic diversity, and percentage of polymorphic loci. Molecular variance analysis confirmed main variance of HMW-GS within species, regions, and locations, respectively. Variance among species and regions was enhanced gradually with the increase of ploidy. Significant non-random distributions between the phylogenic trees of HMW-GS and the locations of accessions were tested by GenGIS software, indicated that geographic factors played an important role along the different orientations in the spread of Triticum species. We found one original diversified center in diploid what located around Elazig, Malatya, Gaziantep, Urfa, and Kiziltepe in Turkey, and three diversified centers in tetraploid wheat, including Turkey–Armenia–Georgia–Iran, Portugal–Spain, and Ethiopia, respectively, and two diversified adjacent areas between Turkey and Switzerland and around Turkey, Georgia, and Armenia. The original center of diploid species located in southeast Turkey, where the unexpressed 1Ay subunit was mainly distributed in T. urartu, could be one of the candidate regions of polyploidization of Triticum L. The regional distribution of HMW-GS and species also provided geographic evidences for the existence of founder effect on the spread of Triticum species. The present study suggests that integrating genetic diversity with geographic characterization in Triticum could very useful for collection, conservation, and utilization, as well as for research microevolution and domestication.     

Characterization of Mexican Creole wheat landraces in relation to morphological characteristics and HMW glutenin subunit composition

One hundred lines derived from 14 wheat landraces collected in Mexico were characterized in relation to spike and grain morphology and HMW-glutenin subunit composition. Up to seven botanical varieties were identified among these materials based in four morphological traits. The remaining nine morphological traits showed wide variation. The allelic variation at the Glu-1 loci was wide, although showed a clear risk of genetic erosion due to the low frequency of some alleles. These genotypes could be used as genetic resources to improve important biotic and abiotic traits as well as to widen genetic diversity controlling the HMW glutenin subunit composition of common wheat.     

Genetic variation of high-molecular-weight glutenin subunit composition in Asian wheat

To clarify the genetic properties of the HMW glutenin subunit composition of Asian endemic wheats, SDS–PAGE analysis was conducted using 1,139 bread wheat accessions that were originally collected in Asia. The samples were divided into six regional groups, Western Asia, Caucasia, Central Asia, Afghanistan, Southern Asia, and Eastern Asia. The genotype Glu-A1c, Glu-B1b, and Glu-D1a encoding subunits null, 7+8, and 2+12 had an overall frequency of 55.2%. Thus, we conclude that it is the typical genotype of the HMW glutenin subunits that characterize Asian endemic wheat. The frequency of the typical Asian genotype was relatively high in the central belt of Asia (Western Asia, Afghanistan, and Eastern Asia) and low in the marginal regions (Caucasia, Central Asia, and Southern Asia). In Southern Asia, the frequency of Glu-B1i, which encodes subunit 17+18, was the highest at the Glu-B1 locus. In Caucasia and Central Asia, the frequency of Glu-D1d, which encodes subunit 5+10 (which is considered to be the most useful for making bread), was high. The level of genetic variation, as estimated using the frequencies of the various alleles, was relatively low in the central belt of Asia and high in the marginal regions. Among the three Glu-1 loci, the highest number of alleles was detected at the Glu-D1 locus. This result was caused by the presence of rare Asian specific alleles at the Glu-D1 locus, in which a newly found allele, Glu-D1bs, encoding subunit 2.1+12 was included.     

Intra- and interpopulation diversity for HMW glutenin subunits in Spanish spelt wheat

The diversity of HMW glutenin subunits in spelt wheat, Triticum aestivum ssp. spelta, was studied electrophoretically in 333 accessions grouped in 50 populations originally collected from Asturias, North of Spain, in 1939. The inter- and intra-population distribution of HMW glutenin alleles at the Glu-A1, Glu-B1 and Glu-D1 loci were investigated. The results show that the genetic variation in HMW glutenin subunits is mainly present within populations, being the variation between populations only 21%. The materials analysed showed a wide polymorphism for the HMW glutenin subunits, although some allelic variants were clearly dominant. This suggests the possibility of a loss of variability before the collection that could have increased with the subsequent reduction of the cultivation area of this species in this Spanish region.     

Characterization of a monoclonal antibody specific for HMW subunits of glutenin and its use to investigate glutenin polymers

A monoclonal antibody, IFRN 1602, has been developed to a synthetic peptide based on the sequence (94)GSVTCPQQV(101) of HMW subunit 1Dx5. The antibody bound strongly to the synthetic peptide based on the cognate sequence of HMW subunit 1Dx2 which contains a serine instead of a cysteine residue. However, it recognized the immunizing peptide by enzyme-linked immunosorbent assay (ELISA) only poorly, probably because the peptide exists as a disulfide-bonded dimer under the assay conditions. From immunoblotting studies against a wide range of wheat varieties, IFRN 1602 was shown to primarily recognize x-type HMW subunits of glutenin encoded on chromosomes 1A and 1D, cross-reacting weakly with the 1A and 1D y-type subunits. It did not bind to any of the 1B-encoded subunits. The Mab also recognized a small number of polypeptides of greater mobility than HMW subunits which were not visible on the stained gels and occurred only in the presence of specific 1A and 1D x-type HMW subunits. Such polypeptides were not present in a preparation of recombinant subunit 2, suggesting that they are modified forms of the subunits which arise in the seed perhaps by processing of the associated subunits. When used to probe partially reduced glutenin, IFRN 1602 bound to 1Dx5-1Dy10 dimers. As the Mab reacted primarily with Cys(97) of 1Dx5 in a reduced form, these data suggest that this residue is not involved in either intra- or intermolecular disulfide bond in the HMW subunit dimers. Thus, Cys(97) of 1Dx5 may be present in gluten in a reduced form, involved in intramolecular disulfide bonds, or linking of the HMW subunit dimers into larger polymers.     

Genetic diversity of HMW glutenin subunit in Chinese common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) landraces from Hubei province

The high molecular weight (HMW) glutenin subunit composition of 111 common landraces of bread wheat collected from Hubei province, China has been determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ninety six of the accessions were homogeneous for HMW glutenin subunit composition and 15 were heterogeneous. For the Glu-1 loci, 16 alleles were detected, 3 at the Glu-A1locus, 9 at the Glu-B1and 4 at the Glu-D1. Three novel alleles were identified, two at the Glu-B1 and one at the Glu-D1locus. Combination of these 16 alleles resulted in 14 different HMW subunit patterns. The distribution of HMW glutenin subunit alleles in a subset of 105 of the 111 accessions representing six populations was assessed both at the individual population and whole population levels. The results demonstrated that the distribution of allelic patterns varied among populations. Taken together, 62.5% of the alleles detected were considered to be rare alleles while the Glu-A1c (null), Glu-B1b (1Bx7 + 1By8) and Glu-D1a (1Dx2 + 1Dy12) alleles were found most frequently in the six populations. The subset exhibited relatively high genetic diversity (A = 5.33, P = 1.00, Ae = 1.352 and He = 0.238) with 81.5% of the diversity being within populations and 18.5% between populations.     

Novel and ancient HMW glutenin genes from Aegilops tauschii and their phylogenetic positions

A pair of novel high-molecular-weight glutenin subunits (HMW-GS) 1Dx3.1^t and 1Dy11*^t were revealed and characterized from Aegilops tauschii Coss. subspecies tauschii accession AS60. SDS-PAGE band of 1Dx3.1^t was between those of 1Dx2 and 1Dx3, while 1Dy11*^t was between 1Dy11 and 1Dy12. The lengths of 1Dx3.1 ^t and 1Dy11* ^t were 2,514?bp and 1,968?bp, encoding 836 and 654 amino acid residues, respectively. Their authenticity was confirmed by successful expression of the coding regions in Escherichia coli. Network analysis indicated that 1Dx3.1 ^t together with other five rare alleles only detected in Asia common wheat populations represented the ancestral sequences in Glu-D1 locus. Neighbor-joining tree analysis of previously cloned x-type and y-type alleles in the Glu-D1 locus supported the hypothesis that more than one Ae. tauschii genotypes were involved in the origin of hexaploid wheat and that different Ae. tauschii accessions contributed the D genome to common wheat and Ae. cylindrical Host, respectively. An Ae. tauschii accession with 1Dx3.1 ^t or a closely related allele probably have involved in the origin of common wheat. Since accession AS60 used in this study belonged to typical ssp. tauschii, present results suggested the possibility that ssp. tauschii was involved in the evolution of common wheat.     

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.     

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.     

Genetic diversity of HMW glutenin subunits in diploid,tetraploid and hexaploid <Emphasis Type="Italic">Triticum</Emphasis> species

The genetic variations of high-molecular-weight (HMW) glutenin subunits in 1051 accessions of 13 Triticum subspecies were investigated using sodium dodecyl sulfate polyacrylamide-gel electrophoresis. A total of 37 alleles were detected, resulting in 117 different allele combinations, among which 20, 68 and 29 combinations were observed in diploid, tetraploid and hexaploid wheats, respectively. Abundance and frequency of allele and combinations in tetraploid wheats were higher than these in hexaploid wheats. Allele Glu-A1c was the most frequent subunit at Glu-A1 locus in tetraploid and hexaploid wheats. Consequently, the results also suggested that the higher variations occurred at Glu-B1 locus compared to Glu-A1 and Glu-D1. Therefore, carthlicum wheat possessing the allele 1Ay could be presumed a special evolutional approach distinguished from other tetraploid species. Furthermore, this provides a convenient approach of induction of the 1Ay to common wheat through direct cross with carthlicum wheat. Alleles Glu-B1c and Glu-B1i generally absent in tetraploid wheats were also found in tetraploid wheats. Our results implied that tetraploid and hexaploid wheats were distinguished in dendrogram, whereas carthlicum and spelta wheats and however displayed the unique performance. In addition, founder effect, no-randomness of diploidization, mutation and artificial selection could cause allele distribution of HMW-GS in Triticum. All alleles of HMW-GS in Triticum could be further utilized through hybrid in the quality improvement of common wheat.     

Assessment of genetic variability in hexaploid wheat landraces of Pakistan based on polymorphism for HMW glutenin subunits

Summary Sixty hexaploid wheat landraces collected from five regions of Pakistan were assessed for genetic variability in terms of high molecular weight (HMW) glutenin subunits as revealed by SDS-PAGE. The germplasm appeared to be diverse and unique on the basis of HMW glutenin subunit compositions. Out of 24 alleles detected at all the Glu-1 loci, four belonged to Glu-A1, 12 to Glu-B1 and eight to Glu-D1 locus. The number of novel HMW glutenin subunits detected were 1, 4 and 6 at the three loci (Glu-A1, Glu-B1, Glu-D1), respectively. The frequency distribution patterns of 24 allelic variants detected at the three Glu-1 loci in 1080 samples analysed for 60 accessions were determined both on the basis of individual accessions and on the basis of regions (accessions pooled across the regions). One allele (null) at the Glu-A1 locus, three alleles (17+18, 7+8, 14) at the Glu-B1 locus and, two alleles (2+12 and 2^**+12) at the Glu-D1 locus were found most frequently distributed in the 60 populations. Maximum variation was observed in the Baluchistan and Gilgit regions of Pakistan in terms of distribution of novel Glu-1 alleles. A higher gene diversity was observed between the populations as compared to the gene diversity within the populations while, a reverse pattern of gene diversity was observed when populations were pooled across the regions (higher within the regions than between the regions). A data base has been generated in this study which could be expanded and usefully exploited for cultivar development or management of gene bank accessions.     

Polymorphism of high molecular weight glutenin subunits in three species of Aegilops

A collection of 136 accessions of Aegilops umbellulata (39), Ae. comosa (75) and Ae. markgrafii (22) was analysed for high-molecular-weight (HMW) glutenin subunits composition. The homogeneity of the accessions was studied and 55.1% of the collection was homogeneous for HMW glutenin subunits (29 Ae. umbellulata, 33 Ae. comosa and 14 Ae. markgrafii). The HMW glutenin subunits of Ae. umbellulata are encoded by the Glu-U1 locus; in Ae. comosa results showed that this proteins are encoded at the 1M chromosome, and the locus was named Glu-M1. In Ae. markgrafii it was assumed that HMW glutenin subunits were encoded by an homoeologous locus and it was named Glu-C1. All the accessions of Ae. umbellulata and Ae. markgrafii expressed both, x-type and y-type subunits. Among the Ae. comosa accessions, only one expressed an x-type subunit alone. All the accessions of Ae. umbellulata and some of Ae. comosa had x-type glutenins of higher molecular weights than those commonly present in bread wheat. A total of 8 alleles were detected at the Glu-U1 locus, 11 at the Glu-M1 and 4 at the Glu-C1. The new HMW glutenin variation found in this work suggests their possible utilisation in breeding for wheat quality.     

The relationship between high-molecular-weight glutenin subunit composition and the quality of Japanese hexaploid wheat lines

To reveal the high-molecular-weight (1-1MW) glutenin subunit composition, the seed storage proteins of 40 Japanese wheat (Triticum aestivum) lines were fractionated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis to determine their HMW glutenin subunit composition. These were identified by comparison of subunit mobility with that previously found in hexaploid wheat. Twelve different, major glutenin HMW subunits were identified. Each line contained three to five subunits, and 11 different glutenin subunit patterns were observed for 11 alleles in Japanese lines. The Glu-1 quality scores were not particularly high for most of the Japanese wheats in the southern part of Japan (Kyushu district). However, the Glu-1 quality scores of several wheat lines in the Hokkaido area (north Japan) were high. South Japanese wheat lines showed specialty allelic variation in the glutenin HMW 145 kfla subunit, different from those in non-Japanese hexaploid wheats.     

Prolamin aggregation, gluten viscoelasticity, and mixing properties of transgenic wheat lines expressing 1Ax and 1Dx high molecular weight glutenin subunit transgenes

The composition of high molecular weight (HMW) subunits of glutenin determines the gluten strength and influences the baking quality of bread wheat. Here, the effect of transgenes coding for subunits 1Ax1 and 1Dx5 was studied in two near-isogenic wheat lines differing in their HMW subunit compositions and mixing properties. The subunits encoded by the transgenes were overexpressed in the transformed lines and accounted for 50-70% of HMW subunits. Overexpression of 1Ax1 and 1Dx5 subunits modified glutenin aggregation, but glutenin properties were much more affected by expression of the 1Dx5 transgene. This resulted in increased cross-linking of glutenin polymers. In dynamic assay, the storage and loss moduli of hydrated glutens containing 1Dx5 transgene subunits were considerably enhanced, whereas expression of the 1Ax1 transgene had a limited effect. The very high strength of 1Dx5 transformed glutens resulted in abnormal mixing properties of dough. These results are discussed with regard to glutenin subunit and glutenin polymer structures.     

真空和面对面条面团谷蛋白大聚合体含量及粒度分布的影响

明确真空和面对低水分面条面团中谷蛋白大聚合体(glutenin macropolymer,GMP)特性的影响,有助于探讨真空和面改善面条质地的化学结构基础,该研究以3个小麦品种(郑麦366、宁春4号、济麦22)磨制的面粉为材料,在不同真空度(0、0.06、0.08 MPa)下和面,和面时间为8 min,测定面团中GMP含量及粒度分布,并采用Ellman试剂比色法分析蛋白质和GMP中游离巯基含量的变化。结果表明,与非真空和面相比,0.06 MPa制作的面团中GMP含量较高,而过高的真空度(0.08 MPa)会导致GMP含量降低。真空和面对面团中GMP粒度分布有显著影响,济麦22和宁春4号面团在0.06 MPa时大粒径GMP所占体积、表面积和数目百分比显著高于0和0.08 MPa(P0.05),而郑麦366在0.08 MPa时大粒径GMP所占体积百分比显著较高。真空度为0.06 MPa时,济麦22和宁春4号面团中的游离巯基含量显著低于0和0.08 MPa(P0.05);而对于郑麦366,0.08 MPa制作的面团中游离巯基含量显著低于非真空和面。对于2种中筋小麦粉(济麦22和宁春4号),适宜真空度和面会使GMP中更多的游离巯基参与二硫键交联。结论认为,适宜真空度和面可以提高面团中蛋白质聚合度;与蛋白质和湿面筋含量高、面团强度大的郑麦366相比,2种中筋小麦粉面团中GMP特性受真空度变化的影响更明显。研究结果为揭示真空和面的作用机制、深入认识面筋蛋白在面条加工中的作用提供参考。     

施氮量对不同小麦品种高分子量麦谷蛋白亚基表达量及品质影响的研究

采用盆栽试验和SDS-PAGE技术,研究了氮肥对强筋和中筋小麦亚基表达量、品质性状及相关关系的影响。结果表明,施用氮肥提高了优质小麦高分子量谷蛋白亚基表达量,对强筋小麦影响较小,而对中筋小麦影响较大。施氮后,强筋小麦的亚基表达量与醇溶蛋白和谷蛋白的含量极显著相关,而中筋小麦的亚基表达量仅与谷蛋白的含量显著相关,说明氮肥对不同亚基组成小麦高分子量谷蛋白表达量及蛋白组分调控有一定差异。施氮后,高分子量谷蛋白亚基表达量与各项加工品质指标呈正相关;强筋小麦的亚基表达量与湿面筋含量、稳定时间、沉降值和评价值相关性达到显著或极显著水平;而中筋小麦的亚基表达量仅与沉降值相关性达到显著水平。