High-Resolution Chloroplast SNV Profiling of 409 Grapevine (Vitis vinifera L.) Cultivars Using Whole-Genome Shotgun Sequencing

The grapevine (<i>Vitis vinifera</i> L.) is one of the most important horticultural crops, with thousands of varieties cultivated worldwide. In this study, we analyzed chloroplast SNV markers using a whole-genome shotgun sequencing approach to investigate the genetic diversity and phylogeny of 409 cultivated <i>V. vinifera</i> accessions originating from nine countries across Southeast and Central Europe, as well as a heterogeneous set of additional accessions maintained by INRAE. Shotgun sequencing allowed high coverage, enabling the detection of 93 SNVs across 24 chloroplast genes, including 11 non-synonymous variants. The <i>ycf1</i> gene showed the highest variability, consistent with its role in species differentiation. Haplotype analysis revealed 102 distinct haplotypes, with clear geographic structuring: ATT predominated in the eastern Mediterranean, ATA in western Europe, and GTA mainly in a heterogeneous group of varieties from a French collection. To validate the shotgun approach, seven SNV markers were analyzed using target capture sequencing, confirming the accuracy of detected variants with only minimal discrepancies, which is mostly attributable to homopolymeric regions and low-frequency alleles. Phylogenetic analyses using both trees and networks delineated three major haplotype clusters, reflecting human-mediated dispersal of grapevine cultivars through historical viticultural practices. This study represents the largest chloroplast genome analysis of cultivated <i>V. vinifera</i> to date, providing a large cpDNA resource for assessing chloroplast diversity and maternal haplotype structure in cultivated grapevine. The results highlight the power of combining high-throughput sequencing and chloroplast genomics for population-level studies in perennial crops.