Positional cloning of fertility restorer genes in sugarbeet and its appliation to molecular breeding

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Sugarbeet plants containing the Owenmitochondria encoding cytoplasmic male sterility are fertile if they carry two dominant nuclear restorer genes (RfX and RfZ). In order to understand the molecular mechanism of fertility restoration mediated by RfX and RfZ, we decided to clone RfX using a positional cloning strategy. Bulked segregant analysis was utilized to identify RAPD and AFLP markers linked to the RfX locus. Out of 812 RADP markers examined, the closest marker was observed to be located 5.7cM away from RfX. A total of 2721 AFLP primer combinations were further tested for polymorphisms, which allows the screening of approximately 27000 loci for linkage to RfX. We obtained eight markers tightly linked to the RfX locus which was restricted within 3.4cM by these markers. It should also be mentioned that three AFLP markers co-segregated with the RfX locus. A sugarbeet BAC library (3.4 genome equivalents), with an average insert size of 98kb, was constructed and screened with the linked markers. The target region around RfX comprises approximately 300kb and is covered with a minimum of eight BAC clones. By nucleotide suquencing and RT-PCR analysis, we identified some open reading frames within the 300kb-region, which were observed to be actively transcribed in anther tissues and to have mitochondrial targeting sequences. It thus seems reasonable to consider that one of the reading frames identified here is a good candidate for RfX gene.