Map-based cloning of a recessive genic male sterility locus in Brassica napus L. and development of its functional marker
Map-based cloning of a recessive genic male sterility locus in Brassica napus L. and development of its functional marker
复制标题
甘蓝型油菜隐性雄性不育基因座的图位克隆及其功能标记的开发
DOI：
10.1007/s00122-012-1827-5
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发表时间：
2012-07-01
影响因子：
5.4
通讯作者：
Yang, Guangsheng
中科院分区：
文献类型：
--
作者：
Li, Ji;Hong, Dengfeng;Yang, Guangsheng
我们先前从广泛使用的甘蓝型油菜隐性核不育系(9012AB)中定位了一个雄性不育基因(Bnms3)到0.14 cM的基因组区域。在这项研究中，利用基于图谱的克隆策略，鉴定了两个可能包含候选BnMs3基因的高度同源的BAC重叠群。一个BnMs3连锁的SCAR标记(DM1)可以区分甘蓝型油菜和甘蓝的亚基因组，辅助将BnMs3定位在甘蓝型油菜C9染色体的重叠群上。从两个重叠群中分别测序了一个代表性的BAC克隆，根据两个克隆之间的序列差异产生了更多的标记。为了分离BnMs3，对另外两个具有不同遗传背景的Bc1群体进行了分析。该方法可以将BnMs3的突变功能区定位为9.3kb的DNA片段。基因预测表明，该片段含有一个完整的开放阅读框架(ORF、ORF2)和ORF1、ORF3的部分CDS片段。序列比较和遗传转化最终表明，BnMs3的唯一候选基因是拟南芥基因AT5G16620的类似物ORF1(命名为BnaC9.Tic40)，该基因编码叶绿体40内膜转运子(Tic40)。此外，从9012A和临时保持系T45中分别发现了两种不同的ORF1突变类型，这两种突变类型都导致了雄性不育表型。并对该雄性不育的分子机制以及BnMs3相关的功能标记和共分离标记在真育种中的应用进行了讨论。
We previously mapped one male-sterile gene (Bnms3) from an extensively used recessive genic male sterility line (9012AB) in Brassica napus to a 0.14-cM genomic region. In this study, two highly homologous BAC contigs possibly containing the candidate BnMs3 gene were identified using a map-based cloning strategy. A BnMs3-linked SCAR marker (DM1) capable of differentiating the subgenomes between B. rapa and the B. oleracea aided mapping of BnMs3 on the contig derived from the B. napus chromosome C9. One representative BAC clone was sequenced from each of the two contigs and resulted in a larger number of markers according to the sequence difference between the two clones. To isolate BnMs3, these markers were then analyzed in another two BC1 populations with different genetic backgrounds. This assay allowed for a delimitation of the mutated functional region of BnMs3 to a 9.3-kb DNA fragment. Gene prediction suggested that one complete open reading frame (ORF, ORF2) and partial CDS fragments of ORF1 and ORF3 reside in this fragment. Sequence comparison and genetic transformation eventually indicated that ORF1 (designated as BnaC9.Tic40), an analogue of the Arabidopsis gene AT5G16620 which encodes a translocon of the inner envelope of chloroplasts 40 (Tic40), is the only candidate gene of BnMs3. Furthermore, two distinct mutation types in ORF1 both causing the male-sterile phenotype were individually revealed from 9012A and the temporary maintainer line T45. The molecular mechanism of this male sterility as well as the application of BnMs3-associated functional and cosegregated markers in true breeding programs was also discussed.