Genetic basis and timing of a major mating system shift in Capsella

• Transitions from outcrossing to self-fertilization have occurred repeatedly in flowering plants, and often involve the breakdown of genetic self-incompatibility (SI). Theory predicts that loss of SI should often occur through dominant loss-of-function mutations that affect the pollen component of SI. • Here, we investigate the genetic basis of loss of SI, and specifically the role of mutations in the pollen SI component, in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of multiple complete S-haplotypes, gene expression analyses, and controlled crosses. • We show that loss of SI in C. orientalis occurred less than 2.6 Mya and maps as a dominant trait to the S-locus. By comparing S-haplotypes, we identify a frameshift deletion in the pollen specificity gene SCR that is fixed in C. orientalis, and we confirm loss of pollen SI specificity using controlled crosses. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility and is conserved with Arabidopsis halleri. • Our results are consistent with theory predicting a role for dominant S-linked mutations in the loss of SI, and improve our understanding of the role of parallel changes for shifts to selfing.