Although recent findings suggest that the F-box genes SFB/SLF control pollen-part S specificity in the S-RNase-based gametophytic self-incompatibility (GSI) system, how these genes operate in the system is unknown, and functional variation of pollen S genes in different species has been reported. Here, we analyzed the S locus of two species of Maloideae: apple (Malus domestica) and Japanese pear (Pyrus pyrifolia). The sequencing of a 317-kb region of the apple S⁹ haplotype revealed two similar F-box genes. Homologous sequences were isolated from different haplotypes of apple and Japanese pear, and they were found to be polymorphic genes derived from the S locus. Since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes are specifically expressed in pollen, and variable regions of the SFBB genes are under positive selection. In a style-specific mutant S haplotype of Japanese pear, the SFBB genes are retained. Apart from their multiplicity, SFBB genes meet the expected characteristics of pollen S. The unique multiplicity of SFBB genes as the pollen S candidate is discussed in the context of mechanistic variation in the S-RNase-based GSI system.

Summary: The S‐RNase‐based gametophytic self‐incompatibility (SI) of Rosaceae, Solanaceae, and Plantaginaceae is controlled by at least two tightly linked genes located at the complex S locus; the highly polymorphic S‐RNase for pistil specificity and the F‐box gene (SFB/SLF) for pollen. Self‐incompatibility in Prunus (Rosaceae) is considered to represent a ‘self recognition by a single factor’ system, because loss‐of‐function of SFB is associated with self‐compatibility, and allelic divergence of SFB is high and comparable to that of S‐RNase. In contrast, Petunia (Solanaceae) exhibits ‘non‐self recognition by multiple factors’. However, the distribution of ‘self recognition’ and ‘non‐self recognition’ SI systems in different taxa is not clear. In addition, in ‘non‐self recognition’ systems, a loss‐of‐function phenotype of pollen S is unknown. Here we analyze the divergence of SFBB genes, the multiple pollen S candidates, of a rosaceous plant Japanese pear (Pyrus pyrifolia) and show that intrahaplotypic divergence is high and comparable to the allelic diversity of S‐RNase while interhaplotypic divergence is very low. Next, we analyzed loss‐of‐function of the SFBB1 type gene. Genetic analysis showed that pollen with the mutant haplotype S^4sm lacking SFBB1‐S⁴ is rejected by pistils with an otherwise compatible S¹ while it is accepted by other non‐self pistils. We found that the S⁵ haplotype encodes a truncated SFBB1 protein, even though S⁵ pollen is accepted normally by pistils with S¹ and other non‐self haplotypes. These findings suggest that Japanese pear has a ‘non‐self recognition by multiple factors’ SI system, although it is a species of Rosaceae to which Prunus also belongs.

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.