Genotyping of Italian regional grapevine accessions by SNPs analysis

The evaluation and characterization of genetic variability in grape germplasm is a key step for cvs. identification, parentage determination, and population structure evaluation for further genome wide association mapping studies. Robust genotyping requires a large number of polymorphic markers and the availability of databases allowing for comparisons with previously characterized genotypes. Up to now, simple sequence repeats (SSR) have been very useful not only for genetic identification but also for parentage analysis. However sequencing projects have generated such a quantity of sequence information to make possible the development of SNPs, bi-allelic markers, very frequent in the genomes, and with almost unlimited availability. Therefore, after the establishment of an international Consortium, a 20K SNP Infinium array has been developed, starting from genomic sequences of 63 accessions of Vitis vinifera, 4 of V. sylvestris, 3 of V. labrusca, 3 of V. cinerea, 3 of V. berlandieri, 3 of V. aestivalis. A panel of SNP controls has been introduced in the array, going into details, 5488 SNPs segregating in V. vinifera, 1300 segregating in Vitis genus, 1200 species-specific, and 735 located in candidate genes have been selected. The results obtaneid from the joining of the genotyping data developed inside two Italian projects (Ager project and Vigneto project) are presented, with the final aim to build a new genetic tool based on a common panel of SNP markers, a large genotypes database of Italian accessions, and a bioinformatics pipeline for robust data analysis (Tumino et al. this conference). About 850 mainly Italian accessions were analyzed, representing more than 500 unique genotypes (including sativa and sylvestris genotypes, and also some no vinifera and no Vitis genotypes and clones of some of the most important varieties in Italy. The AGER group analyzed genotypes (415 accessions) cultivated in Piemonte, Emilia-Romagna, Toscana, Lazio, Basilicata, Puglia, Calabria and Sicilia regions, as well as non vinifera and non Vitis genotypes; the VIGNETO group analyzed vinifera genotypes (443) grown in the Grapevine National Collection in Conegliano (TV). DNA samples were extracted and prepared independently in 6 laboratories (some using a CTAB protocols and further column purification and some just column kits) and Infinium chip hybridization; genotype calling was performed by two providers using Illumina standard protocols. The SNPs data were subjected to identity and parentage analysis by PLINK software, while the genetic distance and UPGMA tree were elaborated with PEAS and MEGA 5 softwares. Finally, population’s analysis was performed with STRUCTURE software. The percentage of failed markers ranged from 3.46% to around 80%, mainly depending on taxonomic groups: Vitaceae accessions not belonging to the Vitis genus showed the highest missingness. At a lower extent, the failure can depend on the DNA quality related to the different DNA extraction protocols and laboratories: in particular, the percentage of failed markers for sativa genotypes only ranged from 4.50% to around 15% for the different laboratories (mean of all sativa samples of each laboratory). Some of the possible relationships among genotypes and regions will be discussed. This genotyping work can have significant implications for grape genetics, association mapping and breeding in Italian wine production chains. In the future a smaller panel of highly informative SNPs will be selected in order to develop a diagnostic tool for varietal identification.