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

含有编码细胞质雄性不育的Owen线粒体的甜菜植株，如果它们携带两个显性的核恢复基因(RfX和RfZ)，则它们是可育的。为了了解RfX和RfZ介导的育性恢复的分子机制，我们决定采用定位克隆策略克隆RfX。采用批量分离分析方法筛选与RfX基因座连锁的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RfX 5.7 cM处。对2721对AFLP引物组合进行了进一步的多态性检测，筛选出了大约27000个与Rfx连锁的基因座。我们获得了8个与RfX基因座紧密连锁的标记，这些标记将其限制在3.4 cM以内。还需要指出的是，三个AFLP标记与RfX基因座共分离。构建了甜菜BAC文库(3.4个基因组当量)，平均插入大小为98kb。RfX周围的靶区大小约为300kb，并被至少8个BAC克隆覆盖。通过核苷酸测序和RT-PCR分析，我们在300kb区域内发现了一些开放阅读框，它们在花药组织中被活跃地转录，并具有线粒体靶向序列。因此，似乎有理由认为，这里确定的读框之一是RfX基因的一个很好的候选者。

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.