Diagnostic KASP Markers for Flower Sex and Stenospermocarpic Seedlessness in Diverse Vitis, Muscadinia, and Wide Hybrid Populations

The genus Vitis is composed of two subgenera, Vitis (2n = 38) and Muscadinia (2n = 40), which are both cultivated for fresh-market, juice, and wine industries. Stenospermocarpic seedless and perfect-flowered vines are highly desired in both Vitis vinifera and muscadine ( Muscadinia rotundifolia ) breeding programs. Stenospermocarpy has recently been introgressed from V. vinifera to muscadines through conventional breeding despite their differing chromosome number, but no molecular markers for this trait have been validated in muscadine germplasm. Wild vines in both subgenera are dioecious, but perfect-flowered forms were independently selected during domestication to enhance reproductive efficiency and fruit production. This study reports the development and validation of two Kompetitive allele-specific PCR (KASP) markers targeting causal polymorphisms within candidate genes for male sterility ( VviINP1 ) and stenospermocarpy ( VviAGL11 ). Sequence alignments with published M. rotundifolia genomes suggested that the seedless_Arg197Leu_site56 and female_INP_indel_site56 KASP markers might be broadly effective across diverse species within Vitis and Muscadinia . Marker performance was evaluated using a validation panel including 918 Vitis × Muscadinia hybrid seedlings from the University of Arkansas Fruit Breeding Program and a diverse set of cultivated and wild accessions (209 accessions evaluated with the seedless marker and 315 accessions with the flower sex marker). After excluding incomplete phenotype and genotype data, the stenospermocarpic marker (seedless_Arg197Leu_site56) accurately predicted seedlessness in 921 of 924 (99.7%) entries. Additionally, 148 of 203 seedlings that failed to produce fruit across both growing seasons were predicted to be stenospermocarpic with the seedless KASP marker, suggesting that seedless Vitis × Muscadinia hybrids may have partial sterility or lack cold hardiness. The flower sex marker (female_INP_indel_site56) correctly predicted flower sex in all 1138 (100%) entries. Together, these KASP markers provide highly accurate and cost-effective tools for early selection of seedless and perfect-flowered genotypes across Vitis , Muscadinia , and hybrid breeding programs.