Abstract
In many flowering plants, self-incompatibility (SI) is a genetically controlled recognition system that prevents self-fertilization, thereby promoting outcrossing and maintaining genetic diversity within populations. The gametophytic SI system in grasses (Poaceae) is governed by two multiallelic loci, S and Z, each locus comprising of three genes governing the initial recognition between pollen and stigma. Identifying these genes establishes the molecular basis of SI in grasses, but how its unique two-locus architecture has shaped the evolution of the underlying loci and recognition genes remains largely unexplored. Here, we report targeted sequence capture of SI genes in 362 genotypes across nine forage grass species, revealing extensive allelic richness and sequence diversity. The SI genes exhibited elevated nucleotide diversity (π) (0.134 < π < 0.403) and great amino acid sequence divergence, ranging between 48.2-58.0% for the female determinants (sS and sZ) and 66.5-83.4% for the male determinants (SI-DUF247). The SI-DUF247 proteins also displayed trans-generic polymorphism, indicating that alleles predate speciation. Phylogenetic analysis of SI determinants showed congruence at both S- and Z-locus, consistent with co-evolution of recognition partners. Our results demonstrate that the unique two-locus SI system in grasses shares evolutionary characteristics with other SI systems, suggesting that their reproductive function is shaping their evolution. These findings also indicate that the grass SI loci are under long-term balancing selection to maintain recognition specificity while creating sufficient allelic diversity. Our study provides comprehensive sequence resources for SI genes and new insights into the evolution of the unique two-locus SI system of grasses.