Screening Of Mitochondrial Genes Associated With Cytoplasmic Male Sterility In Cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait and a ubiquitous phenomenon in plants. It has unique advantages on utilizing heterosis in the case of the hybrid production, and it brings huge economic and social benefits; In theory, it is also a good material on the reasearch of pollen development, cytoplasmic nuclear interaction and genetic transformation of cell organelle. But the molecular mechanism of CMS is not clear, so more and more researches show great interests in it. Most results demonstrated that mitochondrion is correlated with CMS. And some CMS-associated mitochondrial genes or ORFs had been detected in some plants. Cotton is a representative of often cross-pollinated plants and a significant economic crop. But molecular researches on cotton lagged behind other crops. With the development of our lives, the yield and quality of cotton need to be improved. This is a serious challenge for cotton researchers. By using CMS system to produce hybrid seeds is an efficient and potential way to solve this problem. But researches on the molecular mechanism of CMS in cotton are still less. In this research, G. harknessii Brandegee S and its maintainer F were used as materials; and the method of RFLP (Restriction fragment length polymorphism), Northern blot and Genome walking were performed to screen the mitochondrial gene associated with CMS in cotton. Here are the main results and conclutions of the research:1. Ten CMS-associated mitochondrial genes (atpA, atp6, atp9, cob, coxâ… , coxâ…¡, coxâ…¢, nad3, nad6 and nad9) were taken as probes in analysis of RFLP. Three of them, atpA,coxâ…¢and nad6 displayed polymorphism.2. Northern blot was performed to further confirm the different expression pattern between CMS line and its maintainer for these three polymorphic genes. The result demonstrated that atpA and coxâ…¢were detected in maintainer but absent in CMS; one band of nad6 could be detected in maintainer but two bands in CMS line.3. Totally 24368 bp of sequence of atpA,coxâ…¢and nad6 and their flanking sequence were gotten via PCR of flanking sequence, cloning and sequencing (including 12156 bp of maintainer and 12212 bp of CMS line), of which, 7775 bp from atpA and its flanking sequence (including 3868 bp of maintainer and 3907 bp of CMS line), 4647 bp from coxâ…¢(including 2315 bp of maintainer and 2332 bp of CMS line) and 11946 bp from nad6 (including 2315 bp of maintainer and 2332 bp of CMS line).4. The alignments of corresponding DNA sequences between CMS and its maintainer were taken using bioinformatic tool CLASTAL X. The result revealed that most bases of coxâ…¢and nad6 are same except for some bases difference between two materials, while for the atpA gene, a fragment of about 500 bp downstream of the atpA gene were isolated in CMS line, which was significantly different from its maintainer.5. With the analysis of bioinformatics on sequences, a novel ces486 was detected in CMS line, which encoded a COXâ…¡protein of sugar beet with 42% identities. And this novel sequence maybe is associated with CMS in cotton.