Molecular characterization of S-RNase-based self-incompatibility in Aquilaria sinensis and implications for germplasm improvement

Self-incompatibility (SI) is an important reproductive strategy in flowering plants that prevents self-fertilization and promotes genetic diversity. Aquilaria sinensis ,is an endangered tree species of high economic value, producing agarwood that is widely used in medicine, fragrances, and religious practices. In this study, we demonstrate that A. sinensis exhibits a gametophytic self-incompatibility (GSI) system, as self-pollen tubes fail to enter the ovary and arrest at the base of the style by 72 h after pollination. We cloned and characterized the S-locus gene AsS-RNase , which encodes a 229-amino acid protein with a typical T2 RNase domain. AsS-RNase showed strong style-specific expression and was significantly upregulated upon self-pollination. Functional assays confirmed its ability to inhibit pollen germination and pollen tube growth, and subcellular localization assays indicated its presence on the plasma membrane and around the nucleus, suggesting roles in recognition and signalling. We identified 19 allelic variants of AsS-RNase, with hypervariable regions typical of S-RNases. Cross-pollination experiments revealed that accessions sharing the same S-haplotype were incompatible, whereas those with different haplotypes had higher fruit set rates. These findings provide the first molecular evidence for an S-RNase-based GSI system in A. sinensis , offering insights into SI mechanisms in Aquilaria and providing a foundation for future breeding and conservation strategies. • Identified S-RNase as the female determinant of GSI in Aquilaria sinensis for the first time. • Functional assays confirmed AsS-RNase inhibits pollen tube growth in a dosage-dependent way. • Nineteen AsS-RNase alleles were cloned and characterized from diverse germplasms. • AsS-RNase alleles serve as effective molecular markers for breeding and compatibility selection.