A comprehensive next-generation sequencing approach for S-locus genotyping in apricot. Implications for the unification of the S-haplotype nomenclature in Prunus
Most temperate fruit crops exhibit a Gametophytic Self-Incompatibility (GSI) mechanism to prevent incompatible pollen tube growth, promoting outbreeding. 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 crucial for fruit breeders and producers, and often relies on PCR analysis targeting intronic regions of polymorphic length. In apricot (Prunus armeniaca L.), 32 S-RNase and 33 SFB alleles have been identified, but limitations of PCR-based methods, combined with the partial sequencing of most S-alleles, have resulted in many misidentifications. Next generation sequencing (NGS) has provided a large number of genomes, including hundreds of resequenced apricot accessions with numerous unpublished self-incompatibility alleles available at NCBI. However, the S-locus genotypes of most of these accessions remain unknown. In this study, we propose a more accurate and high-throughput methodology for S-locus genotyping than current PCR-based techniques. This approach involves genome filtering of S-locus reads, mapping to a synthetic reference sequence, and an automated and powerful S-allele calling procedure. Using this method S-alleles were successfully identified in 226 apricot cultivars, 187 of them for the first time, confirming 30 and revealing misidentifications in 9. In addition, it resolved 22 synonyms and 20 homonyms of S-locus and identified a total of 51 S-RNase alleles and 28 SFB alleles, thereby expanding the count of complete S-loci from 11 to 20. We recommend introducing a new nomenclature to prevent future misidentifications among apricot cultivars which could be extended to other Prunus species.