Genomic architecture of the self-incompatibility locus in apple provides insights into the evolution of collaborative non-self recognition

Self-incompatibility (SI) systems prevent self-fertilization, thereby maintaining genetic diversity in flowering plants. Among them, collaborative non-self recognition (CNSR) is the most widespread, yet its genomic architecture remains poorly understood. Using 27 haplotype-resolved genomes from wild and domesticated apples (Malus spp.), we dissected the structure and evolution of the S-locus. We identified 17 S-RNase alleles and 500 pollen-expressed S-locus F-box brother (SFBB) genes across 18 families. The S-locus exhibits hallmark features of a supergene, including suppressed recombination, TE accumulation, and an excess of non-synonymous polymorphisms. Nonetheless, long-term balancing selection preserves both the integrity and diversity of entire S-haplotypes. Recurrent associations between SFBB families and transposable elements, coupled with gene conversion signatures, suggest a dynamic mechanism for reshuffling recognition specificities. Our results provide a detailed view of how a multigenic, tightly linked recognition system is maintained in a woody perennial, and highlight the interplay between structural constraints and selection in sustaining functional diversity and reproductive barriers.