A TBP-BASED APPROACH FOR SIMPLE GENETIC PROFILING AND FAST SNPS IDENTIFICATION IN WHEAT GENOTYPES

The issue of food authenti city and traceability is of increasing importance also in the wheat sector, where ancient genotypes (spelta, emmer wheat) or parti cular culti vars (i.e. kamut) are regarded as healthier alternati ves to bread wheat and have reached high commercial value. TBP (B-tubulin based polymorphisms) is a PCR-based, multi locus, co-dominant marker useful for fast fi ngerprinti ng of any higher plant species. Based on intron length polymorphism of members of the B-tubulin gene family, it is able to provide a unique, specie-specifi c geneti c profi le (barcode) using two universal primer pairs. This approach has been successfully applied to the authenti cati on of the compositi on of mixed feed and food matrices. We applied TBP analysis, followed by amplicon separati on by Capillary Electrophoresis, to commercially relevant wheat genotypes (emmer, spelta, einkorn) and to 32 diff erent durum wheat accessions. Specifi c profi les were clearly obtained for the diff erent species, while few intron length polymorphisms were present at the variety level. Hence, we wondered if the limited but highly variable intronic region amplifi ed by TBP primers (5- 10 kbp/haploid genome) was suitable to uncover enough allelic sequence polymorphisms at the variety level. We followed a NGS approach on the Miseq platf orm to sequence TBP amplicons of 47 wheat samples (4 exaploid, 32 tetraploid and 11 diploid wheat relati ves or ancestors) in order to provide an initi al TBP sequence database and to identi fy SNPs among genotypes. Sequence data were processed as a de novo amplicon assembly and all the conti gs from T. aesti vum of the culti var Bilancia were manually refi ned and used as reference for sequence re-assembly. The limited region analyzed makes the bioinformati cs analysis extremely handy suggesti ng the possibility of an applicati on of this technique as a very simple tool to evaluate geneti c diversity in wheat. Applicati on to phylogeneti c analysis is also under evaluation.