Maenosono, T.; Isono, K.; Kuronuma, T.; Hatai, M.; Chimura, K.; Kubo, K.-i.; Kokubun, H.; Greppi, J. A.; Watanabe, H.; Uehara, K.; Tsuchimatsu, T.

Self-incompatibility (SI) is a genetic mechanism to prevent self-fertilization and thereby promote outcrossing in hermaphroditic plant species through discrimination of self and non-self pollen by pistils. In many SI systems, recognition between pollen and pistils is controlled by a single multiallelic locus (called S-locus), in which numbers of alleles (called S-alleles) are segregating. Because of the extreme level of polymorphism of the S-locus, identifications of S-alleles have been a major issue in many SI studies for decades. Here we report an RNA-seq-based method to explore allelic diversity of the S-locus by employing the long-read sequencing technology of the Oxford Nanopore MinION, and applied it for the gametophytic SI system of Petunia (Solanaceae), in which the female determinant is a secreted ribonuclease called S-RNase that inhibits the elongation of self-pollen tubes by degrading RNA. We developed a method to identify S-alleles by the search of S-RNase sequences, using the previously reported sequences as queries, and found in total 62 types of S-RNase including 45 novel types. We validated this method through Sanger sequencing and crossing experiments, confirming the sequencing accuracy and SI phenotypes corresponding to genotypes. Then, using the obtained sequence data together with PCR-based genotyping in a larger sample set of 187 plants, we investigated the diversity, frequency, and the level of shared polymorphism of S-alleles across populations and species. The method as well as the dataset obtained in Petunia will be an important basis for further studying the evolution of S-RNase-based gametophytic SI systems in natural populations.