Summary: Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The α‐gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33‐mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33‐mer in the α‐gliadin genes. Twenty‐one mutant lines were generated, all showing strong reduction in α‐gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene‐free lines were identified, and no off‐target mutations have been detected in any of the potential targets. The low‐gluten, transgene‐free wheat lines described here could be used to produce low‐gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.

Comparative transcriptomics are useful to determine the role of orthologous genes among Triticeae species. Thus they constitute an interesting tool to improve the use of wild relatives for crop breeding. Reverse transcription quantitative real-time PCR (qPCR) is the most accurate measure of gene expression but efficient normalization is required. The choice and optimal number of reference genes must be experimentally determined and the primers optimized for cross-species amplification. Our goal was to test the utility of wheat-reference genes for qPCR normalization when species carrying the following genomes (A, B, D, R, H ^v and H ^ch ) are compared either simultaneously or in smaller subsets of samples. Wheat/barley/rye consensus primers outperformed wheat-specific ones which indicate that consensus primers should be considered for data normalization in comparative transcriptomics. All genes tested were stable but their ranking in terms of stability differed among subsets of samples. CDC (cell division control protein, AAA-superfamily of ATPases, Ta54227) and RLI (68 kDa protein HP68 similar to Arabidopsis thaliana RNase L inhibitor protein, Ta2776) were always among the three most stable genes. The optimal number of reference genes varied between 2 and 3 depending on the subset of samples and the method used (geNorm vs. coefficient of determination between sequential normalization factors). In any case a maximum number of three reference genes would provide adequate normalization independent of the subset of samples considered. This work constitutes a substantial advance towards comparative transcriptomics using qPCR since it provides useful primers/reference genes.

Summary: Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten‐free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from α‐, γ‐, ω‐gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP‐HPLC and mass spectrometry (LC‐MS/MS), whereas gluten immunogenicity was assayed by an anti‐gliadin 33‐mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only γ‐ and α‐gliadins, respectively. Four plasmid combinations were highly effective in the silencing of ω‐gliadins and γ‐gliadins, and three of these also silenced α‐gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC‐MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic α‐ and ω‐gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars.

Summary: The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non‐celiac gluten sensitivity, two pathologies affecting up to 6–8% of the human population worldwide. The wheat α‐gliadin proteins contain three major CD immunogenic peptides: p31–43, which induces the innate immune response; the 33‐mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5‐glia‐α3 epitope which partially overlaps with the 33‐mer. Next‐generation sequencing (NGS) and Sanger sequencing of α‐gliadin genes from diploid and polyploid wheat provided six types of α‐gliadins (named 1–6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31–43 peptide were found in most of the α‐gliadins. Variants of the DQ2.5‐glia‐α3 epitope were associated with specific types of α‐gliadins. Remarkably, only type 1 α‐gliadins contained 33‐mer epitopes. Moreover, the full immunodominant 33‐mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α‐gliadins found only in Aegilops tauschii, the D‐genome donor of hexaploid wheat. Type 3 α‐gliadins seem to be the ancestral type as they are found in most of the α‐gliadin‐expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome‐editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α‐gliadins containing only immunogenic peptides.