Fertility restoration in cytoplasmic male sterile tomato via knockout of either DNA polymerase I or Topoisomerase I, two nuclear-encoded organellar DNA replication genes

Cytoplasmic male sterility (CMS) and restorer-of-fertility (Rf) genes are widely used for F₁ hybrid breeding. To date, most identified Rf genes encode pentatricopeptide repeat (PPR) proteins, whereas several Rf genes encode other proteins. Identification of Rf genes is essential for promoting F₁ hybrid breeding; however, exploring Rf genes that encode proteins other than PPR is challenging because information on such genes remains limited. In this study, we focused on the nuclear genes involved in mitochondrial DNA replication to identify novel tomato Rf genes. Twenty-six tomato genes homologous to 19 Arabidopsis mitochondrial DNA replication genes were screened for mutations using whole-genome sequencing data from fertility-restored tomato suppressor mutants, and several candidate mutations for fertility restoration were identified. CRISPR/Cas9-mediated gene knockout experiments revealed that the loss of function of SlPolI, which encodes an organellar DNA polymerase, and SlTopI, which encodes an organellar topoisomerase I, restored pollen fertility in tomato CMS lines. This study provides new insights into the molecular basis of fertility restoration and opens up opportunities for the efficient discovery of Rf genes to accelerate F₁ hybrid breeding in tomatoes and other crops.