Artificial Mutations in the Nuclear Gene Encoding Mitochondrial <scp>RNA</scp> Polymerase Restore Pollen Fertility in Cytoplasmic Male Sterile Tomato

Cytoplasmic male sterility (CMS) and restorer of fertility (Rf) are important traits in F₁ hybrid breeding. However, the CMS/Rf system has not been used in tomatoes because of the limited resources of Rf lines. In this study, we performed mutagenesis in tomato CMS seeds and successfully obtained 13 suppressor mutants with pollen fertility. Using bulked segregant analysis and whole-genome sequencing for each suppressor mutant, we detected mutations associated with fertility restoration in the nuclear-encoded gene for the mitochondrial RNA polymerase termed SlRPOTm in four independent mutants created through mutagenesis. Furthermore, we found that the loss of function of SlRPOTm was associated with fertility restoration in the tomato CMS line. Expression analysis of orf137, a tomato CMS-causing gene, revealed that reduced expression of orf137 was associated with fertility restoration in tomato CMS. In addition, F₁ plants carrying mutations in SlRPOTm were generated, and tomato fruit formation was comparable to that of normal F₁ plants. This study demonstrates for the first time that the loss of function of mitochondrial RNA polymerase contributes to fertility restoration in CMS lines. Furthermore, it is possible to replace various tomato varieties with Rf lines using genome editing technology, which will promote tomato F₁ breeding in the future.