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.     

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.     

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.     

Isozyme variation in species of the section Comopyrum of Aegilops

Genetic variation at 21 enzyme loci was studied in Aegilops comosa and Ae. uniaristata, the two species belonging to section Comopyrum of Aegilops. In Ae. comosa, the mean number of alleles per locus was 2.00 and the proportion of polymorphic loci was 0.667; in Ae. uniaristata they were 1.19 and 0.143, respectively. The two species were genetically distant from each other (I=0.561) supporting the previously assigned different genome symbols, M and N.     

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.     

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.     

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.     

Rheological Properties of Full-Formula Doughs Derived from Near-Isogenic 1BL/1RS Translocation Lines

Four pairs of near-isogenic wheat lines, with and without the 1BL/1RS translocation, and differing at the Glu-1 loci (coding for high molecular weight [HMW] glutenin subunits) were evaluated for their dough mixing properties, dough stickiness, and baking performance. In all 1BL/1RS translocation lines, weakening of the dough consistency occurred within 2 min past peak time. The full-formula dough from every 1BL/1RS translocation line exhibited poor dough mixing characteristics and increased stickiness compared to the corresponding wheat control. The HMW glutenin subunits coded by the Glu-A1 locus had no apparent effect on mixing properties, but did have a slight effect on the dough stickiness at two of the four stages of dough mixing. Glu-B1 and Glu-D1 loci encoded glutenin subunits produced significant changes in dough mixing properties and dough stickiness, respectively. With respect to baking performance, there was no significant difference between loaf volumes of 1BL/1RS versus control wheats for three of four near-isogenic pairs. Within the 1RS-group, the translocation lines containing HMW glutenin subunits 5+10 produced bread with greater loaf volumes than the pairs containing its allelic counterpart 2+12. Loaf volume was not influenced by the subunits associated with the Glu-B1 loci. In general, the breads baked from 1BL/1RS translocation lines had a relatively poor crumb and crust quality and contained larger gas cells than the wheat controls. In comparing isogenic pairs, the magnitude of the difference in loaf volume between the control wheat and the corresponding 1BL/1RS translocation line was greater in the pair unique for HMW subunits 5+10; the difference was primarily due to the stronger mixing properties of the wheat control.     

Polymorphism of high M r glutenin subunits in wild emmer Triticum turgidum subsp. dicoccoides: chromatographic,electrophoretic separations and PCR analysis of their encoding genes

Triticum turgidum subsp. dicoccoides (Körn. ex Asch. et Graebn.) Thell. (AABB), the immediate progenitor of tetraploid and hexaploid wheats, is a species characterised by a wide range of protein polymorphism and by high protein content. Surveys on polymorphism and genetic control of the high molecular weight glutenin subunits (HMW-GS) present in this species, in two forms x- and y-type at the Glu-A1 and Glu-B1 loci, are still considered useful, both to improve technological properties of breeding varieties and to study the genome evolutionary process in wheats. Comparative Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoretic and Reversed Phase High Performance Liquid Chromatographic analyses (SDS-PAGE, RP-HPLC) of the HMW-GS present in several accessions of T. turgidum subsp. dicoccoides allowed the detection of new alleles of Glu-A1 and Glu-B1 loci, with x- and y-type glutenin subunits, apparently similar to those present in cultivated wheats in molecular weight, but different in surface hydrophobicity. In addition, changes in the number of x- and y-type subunits at the glutenin loci were also ascertained. The y-type subunits at the Glu-A1 locus, which are never expressed in cultivated bread and durum wheats, and single y-type expressed glutenin subunits at the Glu-B1 locus were also identified in several accessions. DNA extracted from samples, differing in number or type of HMW-GS and corresponding to x- and y-type genes at Glu-1 loci, were amplified using specific primers, two of which were constructed within the transposon-like sequence of Chinese Spring DNA and analysed by polymerase chain reaction. The results showed this insertion in some accessions of T. turgidum subsp. dicoccoides and also the presence of silent Ax, Bx and By type genes. The usefulness for breeding of these comparative analyses carried out on different HMW-GS alleles detected in Triticum turgidum subsp. dicoccoides, is discussed.     

Towards allelic diversity in the storage proteins of old and currently growing tetraploid and hexaploid wheats in Portugal

Using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the different alleles encoded at the 6 glutenin loci and 3 ω-gliadin loci were identified from a set of 134 hexaploid and 128 tetraploid wheat accessions mainly grown in Portugal. In the hexaploid wheats (T. aestivum L.), a total of 56, 42 and 36 patterns were observed for high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS) and ω-gliadins respectively. For HMW-GS encoded at Glu-A1, Glu-B1 and Glu-D1 loci, 4, 10 and 6 alleles were observed, respectively. LMW-GS displayed similar polymorphism, as Glu-A3, Glu-B3 and Glu-D3 loci, which comprises 5, 9 and 3 alleles. Twenty-four alleles were observed for ω-gliadins found at Gli-A1, Gli-B1 and Gli-D1 loci with, 5, 16 and 3 alleles respectively. For tetraploid collection fifty different alleles were identified for the seven loci studied Glu-A1 (3), Glu-B1 (13), Glu-A3 (6), Glu-B3 (7), Glu-B2 (2), Gli-A1 (5) and Gli-B1 (14). The genetic distances within hexaploid and tetraploid wheats were presented using cluster representation. The mean value of genetic variation indices (H) for wheat storage protein loci was slightly lower in current commercially available varieties (0.592) and highest for old varieties (0.574).     

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.     

HMW-glutenin and gliadin variations in Tibetan weedrace, Xinjiang rice wheat and Yunnan hulled wheat

Nine Tibetan weedrace, 9 Xingjiang rice wheat and 14 Yunnan hulled wheat accessions were evaluated for the variability of HMW-glutenins and gliadins. Higher variability was observed for both HMW-glutenins and gliadins in Tibetan weedrace and Xingjiang rice wheat, while lower variability was observed in Yunan hulled wheat. There were 4 HMW-glutenin and 9 gliadin patterns in 9 Tibetan weedrace accessions, 5 HMW-glutenin and 8 gliadin patterns in 9 Xingjiang rice wheat accessions, and 3 HMW-glutenin and 8 gliadin patterns in 14 Yunnan hulled wheat accessions. In Xinjiang rice wheat, one accession (i.e. Daomai 2) carried subunits 2.1 + 10.1 encoded by Glu-D1, which is very rare in common wheat.     

Morphological and isozyme variation in genebank accessionsof Aegilops umbellulata Zhuk., a wild relative of wheat

Morphological variation and genetic variation at 15 enzyme lociwere studied in genebank accessions of Aegilopsumbellulata Zhuk., the diploid genome donor to all thepolyploid species of the section Aegilops ofAegilops. Accessions from the Greek Islands hadshorter spikes with smaller number of spikelets and smaller number ofawns on the empty glume. The number of alleles per locus (A= 2.01) and the proportion of polymorphic loci (P= 0.627) were similar to those of the other species ofAegilops so far reported. Genetic distancescalculated from isozyme variations among five regions revealed thataccessions from the Greek Islands are more distantly related to theother four continental regions, Iran and Iraq, Southeast Turkey,Central Turkey and South and West Turkey. The present resultsindicated the importance of collection covering the whole range ofgeographical distribution to capture the genetic variation present inAe. umbellulata.     

High Molecular Weight (HMW) Glutenin Subunit Composition of Some Nordic and Middle European Wheats

A collection of 123 winter and 106 spring wheat (Triticum aestivum L.) cultivars and breeding lines commonly grown in Nordic and Middle European countries were characterised for the composition of high-molecular-weight (HMW) glutenin subunits on the ground of data from literature and experiments of author. HMW glutenin subunit composition was determined by one-dimensional sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). The present database includes data for cultivars and breeding lines from Finland (FIN – 56), Estonia (EST – 27), Sweden (SW – 27), Germany (D – 24), Lithuania (LIT – 17), United Kingdom (UK – 6), France (F – 3), Poland (POL – 5), Italy (IT – 7), Netherlands (NL – 6), Norway (NOR – 12), Russian Federation (RUS – 14 items). The occurrence of individual alleles and corresponding HMW glutenin subunits in surveyed cultivars is demonstrated. Special attention has been paid to cultivars growing in neighbouring countries with the aim to apply them in Estonian plant breeding. The database consists of 4 tables with data of HMW glutenin subunits, encoding them Glu-1 alleles, quality score and sources of data. The database could be useful for varietal identification and for plant breeders to improve wheat quality and accelerate the breeding process.     

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.     

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.     

Allelic Variation at Glu-D1 Locus for High Molecular Weight (HMW) Glutenin Subunits: Quantification by Multistacking SDS-PAGE of Wheat Grown Under Nitrogen Fertilization

Two biotypes of an Australian wheat cultivar, Warigal, differing only in the Glu-D1 high molecular weight (HMW) glutenin subunits 5+10 and 2+12 were used in this study. The objective was to examine the effects of nitrogen fertilization and allelic variation at the Glu-D1 locus on the characteristics of glutenin polymers. Unreduced proteins containing the SDS-soluble glutenins and the other protein classes were analyzed by multistacking SDS-PAGE which separates the glutenin into six distinctly different-sized aggregates. The results showed that nitrogen fertilization significantly increased protein quantity, ratio of polymers to monomeric proteins, and sizes of SDS-soluble glutenins. Nitrogen fertilization affected the proportions of HMW subunits in both SDS-soluble and SDS-insoluble glutenin polymers and the ratio of x to y subunits in SDS-insoluble glutenin polymers. Nitrogen fertilization, however, did not cause a significant change in ratio of SDS-soluble to SDS-insoluble glutenins. SDS-insoluble glutenins had a greater ratio of HMW to LMW and x to y subunits, especially with a higher increase of 1Dx subunits, than SDS-soluble glutenins. The HMW/LMW subunit ratio and the x/y subunit ratio may be used to predict sizes of glutenin polymers. The biotype with 5+10 subunits had a greater x/y subunit ratio in the SDS-insoluble glutenins than the 2+12 type. A greater proportion of subunit 5 was formed than subunit 2 in the SDS-insoluble glutenin polymers. Both nitrogen fertilization and allelic variation at Glu-D1 loci could affect the characteristics of glutenin polymers.     

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 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.