Artificial mutations in nuclear gene encoding mitochondrial RNA 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 due to the limited resources of Rf lines. In this study, we performed mutagenesis in tomato CMS seeds and successfully obtained 13 fertile revertants. By bulked segregant analysis and whole genome sequencing for each revertant, we detected mutations associating with fertility restoration in the nuclear-encoded gene for the mitochondrial RNA polymerase named SlRPOTm in four independent mutants created by mutagenesis. Furthermore, we found that the loss-of-function of SlRPOTm is associated with fertility restoration in the tomato CMS line. Expression analysis for orf137, which is a tomato CMS-causing gene revealed that a reduced expression level of orf137 is related to fertility restoration in the tomato CMS. In addition, F1 plants carrying mutations in SlRPOTm were created, and tomato fruit formation was found to be comparable to those of usual F1 plants. This study demonstrates that loss-of-function of mitochondrial RNA polymerase contributes to fertility restoration in CMS lines for the first time. Furthermore, it is possible to replace various tomato varieties with Rf lines by utilizing genome editing technology, which will promote tomato F1 breeding in the future.