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.     

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.     

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.     

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.     

Geographical comparison of genetic diversity in Asian landrace wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) germplasm based on high-molecular-weight glutenin subunits

Glutenin largely determines wheat bread baking quality. As high-molecular-weight glutenin subunit (HMW-GS), related to Glu-1 loci, determines wheat flour elasticity, it correlates strongly with bread-making quality. This study was aimed at clarifying genetic variations in bread-making characteristics between East and West Asian wheat landrace germplasms, by investigating HMW-GS allelic composition of 1068 wheat accessions. Herein, the accession number having reported HMW-GS pattern in previous studies was 855. However, the accession number with newly detected HMW-GS patterns was 114. These new HMW-GS patterns were classified into 4 types based on similarity. Eight Korean accessions with these four types were identified. Concerning landrace germplasm nature, 99 accessions showed heterogeneous patterns caused by seed mixture. The Glu-1 loci allelic variation analysis, revealed that the percentages of Glu-A1c (73.6%), Glu-B1b (60.2%), and Glu-D1a (68.5%) were highest at Glu-A1, Glu-B1, and Glu-D1 loci, respectively. The incidence of preferable alleles for bread baking was high in Chinese accessions. In bread-making quality evaluation using Glu-1 score, 24 among 35 accessions with full score were from China. The polymorphic information content index of each origin based on HMW glutenin subunit combination showed that West Asian and neighboring-regional landraces, excluding Afghanistan ones, were more diverse than East Asian landraces excluding Chinese ones. Cluster analysis based on Glu-1 allelic combination showed that many Korean, Japanese, and Afghan accessions were in the same group. However, many Chinese and other West Asian accessions were in the other group despite geographical distance.     

Use of near-isogenic wheat lines to determine the glutenin composition and functionality requirements for flour tortillas

In wheat ( Triticum aestivum L), the synthesis of high molecular weight (HMW) glutenins (GS) is controlled by three heterologous genetic loci present on the long arms of group 1 wheat chromosomes. The loci Glu-A1, Glu-B1, and Glu-D1 and their allelic variants play important roles in the functional properties of wheat flour. This study focused on understanding the functionality of these protein subunits on tortilla quality. Near-isogenic wheat lines in which one or more of these loci were absent or deleted were used. Tortillas were prepared from each deletion line and the parent lines. The elimination of certain HMW-GS alleles alter distinct but critical aspects of tortilla quality such as diameter, shelf stability, and overall quality. Two deletion lines possessing HMW-GS 17 + 18 at Glu-B1 and deletions in Glu-A1 and Glu-D1 had significantly larger tortilla diameters, yet tortilla shelf life was compromised or unchanged from the parent lines used to develop the deletion lines or the commercial tortilla flour used as a control. Alternatively, a deletion line possessing Glu-A1 and Glu-D1 (HMW-GS 1, 5 + 10) and a deletion in Glu-B1 also significantly improved tortilla diameters. Whereas the increase in diameter was less than the line possessing only HMW-GS 17 + 18 at Glu-B1, the stability of the tortillas were, however, maintained and improved as compared to the parent lines containing a full compliment of HMW-GS. Thus, the presence of subunits 5 + 10 at Glu-D1 alone or in combination with subunit 1 at Glu-A1 appears to provide a compromise of improvement in dough extensibility for improved tortilla diameters while also providing sufficient gluten strength to maintain ideal shelf stability.     

我国不同时期小麦品种高分子量麦谷蛋白亚基组成比较

用SDS-PAGE方法测定了我国10个小麦主产省份171份小麦品种和高代品系的高分子量麦谷蛋白亚基(HMW-GS)组成。鉴定出18种HMW-GS,40种HMW-GS组成形式,其中20种亚基其组成形式只在一个品种(系)中出现。Glu-A1位点亚基1和Null出现最多,Glu-B1位点7 8和7 9亚基对占绝对优势,Glu-D1位点2 12亚基对出现频率最高。Null、7 9、2 12、Null,7 8,2 12,1、7 8,2 12,1、7 9、2 12等亚基组成形成出现频率最高,占分析品种的49.71%。与前人研究相比,新育成品种HMW-GS亚基组成发生了明显变化,面包优质亚基(对)1、5 10出现的频率显著升高,亚基多态性增加,组成形式明显改善,这些对于品质改良和品种选育是非常有利的,新育成品种Glu-1品质评分已超过7。尽管个别品种亚基组成好,品质优良,但总体上看,我国小麦品种与其它国家相比品质还存在一定差距,提高5 10、17 18等优质亚基的频率是改善我国小麦面包品质的重要措施。     

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.     

Genetic variability in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) of Pakistan based on polymorphism for high molecular weight glutenin subunits

Variation in bread wheat including pre and post green revolutions varieties of Pakistan along with landraces was investigated for high molecular weight Glutenin subunits (HMW Gs) encoded at three genes (Glu-A1, Glu-B1, Glu-D1) with SDS-PAGE. The germplasm was diverse and unique on the basis of HMW Gs compositions and out of 14 alleles detected at all the Glu-1 loci, three belonged to Glu-A1, nine to Glu-B1 and two to Glu-D1 locus. High variation was observed in the landraces and higher gene diversity was observed between the populations as compared to the gene diversity within populations, whereas a reverse pattern of gene diversity was observed when populations were pooled across the region (higher within the regions than between the regions). A lack of relationship between the HMW Gs diversity and the altitude of collection site was observed. A data base has been generated in this study which could be expanded/exploited for cultivar development or management of gene bank.     

新疆小麦地方品种资源HMW-GS的遗传多样性组成分析

为了明确新疆小麦地方品种高分子量麦谷蛋白亚基的遗传多样性,并为小麦品质改良提供基础材料,利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE) 技术,分析了源自新疆地区的282份小麦地方品种的高分子量麦谷蛋白亚基组成。结果表明,在Glu-A1、Glu-B1和Glu-D1位点上的等位变异分别为3、6和 5种,三个位点上的优势亚基依次为null、7+8和2+12,其频率分别是75.5%、90.8%和72.0%。在Glu-1位点共检测到20种亚基组合,其中(null, 7+8, 2+12)组合的频率最高,为52.8%, 其次是(null, 7+8, 2.6+12)和(2*, 7+8, 2+12)组合,其频率分别为14.1%和11.0%,其它亚基组合的频率均低于10%。另外,在Glu-D1位点上还检测到一个新的亚基2.6+12。在供试的282份新疆地方品种中发现了两份具有优质亚基组合的材料,它们的亚基组成为(2*, 7+9, 5+10)和(1, 7+9, 5+10),这些地方品种可作为改良小麦品质性状的重要遗传资源。     

Protein heterogeneity in European wheat landraces and obsolete cultivars

Identity and present degree of genetic homogeneity and heterogeneity, respectively of 52 European wheat accessions, maintained in the collection of wheat genetic resources, have been characterized using analyses of glutenins by sodiumdodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Six of the analyzed wheat accessions were observed to be homogeneous, while 46 (88.5%) of them were heterogeneous in protein profiles. Heterogeneous accessions possessed 2 to 13 different protein lanes. Together, 17 high molecular weight glutenin subunit (HMW-GS) alleles have been found. The most frequent HMW-GS alleles at the Glu-A1, Glu-B1, and Glu-D1 complex loci were 1, 7+9, and 2+12, respectively. However, also low frequented HMW-GS alleles or allelic combinations, such as 7+15, 13+16, 20, 6, 7, and 9 were observed. Furthermore, another new allele encoding HMW glutenin subunit with relative molecular weight 98.6 kDa has been found in one of the lines of the cultivar Eritrospermum 917. The Glu-score in the examined accessions varied in broad range, some of the lines reached the maximum value 10.     

(英文)

用SDS-PAGE方法测定了我国10个小麦主产省份171份小麦品种和高代品系的高分子量麦谷蛋白亚基（HMW-GS）组成。鉴定出18种HMW-GS，40种HMW-GS组成形式，其中20种亚基其组成形式只在一个品种（系）中出现。Glu-A1位点亚基1和Null出现最多，Glu-B1位点7＋8和7＋9亚基对占绝对优势，Glu-D1位点2＋12亚基对出现频率最高。Null、7＋9、2＋12、Null，7＋8，2＋12，1、7＋8，2＋12，1、7＋9、2＋12等亚基组成形成出现频率最高，占分析品种的49.71％。与前人研究相比，新育成品种HMW-GS亚基组成发生了明显变化，面包优质亚基（对）1、5＋10出现的频率显著升高，亚基多态性增加，组成形式明显改善，这些对于品质改良和品种选育是非常有利的，新育成品种Glu-1品质评分已超过7。尽管个别品种亚基组成好，品质优良，但总体上看，我国小麦品种与其它国家相比品质还存在一定差距，提高5＋10、17＋18等优质亚基的频率是改善我国小麦面包品质的重要措施。     

(英文)

对来源于美、中、俄及埃塞阿比亚等22个国家的142份硬粒小麦材料的种子贮藏蛋白位点及遗传变异进行了研究。供试的硬粒小麦(Triticum durum Desf )材料共检测出37条醇溶蛋白条带，无1条带纹为所有材料共有，多态性达到100％，说明硬粒小麦具有丰富的醇溶蛋白等位变异。聚类分析将142份供试材料分为3个大类，材料间遗传差异大小在不同的国家有所不同，表明醇溶蛋白带型与地理来源有一定关系。高分子量谷蛋白电泳共分离出14种亚基和15种亚基组合，但是优质亚基所占比例不高，这可能是因为硬粒小麦加工用途的特殊性，使得多年的育种并未太多改变硬粒小麦高分子量谷蛋白亚基等位变异的频率，促成优质亚基的累计。     

Protein Heterogeneity in European Wheat Landraces and Obsolete Cultivars: Additional Information II

The endosperm storage protein of 46 European wheat (Triticum aestivum L.) landraces and obsolete cultivars have been fractionated by SDS-PAGE to determine the composition of high molecular weight glutenin subunits (HMW-GS) composition. It has been discovered that about 46% of the wheats were heterogeneous, comprising 2–11 different glutenin profiles. Eighteen of them were observed to be homogeneous. A total of 13 HMW-GS alleles, including 3 at the Glu-A1, 8 at the Glu-B1, and 3 at the Glu-D1 loci were revealed. HMW-GS null controlled by locus Glu-A1, subunits 7 + 8 by Glu-B1, and 2 + 12 by Glu-D1 predominated. However low frequented alleles such as 17 + 18, 20, 6, and 7 were observed. Furthermore, other new alleles encoding HMW-GS at the locus Glu-B1 have been found in one of France cultivar (Saumur d’Automne). The glutenin-based quality score ranged from 4 to 10.     

Frequency of the high-molecular-weight glutenin allele in Asian hexaploid wheat (Triticum aestivum L.) and the transmission route through which the wheat may have reached Japan,the most geographically remote region of wheat production in the world

The frequency of the Glu-D1f allele in Japanese, Chinese, and other Asian hexaploid wheat varieties was analyzed in order to investigate a possible transmission route for hexaploid wheat to the Far East, Japan. The 1380 published data sets were compared to the results for 1107 hexaploid Asian wheat varieties which were determined in this study. The frequency of the Glu-D1f allele was clearly different between areas; the allele was present from northern and southern Japan, from Xinjiang, Jiangsu, Zhejiang, and Beijing in China, and from Afghanistan. A high frequency of the high-molecular-weight glutenin Glu-D1f allele was found predominantly in southern Japan. This distribution of an adaptively neutral character suggests a specific route of transmission for hexaploid wheat to eastern China and the Far East, Japan. It was introduced from Afghanistan, carried to Xinjiang (in northwest China), Jiangsu, and Zhejiang (in southeast China), and then to southern Japan along the so-called Silk Road. It is believed that cultivated hexaploid wheat originated in the Middle East and the Near East and was carried along the Silk Road through China to the Far East, Japan. Japan is the most geographically remote region of wheat production in the world. During the course of its long journey and its adaptation to diverse local environments, Japanese hexaploid wheat has developed a unique composition of glutenin Glu-D1 alleles. The frequency of this allele in different wheat varieties allowed us to hypothesize a possible route for the transmission of hexaploid wheat into the Far East, Japan.     

Protein heterogeneity in European wheat landraces and obsolete cultivars: Additional information

The objective of this study was to determine the composition of high molecular weight glutenin subunits of landraces and obsolete cultivars. Altogether glutenin profiles of 67 European wheats were analyzed by sodiumdodecylsulphate polyacrylamide gel electrophoresis. Nineteen of them were observed to be homogeneous, whereas 48 (71%) were heterogeneous in glutenin profiles. Heterogeneous accessions possessed from 2 to 9 different glutenin phenotypes. Seventeen high molecular weight (HMW)-glutenin subunits have been found, three belonged to Glu-1A, 11 to Glu-1B, and three to Glu-1D locus. The most frequented HMW-GS at the Glu-A1, Glu-B1, and Glu-D1 complex loci were 0, 7+9, and 2+12, respectively. However, allele low frequented in wheat such as 13+16, 20, 6, 7, 8, and 9 were observed also. Furthermore, other new alleles encoding HMW-GS at the locus Glu-1B with relative molecular weight 120 and 104 kDa have been found in one of the line of the Swedish cultivar Kotte. TheGlu-1 quality score in the examined accessions varied broadly with some lines reaching the maximum value of 10.     

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.     

Detection of high molecular weight glutenin subunits in triticale (x Triticosecale Wittm.) Cultivars by capillary zone electrophoresis

An improved method for separating and characterizing high molecular weight glutenin subunits (HMW-GS) in hexaploid triticale by capillary zone electrophoresis (CZE) was developed. A low-concentrate mixture of hydrophilic polymers, poly(vinylpyrrolidone) (PVP) and hydroxypropylmethylcellulose (HPMC), in an isoelectric buffer was employed for dynamic coating of the capillary inner wall. In separation buffer PVP with lower concentrated poly(ethylene oxide) (PEO) was replaced. The CZE electropherograms of HMW-GS showed two group peaks in accordance with x- and y-type subunits with migration times of 6.8-7.8 and 8.4-11.5 min, respectively. In total, 14 HMW subunits (2 subunits encoded by Glu-A1 locus and 12 by Glu-B1) were identified. The CZE analyses revealed that each of the subunits Bx7 and By8 determined by SDS-PAGE makes up three subunits (Bx6.8, Bx7, and Bx7* and By8, By8*, and new By8**, respectively), with different migration times. It was also shown that the subunits By18 and By20 in triticale determined by SDS-PAGE have different migration times in comparison with the same subunits in bread wheat. For these new HMW-GS, the following names were assigned: By18* instead of By18 and By20* instead of By20. The presented CZE method is an efficient alternative to the SDS-PAGE procedure for early selection of useful triticale genotypes with good breadmaking quality.     

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 variability at the Glu-1 loci in old and modern wheats (triticum aestivum L.) cultivated in Slovakia

The high molecular weight glutenin subunits (HMW-GS) composition at the Glu-1 complex loci, in 23 old original wheat genotypes cultivated in Slovakia several decades ago and 32 modern Slovak and Czech wheat cultivars growed in Slovakia at present were studied by SDS-PAGE. Some of the HMW-GS – subunit pairs 3+12, 17+18, and subunit 20, present in old historical wheats were missing in modern cultivars utilized in Slovakia nowadays. There were observed 15 different HMW-GS encoded by 11 alleles or allelic pairs in old genotypes. Lower number of different HMW-GS and competent alleles were observed in a set of modern wheat cultivars – 11 different HMW glutenin subunits encoded by 8 alleles or allelic pairs. The same number of different HMW-GS patterns was revealed in both sets of wheats. From the point of view of genetic variability, it could be concluded that long-term effort of breeders and decreasing of cultivation of landraces and old cultivars are associated with the loss of several HMW-GS alleles and decreasing of genetic variability of wheats. Molecular characterization can reveal broad allelic variability of old wheat genotypes and landraces. Their maintenance in genetic resource collections can prevent losses of these interesting genes.