A survey of the Sli gene in wild and cultivated potato.

Inbred-hybrid breeding of diploid potatoes necessitates breeding lines that are self-compatible. One way of incorporating self-compatibility into incompatible cultivated potato (Solanum tuberosum) germplasm is to introduce the S-locus inhibitor gene (Sli), which functions as a dominant inhibitor of gametophytic self-incompatibility. To learn more about Sli diversity and function in wild species relatives of cultivated potato, we obtained Sli gene sequences that extended from the 5'UTR to the 3'UTR from 133 individuals from 22 wild species relatives of potato and eight diverse cultivated potato clones. DNA sequence alignment and phylogenetic trees based on genomic and protein sequences show that there are two highly conserved groups of Sli sequences. DNA sequences in one group contain the 533 bp insertion upstream of the start codon identified previously in self-compatible potato. The second group lacks the insertion. Three diploid and four polyploid individuals of wild species collected from geographically disjointed localities contained Sli with the 533 bp insertion. For most of the wild species clones examined, however, Sli did not have the insertion. Phylogenetic analysis indicated that Sli sequences with the insertion, in wild species and in cultivated clones, trace back to a single origin. Some diploid wild potatoes that have Sli with the insertion were self-incompatible and some wild potatoes that lack the insertion were self-compatible. Although there is evidence of positive selection for some codon positions in Sli, there is no evidence of diversifying selection at the gene level. In silico analysis of Sli protein structure did not support the hypothesis that amino acid changes from wild-type (no insertion) to insertion-type account for changes in protein function. Our study demonstrated that genetic factors besides the Sli gene must be important for conditioning a switch in the mating system from self-incompatible to self-compatible in wild potatoes.

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 (Solanum tuberosum 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 S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out S-RNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HT-B-only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase-only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B 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.

The Effect of Self-Compatibility Factors on Interspecific Compatibility in Solanum Section Petota.

The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex. Inquiry into these relationships in tomato and its wild relatives has elucidated the pleiotropic and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection. Our findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection. Statistical analyses also demonstrated that HT-B alone is not a significant factor in these pollinations; demonstrating the overlap in gene function between HT-A and HT-B, as HT-A, was present and functional in all genotypes used. We were not able to replicate the general absence of prezygotic stylar barriers observable in S. verrucosum, which has been attributed to the lack of S-RNase, indicating that other non-S-RNase factors play a significant role. We also demonstrated that Sli played no significant role in these interspecific pollinations, directly conflicting with previous research. It is possible that S. chacoense as a pollen donor is better able to bypass stylar barriers in 1EBN species such as S. pinnatisectum. Consequently, S. chacoense may be a valuable resource in accessing these 1EBN species regardless of Sli status.