二倍体和四倍体棉花线粒体基因组atpA重排位点分析

Plant mitochondrial genomes (mtDNA) undergo frequent rearrangements that result in cytoplasmic male sterility (CMS).The gene encoding the α subunit of F1-ATPase (atpA) is one of the rearrangement hotspots, but the rule and mechanism are not clear. Previous study indicates that there are significant RFLP polymorphisms at the mitochondrial atpA sites, not only between cytoplasmic male sterile (CMS) line and maintainer line of Upland cotton, but also among various diploid and tetraploid cotton. This means that atpA gene is an active rearrangement site during cotton evolution. Our group cloned the sequences of the double-copy atpA genes in Upland cotton and its CMS line. The total sequences cloned are more than 40 kb. And we found that the rearrangement site located at the 3' coding and flanking regions of atpA gene. In this research, we plan to perform RFLP and chromosome-walking analysis upon 28 cultivars of diploid cotton and 5 cultivars of tetraploid cotton based on the sequences (more than 40 kb) mentioned above. Our aim is to obtain the sequences of all atpA copies in 33 cultivars of cotton. Our hope is that by surveying dozens of diverse cultivars of cotton through comparative analysis of the sequences at the atpA sites, we could discern the basic pattern of rearrangement of mitochondrial genomes in cotton and, furthermore, learn about the mechanisms underlying these kinds of structural changes.

植物线粒体基因组（mitochondrial DNA, mtDNA）频繁发生重排，并可能导致细胞质雄性不育（cytoplasmic male sterility，CMS），其中F1-ATP合酶α亚基基因（atpA）位点是发生重排的热点之一，但其规律与机理尚不清楚。研究发现atpA基因在陆地棉和其CMS系，以及不同二倍体和四倍体棉中都存在明显RFLP多态性，说明该基因是棉属mtDNA进化过程中的一个重排热点。本研究组首次解析了陆地棉及其CMS系线粒体双拷贝atpA基因区域序列（40 kb），发现重排位点位于atpA 3'编码及侧翼区。本项目拟用上述序列为参照，对28个二倍体和5个四倍体棉种atpA基因进行RFLP及染色体步移分析，确定每个棉种atpA的拷贝数，及每个拷贝的具体序列，针对重排位点进行全面比较分析，以期揭示棉属进化过程中mtDNA的重排规律，探究其重排机理。

atpA基因区是棉属植物线粒体基因组中的一个重排活跃位点。本研究通过RFLP、SSR、染色体步移测序等方法，分析了34种不同二倍体和四倍体棉种atpA基因的拷贝数及序列差异。结果发现：（1）不同棉种线粒体atpA基因都是双拷贝，一个拷贝含有全长atpA基因，一个拷贝含有截短型atpA基因。（2）之前发现的所谓的“棉花细胞质雄性不育相关线粒体DNA序列”（“S555”）存在于瑟伯氏棉（D1）、辣根棉（D2-1）、哈克尼西棉（D2-2）、三裂棉（D8）、特纳氏棉（D10）等5种D染色体型二倍体野生棉中，且五者之间存在6个SNP位点。（3）之前发现的所谓的“棉花可育胞质特异线粒体DNA序列”（“N515”）广泛存在于其它29种棉花（包括所有的A、B、C、E、F、G、AD染色体型棉种和部分的D染色体型棉种）中，且存在55个SNP位点。（4）全长atpA基因3’下游的微卫星序列区在34种棉花间存在丰富多态性，利用该位点的序列差异可将34种棉花分成20个组。（5）同向长重复序列（rrn18-rrn5-atpA）和短重复序列（nad6部分编码序列）共同参与了棉花线粒体atpA基因区的重排；且截短型atpA拷贝存在于线粒体基因组亚环中。这些结果显示，线粒体基因组重排的“接点”在34种二倍体和四倍体棉花间比较保守，共有两种：一种是S555型；一种是N515型。但是不同棉种间的SNP位点却比相像中丰富的多，发现的这些多态性有助于我们更好地了解棉属植物的起源和进化，并有助于育种家更好地利用野生棉。本研究从34种棉花中筛查出5种含有S555序列的野生棉，这也许将有助于育种家培育出新型的棉花细胞质雄性不育系；并且将提示相关研究人员重新审视S555序列与棉花细胞质雄性不育之间的关系。