Restricted distribution of orf687 as the pollen fertility restorer gene for Ogura male sterility in Japanese wild radish

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In radish, the mitochondrial gene orf138 found in Ogura cytoplasm is responsible for male sterility, whereas the nuclear restorer gene (Rf gene) inhibits the expression of orf138. To investigate the distribution of the Rf gene for Ogura male sterile cytoplasm in Japanese wild radish, 226 plants collected from 15 regions in Japan and two additional sites of Korea were used as materials. PCR analysis indicated that 42% of the wild radishes possessed orf138. On the other hand, 207 plants (91.6%) were judged to have an Rf gene by the observation of pollen fertility in wild radishes themselves and in hybrids obtained by crosses with a male sterile variety having Ogura cytoplasm. For plants having restorer function, we analyzed the nucleotide sequence of the Rf gene, orf687, identified to date. Among the 207 plants having the Rf gene, 199 amplified the orf687 fragment of the expected size by PCR. PCR products were further analyzed by mismatch-specific endo-nuclease digestion and PCR-RFLP analysis. As a result, plants that showed an identical RFLP pattern to the known Rf type of orf687 were restricted to 30 plants (14.5% of the 207 plants) collected mainly in populations of southern region of Japan. It was also found that most of the plants (148 plants; 71.5% of 207 plants) possessed the sequence of the orf687 corresponding to the rf genotype lacking the restorer function. We conclude that the Rf gene widely distributed in Japanese wild radish is not orf687.