A multi-species DArTag genotyping panel for grapes: design and validation across Vitis and Muscadinia

Abstract

Grapevine breeding and germplasm curation require a scalable, cost-effective genotyping solution that performs robustly across wine, table, raisin, juice, and muscadine grapes from diverse Vitis species. We designed and validated a targeted genotyping panel covering germplasm from both Muscadinia and Euvitis subgenera using the DArTag platform (http://www.diversityarrays.com/). To ensure multi-species performance, candidate variants were filtered to exclude sites in transposable elements, within 5 bp of indels, and low-quality orlow-frequency variants, and to include variants in conserved genic regions that passed in-silico specificity checks via BLAST alignments of the 75 bp flanking sequences. The resulting 3K panel comprises 1000 high-quality rhAmpSeq core genome loci, 1000Muscadinia-polymorphic loci, 1000 Vitis vinifera-polymorphic loci, and 40 trait-related markers, which include powdery mildew resistance (RUN1, REN3/4/6/9/11/12), downy mildew resistance (RPV3 and RPV10.3), sex determination (YABBY, APT3), seedlessness (VviAGL11), and muscat aroma (VvDXS1). We evaluated its performance on a broad, taxonomically diverse sample set that included Muscadinia, Muscadinia × Vitis hybrids, V. vinifera cultivars, wild Vitis species, and Euvitis interspecific hybrids.

Across samples, the panel delivered consistent, high-quality genotypes with low marker missingness and high replicate concordance.The results file (missing allele discovery count, MADC) reports read counts of 109 bp microhaplotypes for all marker loci. Thesemicrohaplotypes combine target variants (used in assay design) with tightly linked off-target variants within the same short sequencing fragment. Using microhaplotypes and target and off-target SNPs independently, we created linkage maps for a Muscadinia × V. viniferaintrogression population. Additionally, high polymorphism in V. vinifera confirms its utility for cultivar breeding. The DArTag panel isavailable to the grape community through Diversity Arrays.

Because this panel is modular, it can be updated as new markers and targets emerge, supporting continued community adoption and interoperable, multi-species grape genotyping.