First, sequences were sorted by decreasing abundance since the most abundant sequence is likely to be a correct sequence, while less common sequences may include artefacts due to sequencing errors or PCR artefacts. 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 unique deduced protein fragments of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. == Conclusions == The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains. == Background == Wheat-containing products are worldwide an important part of the human daily menu. Hexaploid bread wheat (T. aestivumL., ABD genomes) and tetraploid durum wheat (T. turgidumL., AB genomes) are the most common wheat species grown for food production. The differences in food-technological qualities between both wheat species are largely determined by the composition of the gluten fraction in the grains. Gluten, Rabbit polyclonal to ZBED5 the water-insoluble fraction of wheat seed-storage proteins, consists of the high- and low molecular weight subunit glutenins (HMW-GS and LMW-GS) and the monomeric gliadins (/-, – and -gliadins) [1]. Gluten proteins are relatively resistant to proteolysis. Several specific bioactive gluten peptides have been identified Valemetostat tosylate that survive proteolysis in the human intestine and that can stimulate T cells [2-5] and trigger celiac disease (CD) in genetically susceptible individuals. CD is a T cell mediated chronic inflammatory condition of the small intestine [5,6] with prevalence between 0.5 and 2% in human populations [7,8]. The immunogenic peptide sequences have highly specific cores of at least nine amino acids length [5,9], and become active after deamidation by the enzyme tissue transglutaminase in the intestine [10]. There are natural epitope variants that lack immunogenicity due to single or multiple amino acid substitutions. For instance, a P to S substitution at the epitope core position 8 was shown to be sufficient to abolish T cell stimulation [11]. T cell clones isolated from intestinal celiac lesions showed differential responses to diploidAegilopsandTriticumspecies that are related to the ancestors of the A, B, and D genomes [12-14]. These differences in T cell responses between diploidAegilopsandTriticumspecies especially related to the presence of three CD epitopes derived from alpha-gliadins, DQ2.5-Glia-1 (PFPQPELPY), DQ2.5-Glia-2 (PQPELPYPQ) and DQ2.5-Glia-3 (FRPEQPYPQ); in these epitopes glutamic acid (E) is originating from deamidated glutamine (Q) [5,9,15,16]. The source of these epitopes, the alpha-gliadins, are encoded by a multigene family located on three homoeologous loci,Gli-A2,Gli-B2andGli-D2on the short arms of wheat group 6 chromosomes (6AS, 6BS and 6DS). Estimates of the copy number of alpha-gliadins range from Valemetostat tosylate 25 copies to Valemetostat tosylate even 150 copies per haploid genome, reflecting the large complexity of this gene family [17-19]. The large majority (up to 87% in hexaploid wheat) of the genes contain internal stop codons and are presumably pseudogenes [20,21]. Limiting the abundance of CD epitopes in food products may reduce the risk of sensitization of the immune system of the group of people that are genetically susceptible for CD. In order to breed and select for wheat varieties with significantly reduced immunogenic potential to cause CD it is necessary to accurately estimate the quantity and quality of the CD epitope load in gluten. Up to now, the ability for high throughput quantification of CD epitopes by presently available assays based on T cell clones and on monoclonal antibodies is very limited, mainly because of the high complexity of the wheat material on the one hand, and the laboriousness ofin vitroT cell assays and the promiscuity of the monoclonal antibodies on the other hand [22,23]. In addition, most commercial kits with monoclonal antibodies detect gluten, not CD epitopes. Next-generation sequencing platforms offer now the possibility of efficient and accurate deep sequencing of genetic variation at moderate costs [24,25]. Still, the application of such technologies in bread wheat is a big challenge due to the large genome (17 Gbp, five times the size of the human genome), the allohexaploid nature and the abundance of repetitive sequences [26]. To reduce difficulties with the alignment of sequences for the detection of single nucleotide polymorphisms (SNPs),.