Self-incompatibility (SI) is a pollen-stigma recognition system controlled by a single and highly polymorphic genetic locus known as the S-locus. The S-locus exists in all Brassica napus (B. napus, AACC), but natural B. napus accessions are self-compatible. About 100 and 50 S haplotypes exist in Brassica rapa (AA) and Brassica oleracea (CC), respectively. However, S haplotypes have not been detected in B. napus populations. In this study, we detected the S haplotype distribution in B. napus and ascertained the function of a common S haplotype BnS-6 through genetic transformation. BnS-1/BnS-6 and BnS-7/BnS-6 were the main S haplotypes in 523 B. napus cultivars and inbred lines. The expression of SRK in different S haplotypes was normal (the expression of SCR in the A subgenome affected the SI phenotype) while the expression of BnSCR-6 in the C subgenome had no correlation with the SI phenotype in B. napus. The BnSCR-6 protein in BnSCR-6 overexpressed lines was functional, but the self-compatibility of overexpressed lines did not change. The low expression of BnSCR-6 could be a reason for the inactivation of BnS-6 in the SI response of B. napus. This study lays a foundation for research on the self-compatibility mechanism and the SI-related breeding in B. napus.

Most flowering plants have evolved a self-incompatibility (SI) system to maintain genetic diversity by preventing self-pollination. The Brassica species possesses sporophytic self-incompatibility (SSI), which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK. However, the mysterious molecular mechanism of SI remains largely unknown. Here, a new class II S haplotype, named BrS-325, was identified in a pak choi line ‘325’, which was responsible for the completely self-compatible phenotype. To obtain the entire S locus sequences, a complete pak choi genome was gained through Nanopore sequencing and de novo assembly, which provided a good reference genome for breeding and molecular research in B. rapa. S locus comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs (BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II S haplotypes showed that a number of amino acid variations were present in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the binding and interaction between the ligand and the receptor. Thus, these results suggested that amino acid variations in contact sites may lead to the SI destruction of a new class II S haplotype BrS-325 in B. rapa. The complete SC phenotype of ‘325’ showed the potential for practical breeding application value in B. rapa.