Genetic Relatedness Is Uncoupled from Fruit Color in Sour Cherry: Evidence from SSR, S-RNase, and Expression Profiling

Sour cherry (Prunus cerasus L.) exhibits remarkable phenotypic and genetic diversity, historically classified into morello and amarelle groups based on fruit pigmentation. However, the genetic foundations of these categories remain unclear. Here, we combine 10 SSR loci with S-RNase genotyping to evaluate genetic diversity, phylogenetic relationships, and population structure across 27 Hungarian and internationally relevant sour cherry cultivars. The marker panel proved highly informative, yielding 78 SSR alleles and 17 S-alleles, with a multilocus probability of identity of 3.97 × 10−7. Phylogenetic reconstruction, minimum spanning networks, Bayesian clustering, and PCoA consistently resolved five genetically coherent groups that largely reflect known breeding histories and regional selection rather than fruit color classes. High- and low-anthocyanin cultivars frequently co-occurred within clades, demonstrating that pigmentation does not track genome-wide relatedness. To investigate proximate molecular mechanisms, we profiled flavonoid-pathway gene expression in contrasting accessions (VN-1 and ‘Pipacs 1’). VN-1 exhibited strong late-ripening induction of structural genes and MYB10, whereas ‘Pipacs 1’ showed attenuated late activation and higher early expression of ANR, LAR, and UFGT, suggesting divergent transcriptional regulation and pathway flux between the two genotypes. Together, these results indicate that fruit color variation is largely independent of the multilocus relatedness patterns captured by our marker set, and is likely influenced by lineage-specific regulatory differences.