| Summary: | Abstract Background Cytoplasmic male sterility (CMS) is a widely used trait for hybrid seed production in many crops. Sugar beet CMS is associated with a unique mitochondrial protein named preSATP6 that forms a 250-kDa complex. Restorer-of-fertility 1 (Rf1) is a nuclear gene that suppresses CMS and is, hence, one of the targets of sugar beet breeding. Rf1 has dominant, semi-dominant and recessive alleles, suggesting that it may be a multi-allelic locus; however, the molecular basis for differences in genetic action is obscure. Molecular cloning of Rf1 revealed a gene (orf20) whose protein products produced in transgenics can bind with preSATP6 to generate a novel 200-kDa complex. The complex is also detected in fertility-restored anthers concomitant with a decrease in the amount of the 250-kDa complex. Molecular diversity of the Rf1 locus involves organizational diversity of a gene cluster composed of orf20-like genes (RF-Oma1s). We examined the possibility that members of the clustered RF-Oma1 in this locus could be associated with fertility restoration. Results Six yet uncharacterized RF-Oma1s from dominant and recessive alleles were examined to determine whether they could generate the 200-kDa complex. Analyses of transgenic calli revealed that three RF-Oma1s from a dominant allele could generate the 200-kDa complex, suggesting that clustered RF-Oma1s in the dominant allele can participate in fertility restoration. None of the three copies from two recessive alleles was 200-kDa generative. The absence of this ability was confirmed by analyzing mitochondrial complexes in anthers of plants having these recessive alleles. Together with our previous data, we designed a set of PCR primers specific to the 200-kDa generative RF-Oma1s. The amount of mRNA measured by this primer set inversely correlated with the amount of the 250-kDa complex in anthers and positively correlated with the strength of the Rf1 alleles. Conclusions Fertility restoration by sugar beet Rf1 can involve multiple RF-Oma1s clustered in the locus, implying that stacking 200-kDa generative copies in the locus strengthens the efficacy, whereas the absence of 200-kDa generative copies in the locus makes the allele recessive irrespective of the copy number. We propose that sugar beet Rf1 is a complex locus.
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