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

含有Owenmitochondrial编码细胞质雄性不育的甜菜植株如果携带两个显性核恢复基因（RfX和RfZ），则是可育的。为了了解RfX和RfZ介导的育性恢复的分子机制，我们决定使用定位克隆策略克隆RfX。利用混合分离分析来鉴定与RfX位点连锁的RAPD和AFLP标记。在检测的812个RADP标记中，观察到最近的标记位于距离RfX 5.7cM处。共2721 AFLP引物组合进行了进一步测试的多态性，这使得约27000个位点的连锁RfX的筛选。获得了8个与RfX紧密连锁的标记，这些标记将RfX位点限制在3.4cM以内。还应该提到的是，三个AFLP标记与RfX基因座共分离。构建了平均插入片段大小为98 kb的甜菜BAC文库（3.4个基因组当量），并利用连锁标记进行了筛选。RfX周围的靶区域包含约300 kb，并且覆盖有最少8个BAC克隆。通过核苷酸序列测定和RT-PCR分析，我们在300 kb区域内发现了一些开放阅读框，这些开放阅读框在花药组织中转录活跃，并含有线粒体靶向序列。因此，认为此处鉴定的其中一个阅读框是RfX基因的良好候选者似乎是合理的。

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.