Characterization of the variability and the expression of α-gliadin genes from spelt [Triticum aestivum ssp. spelta (L.) Thell.] in the prospect of addressing the celiac disease issue

Gluten proteins are found in the seeds of cereals like bread wheat (Triticum aestivum L. ssp. aestivum) and spelt [Triticum aestivum ssp. spelta (L.) Thell.], and they play a major role in food processing thanks to the viscoelasticity properties they confer to the dough. They are, however, the source of gluten-related disorders when they are consumed by susceptible individuals. Among them, celiac disease (CD) has the highest clinical relevance and its pathophysiology is well understood, which led to focus this research on this disease only. CD is a pathology affecting genetically predisposed subjects where dietary gluten proteins trigger an inflammatory reaction in the upper small intestine. Among these proteins, the α-gliadins constitute the most immunogenic protein fraction as they display the highest number of peptides (epitopes) recognized by the immune system of CD patients. One of the strategies currently investigated to face the CD issue is to make use of the high genetic variability found in bread wheat and related species and sub-species with the aim of obtaining varieties consumable by CD patients. Among them, spelt belongs to the same species than bread wheat but it has been less studied. It displays interesting nutritional and agronomical properties and spelt germplasm collections hold a high genetic diversity. The objective of this research was thus to evaluate the potential of spelt to respond to the problematic of celiac disease by studying its genetic diversity in CD-related expressed epitope sequences. A cloning and sequencing strategy enabled to study the epitope composition of expressed α-gliadin sequences from a few contrasted spelt accessions selected through a genetic diversity analysis. This revealed a high variability in the epitope amino acid composition and it was linked to the genome from which they were expressed (A, B or D). No clear separation between spelt and bread wheat sequences was observed but different frequencies of some amino acid substitutions were still pointed out. TaqMan probes able to discriminate immunogenic from non-immunogenic epitope variants have then been developed and their specificity has been demonstrated. These probes were subsequently applied to a wide set of spelt accessions in order to study the variability in their epitope expression levels as a function of both genetic and environmental factors. Results showed a very high variability in the α-gliadin epitope composition and expression. Significant differences in the epitope expression were highlighted according to the accession provenance and habit (winter vs spring accessions), whereas spelt breeding did not seem to have influenced this expression. In addition, environmental factors, studied by harvesting samples during four consecutive years and by applying different N fertilization strategies, did not have a major impact on the epitope expression. In conclusion, this thesis provides important information about the expression of CD-related α-gliadin epitopes in spelt and about its potential to develop varieties consumable by CD patients. Perspectives about the different breeding strategies available to obtain such celiac-safe variety are developed.