Minor varieties represent a significant part of the grapevine germplasm (Vitis vinifera L. subsp. vinifera) in Croatia. During the long history of grape cultivation in insular, coastal and continental Croatia, numerous local varieties were cultivated, many of which are still used today. There are also several populations of wild grapevine (Vitis vinifera subsp. sylvestris Hegi Gmel) that have survived until the present in natural sites. Here, we developed a single primer enrichment technology (SPET) panel consisting of 61,308 probes for targeted sequencing of the V. vinifera gene space. In doing so, we examined a total of 28,092 gene models, 88.2% of all predicted genes, interrogating an average of 19 Mb nucleotides per individual genome, which corresponds to 4% of the haploid genome length. This is the first time that SPET-based sequencing has been applied to Croatian grapevine germplasm to generate multilocus genotype data of 126 cultivated accessions and 50 wild specimens. We identified 531,900 variants sites, 208,802 of which reside in coding sequences, 140,836 in introns, 118,416 in UTRs, and 63,424 in the nearby intergenic space that revealed the presence of 33 clonally propagated specimens representing both synonymies and clones within local varieties (22) as well as synonymies between local and international varieties (11), mostly from neighbouring countries. To investigate possible contribution of wild grapevine (Vitis vinifera subsp. sylvestris Hegi Gmel) to the development of present cultivars, four wild grapevine populations were included. None of the cultivated varieties showed close kinship with local wild specimens. This study opens new possibilities for studying the genetic diversity of Croatian grapevine germplasm and provides additional information with respect to SSR genotyping and phenotyping
