Identification of Sex Determination Locus and Development of Marker Combination in <i>Vitis</i> Based on Genotyping by Target Sequencing

The grapevine is an important and economically valuable fruit crop, with flower sex being a key genetic trait that directly affects grapevine yield and quality. Despite its significance, there is a lack of studies on sex-linked molecular markers that can assist in grapevine breeding. In this study, we developed a grapevine single nucleotide polymorphism (SNP) marker array using a combination of genotyping by target sequencing (GBTS) and capture-in-solution technology, and applied it to marker-assisted selection (MAS) of grapevine gender. The SNP array could detect a total of 20,597 core SNPs and 97,453 multiple SNPs (mSNPs), covering over 99% of the grapevine genome on each chromosome. A total of 131 progenies from a cross between Vitis vinifera 'Cabernet Sauvignon' and Vitis pseudoreticulata 'Huadong1058' that exhibited segregated sex phenotypes were sequenced using this array. Through locus mapping and a genome-wide association study (GWAS), a locus on chromosome 2 (54.74-58.80 cM) that explained 98.6% of the phenotypic variation was identified. To the further utilize this locus, a sex prediction marker combination consisting of two SNPs was developed, which accurately predicted the sex of 34 natural grapevine varieties/accessions. This study demonstrates the application of GBTS in grapevine breeding and provides a reliable MAS marker set for early-stage sex selectio