Pan-S-locus analysis reveals insights into the origin and evolution of self-incompatibility in the orange subfamily

BACKGROUND: Self-incompatibility is controlled by a highly polymorphic supergene complex, the S-locus, which is structurally complex, rich in repetitive sequences, and varies in length from hundreds of kilobases to tens of megabases across different plant families. Due to these challenges, the S-locus has been fully reconstructed in only a few species, limiting our understanding of its evolutionary dynamics. RESULTS: This study systematically explores the evolutionary mechanisms and structural characteristics of the self-incompatibility system mediated by S-RNase in the Aurantioideae (orange subfamily) of Rutaceae. We construct a pan-S-locus framework spanning 11 genera within the orange subfamily from 14 newly assembled genomes from representative accessions and the analysis of 41 published citrus genomes. Our analyses reveal significant structural variations and transposable element (TE)-driven pseudogenization of SLF genes. By constructing a comprehensive library of S-RNases and developing a novel genotyping pipeline, we reveal divergent frequencies of S-RNases between self-incompatible and self-compatible populations. Comparative analyses demonstrate a unique evolutionary trajectory marked by asynchronous core nonS-genes duplication and TE-mediated structural diversification, distinct from other families. Innovatively, we identified TE-induced self-incompatibility loss as the primary driver of self-compatibility transition, which implies the uniqueness of the origin and evolution of self-incompatibility in the orange subfamily. CONCLUSIONS: Through the construction and characterization of the pan-S-locus, this study provides profound insight into the origin and evolution of the S-locus in the orange subfamily. These findings not only advance our understanding of self-incompatibility mechanisms, but also establish a foundational framework for investigating the evolution of the gametophytic self-incompatibility systems in other families.