Positional cloning of the rice Rf-1 gene, a restorer of BT-type cytoplasmic male sterility that encodes a mitochondria-targeting PPR protein

The combination of cytoplasmic male sterility (CMS) in one parent and a restorer gene (Rf) to restore fertility in another are indispensable for the development of hybrid varieties. We have found a rice Rf-1 gene that restores BT-type CMS by applying a positional cloning strategy. Using linkage analysis in combination with 6,104 BC1F3 progeny derived from a cross between two near-isogenic lines (NILs) differing only at the Rf-1 locus, we delimited the Rf-1 gene to a 22.4-kb region in the rice genome. Duplicate open reading frames (Rf-1A and Rf-1B) with a pentatricopeptide (PPR) motif were found in this region. Since several insertions and/or deletions were found in the regions corresponding to both the Rf-1A and Rf-1B genes in the maintainer’s allele, they may have lost their function. Rf-1A protein had a mitochondria-targeting signal, whereas Rf-1B did not. The Rf-1B gene encoded a shorter polypeptide that was determined by a premature stop codon. Based on the function of the Rf-1 gene, its product is expected to target mitochondria and may process the transcript from an atp6/orf79 region in the mitochondrial genome. Since the Rf-1A gene encodes a 791-amino acid protein with a signal targeting mitochondria and has 16 repeats of the PPR motif, we concluded that Rf-1A is the Rf-1 gene. Nine duplications of Rf-1A homologs were found around the Rf-1 locus in the Nipponbare genome. However, while some of them encoded proteins with the PPR motif, they do not restore BT-type CMS based on the lack of co-segregation with the restoration phenotype. These duplicates may have played diversified roles in RNA processing and/or recombination in mitochondria during the co-evolution of these genes and the mitochondrial genome.