HT-B and S-RNase CRISPR-Cas9 double knockouts show enhanced self-fertility in diploid Solanum tuberosum .

The Gametophytic Self-Incompatibility (GSI) system in diploid potato ( L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate self-compatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The and genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible lines have been generated by knocking out gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout either individually or in concert with in the diploid self-incompatible clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, only knockouts produced little or no seed. In contrast, double knockout lines of and displayed levels of seed production that were up to three times higher than observed in the -only knockout, indicating a synergistic effect between and in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where and did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding.