A next-generation sequencing approach for high-resolution S-locus genotyping in apricot.

Most temperate fruit crops exhibit a Gametophytic Self-Incompatibility (GSI) mechanism that prevents incompatible pollen tube growth and promotes outbreeding. In Prunus species, this system is governed by the multiallelic S-locus, which contains the S-haplotype-specific F-box (SFB) and S-RNase genes. Accurate determination of S-haplotypes is important for fruit breeding and orchard design and has traditionally relied on PCR-based analysis. However, PCR-based methods, combined with the partial sequencing of many S-alleles, may lead to ambiguous or incorrect allele identification. Next-generation sequencing (NGS) has generated numerous apricot genome datasets and revealed additional self-incompatibility alleles, yet S-locus genotypes remain unknown for many accessions. Here, we present a high-resolution NGS-based approach for S-locus genotyping based on genome filtering, mapping to a synthetic reference sequence, and automated S-allele calling. This approach is not intended to replace routine PCR-based S-genotyping, but rather to complement it in cases requiring sequence-level validation, clarification of ambiguous genotypes, or identification of previously uncharacterized alleles. Using this approach, S-haplotypes were inferred in 226 apricot cultivars, including 187 new genotype assignments, 30 confirmations of previously reported genotypes, and 9 cases that differed from previous reports. These results expanded the available information on pollination requirements to 422 apricot varieties. Sequence-based comparison of reported alleles documented 22 potential cases of synonymy and 20 cases of homonymy and supported the curation of 52 S-RNase and 28 SFB allele groups, increasing the number of reconstructed complete S-loci from 11 to 19. Furthermore, 129 cultivars were identified as carrying the S