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Durum wheat (T. durum Desf., AABB, 2n = 2x = 28 ) is an economically important and traditional crop for Bulgaria, due to its unique qualities and final products. The storage endosperm proteins – glutenins and gliadins, which form gluten, have significant effect on the qualitative parameters of T. durum. In this relation, by using the method of electrophoresis, the allele composition of the γ-gliadins, the high molecular (HMWGS) and low molecular (LMW-GS) glutenins was studied in durum wheat cultivars grown in Bulgaria – Malena, Mirabel, Severina, Mirela and Melina. The obtained results show that three alleles of HMW-GS, coded for by loci Glu-A1 and Glu-B1 and seven alleles of LMW-GS, coded for by loci Glu-A3, Glu-B3 and Glu- B2 were identified. The genotypes possessing gliadin subunits γ-42 (Malena) and γ-45 (Severina, Mirela and Melina) in locus Gli-B1, which are markers for poor and high gluten quality, respectively, were determined. Cultivar Mirabel was characterized by subunit γ-47 (locus Gli- B1). The influence of the subunit γ-47 on gluten strength has not been investigated yet. According to their respective allelic forms in loci GliB1 / GluB3, the studied varieties were divided into two main types (LMW-1 and LMW-2), on which the quality of the ground semolina from durum wheat depended. The quality scores and the genetic variability were calculated for the separate loci.. The obtained results showed that cultivars with LMW-2 type of the low-molecular weight glutenins were predominant in our study, which was due to the higher frequency of gliadin subunit γ-45. [ABSTRACT FROM AUTHOR]

The previously defined pairs of primers GliB1.1 and GliB1.2 were found to produce three and four principal variants, respectively, of PCR sequence length for the γ-gliadin pseudogene in 46 Triticum aestivum L. cultivars from 15 countries carrying 19 known alleles at the Gli-B1 locus. A congruity was established between this polymorphism, allelic sets of the Gli-B1-produced gliadins (especially of the electrophoretic mobility in acid gels of the encoded γ-gliadin) and the presence in the wheat genotype of the Gli-B5b + Rg-1 allelic combination. Six different alleles at the Gli-B1 locus encoding an identical γ-gliadin produced a PCR sequence of about 400 bp (GliB1.1). Nine Gli-B1d-carrying genotypes from four countries produced an identical sequence of about 409 bp (GliB1.2), while three cultivars with Gli-B1h and four with Gli-B1b produced three and two specific sequences, respectively, of slightly different length. Allele Gli-B1j might be the result of recombination between coding and noncoding DNA sequences within the Gli-B1 locus. These observations imply that genetic diversity of the agriculturally important region of chromosome 1B marked by variants of the Gli-B1 locus is rather limited among common wheat cultivars of the 20th century, specifically to eight principal versions. These might have been incorporated into common wheat from diverged genotypes of diploid donor(s), and, due to the scarcity of recombination, subsequently maintained relatively intact. As well as its evolutionary significance, this information is of potential use in wheat breeding and we consider it likely that novel variants of the Gli-B1 locus will be found in hitherto unstudied germplasm.

Gluten is an important protein source for human beings and a wide diversity of foods has been developed to take advantage of this protein in wheat flour. However, some individuals, suffering from Celiac Disease (CD), cannot tolerate proline and glutamine-rich gluten peptides including γ-gliadins. A life-long gluten free diet is the only known effective treatment for such patients. However, the sensitivity intension in CD patients can be controlled by RNA interference (RNAi) technology. The main aim of the present study is to develop an efficient and specific siRNA-inducing cassette, as a first and critical step for an effective targeting of mRNAs of wheat γ-gliadins. To achieve this aim, we have followed the strategy based on 200 bp in sense and antisense orientation with the ~160bp sequence of none potent siRNA-containing region from γ- gliadin gene as a spacer in between. The endosperm-specific γ-gliadin promoter was sub-cloned into upstream the cassette. The nucleotide alignment results validated the sequence data of γ-gliadin promoter and the direct inserts with high homology identities of 99% and 99.97%, respectively. Here, six potential and consecutively arranged-siRNA sites were predicted using computational approaches. All of these sites covered the inverted repeats region with high efficacy and performance values for triggering RNAi. [ABSTRACT FROM AUTHOR]

Background: Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains. Results: A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γgliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone. Conclusions: Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs). [ABSTRACT FROM AUTHOR]

Abstract: Nine new full-length cDNA sequences for γ-gliadins were generated from total mRNA from bread and durum wheat. The length of the open reading frames (ORFs) ranged from 720 to 936bp, which corresponds to putative proteins of 239–311 amino acid residues, respectively. All nine sequences, reconstructed from phylogenetic data showed sequences similar to the γ-gliadins registered at GenBank. All known γ-gliadin sequences fall into four major groups in the tree constructed by the neighbour-joining method. Primer pairs were designed to discriminate between these four γ-gliadin groups during grain development in bread wheat using quantitative real time polymerase chain reaction (QRT-PCR). Expression profiles of two lines of bread wheat showed higher levels of gene transcription for γ-gliadins from group II, whereas γ-gliadins from group I were expressed at the lowest level. Thus, the γ-gliadins from group II are expressed four- and 10-fold higher in ‘Perico’ and ‘Anza’, respectively, than the other γ-gliadins. The results reported may help to understand the order of synthesis and deposition of γ-gliadins in the grain of developing bread wheat. In addition, the differences found in the expression levels among the four groups could be used to select genotypes with specific profiles of γ-gliadins. These findings will also facilitate the up- and down-regulation of γ-gliadin gene expression in transgenic wheat. [Copyright &y& Elsevier]

Polymerase chain reaction (PCR) primers GAG5 and GAG6 were designed based on published γ-gliadin gene sequences and applied to 35 cultivars of closely related spelt (Triticum spelta L.) and hexaploid wheat (T. aestivum L.). Eight tetraploid durum wheat (T. durum Desf.) cultivars were included in the analysis. The obtained PCR products originated from two γ-gliadin genes which were mapped to homeologous chromosomes 1B and 1D and termed GAG56B and GAG56D, respectively. Two alleles of GAG56D differing in a 9-bp deletion/duplication and single nucleotide polymorphism were found. The 18 spelts tested and wheat cultivar ’Chinese Spring’ were discovered to carry a previously unknown γ-gliadin gene, while 16 wheat cultivars possessed its longer, already published allele. Two PCR-based detection systems for the diagnostic alleles were developed and applied. The occurrence of two alleles of GAG56B among the investigated durum wheats correlated with their expression of gluten quality markers γ-gliadins 42 or 45. [ABSTRACT FROM AUTHOR]

Abstract Background Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the α-gliadin fraction of gluten, γ-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic γ-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in γ-gliadin transcripts of developing bread wheat grains. Results A detailed analysis of the genetic variation present in γ-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic γ-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic γ-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the γ-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from γ-gliadins, DQ2-γ-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-γ-I specific and HLA-DQ2 restricted T-cell clone. Conclusions Evaluating the CD epitopes derived from γ-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).

The previously defined pairs of primers GliB1.1 and GliB1.2 were found to produce three and four principal variants, respectively, of PCR sequence length for the γ-gliadin pseudogene in 46 Triticum aestivum L. cultivars from 15 countries carrying 19 known alleles at the Gli-B1 locus. A congruity was established between this polymorphism, allelic sets of the Gli-B1-produced gliadins (especially of the electrophoretic mobility in acid gels of the encoded γ-gliadin) and the presence in the wheat genotype of the Gli-B5b + Rg-1 allelic combination. Six different alleles at the Gli-B1 locus encoding an identical γ-gliadin produced a PCR sequence of about 400 bp (GliB1.1). Nine Gli-B1d-carrying genotypes from four countries produced an identical sequence of about 409 bp (GliB1.2), while three cultivars with Gli-B1h and four with Gli-B1b produced three and two specific sequences, respectively, of slightly different length. Allele Gli-B1j might be the result of recombination between coding and noncoding DNA sequences within the Gli-B1 locus. These observations imply that genetic diversity of the agriculturally important region of chromosome 1B marked by variants of the Gli-B1 locus is rather limited among common wheat cultivars of the 20th century, specifically to eight principal versions. These might have been incorporated into common wheat from diverged genotypes of diploid donor(s), and, due to the scarcity of recombination, subsequently maintained relatively intact. As well as its evolutionary significance, this information is of potential use in wheat breeding and we consider it likely that novel variants of the Gli-B1 locus will be found in hitherto unstudied germplasm. [ABSTRACT FROM AUTHOR]