SNP-Discovery by RAD-Sequencing in a Germplasm Collection of Wild and Cultivated Grapevines ( V-vinifera L.)

Whole-genome comparisons of Vitis vinifera subsp. sativa and V. vinifera subsp. sylvestris are expected to provide a better estimate of the valuable genetic diversity still present in grapevine, and help to reconstruct the evolutionary history of a major crop worldwide. To this aim, the increase of molecular marker density across the grapevine genome is fundamental. Here we describe the SNP discovery in a grapevine germplasm collection of 51 cultivars and 44 wild accessions through a novel protocol of restriction-site associated DNA (RAD) sequencing. By resequencing 1.1% of the grapevine genome at a high coverage, we recovered 34K BamHI unique restriction sites, of which 6.8% were absent in the 'PN40024' reference genome. Moreover, we identified 37,748 single nucleotide polymorphisms (SNPs), 93% of which belonged to the 19 assembled chromosomes with an average of 1.8K SNPs per chromosome. Nearly half of the SNPs fell in genic regions mostly assigned to the functional categories of metabolism and regulation, whereas some nonsynonymous variants were identified in genes related with the detection and response to environmental stimuli. SNP validation was carried-out, showing the ability of RAD-seq to accurately determine genotypes in a highly heterozygous species. To test the usefulness of our SNP panel, the main diversity statistics were evaluated, highlighting how the wild grapevine retained less genetic variability than the cultivated form. Furthermore, the analysis of Linkage Disequilibrium (LD) in the two subspecies separately revealed how the LD decays faster within the domesticated grapevine compared to its wild relative. Being the first application of RAD-seq in a diverse grapevine germplasm collection, our approach holds great promise for exploiting the genetic resources available in one of the most economically important fruit crops.