SNP in tobacco mitochondrial gene atp6 and its correlation with CMS.

The cytoplasmic male sterility(CMS) of higher plants has great significance for the use of heterosis in crop production and the researches of cytoplasmic heredity and the interaction between cytoplasm and nucleolus.The mitochondrial genome is considered as the carrier of sterility gene of cytoplasm,and its mutation and recombination are closely related to CMS.ATP synthase participates in the reactions of oxidative phosphorylation and photophosphorylation and provides energy for the plant growth and development.The atp6 gene is one of the subunits of ATP synthase in mitochondria.The base mutation of DNA sequence of atp6 gene results in the abnormal synthesis of amino acids,causing the dysfunction of ATP synthase and the shortage of energy in plant cells.So the normal physiological function of cells is difficulty to be maintained,which results in the male sterility in higher plant.Single nucleotide polymorphism(SNP) is a frequent sort of genetic variation in organism genome DNA and this genetic variation is generally the direct reason of the changes of biologic traits.To better use the male-sterile lines to breed tobacco varieties,we investigated the molecular mechanism of tobacco CMS.The atp6 is a significant candidate gene carried by chondriosome related to CMS in higher plants.In this study,using specific primers designed,the mitochondrial atp6 gene was distinctively amplified by PCR from seven tobacco(Nicotiana tabacum) CMS lines(named as MS Yunyan 85,MS Zhongyan 90,MS K346,MS K326,MS G28,MS Nordel,and MS Jingyehuang) and their corresponding maintainer lines.Six nucleotide variations at A-59C,T-92C,T-185G,T-253C,T-418C,and T-768C in mitochondrial atp6 gene of CMS were detected by sequencing directly and comparing the sequences of atp6 gene.Four nucleotide variations at 59,92,185,and 418 resulted in the changes of amino acids coded by the changed nucleotide.The rest two nucleotide variations at 253 and 768 didn't cause the changes of amino acids,which was a synonymy mutation.The 59 A→C mutation of atp6 gene was detected and analysed by polymerase chain re-action and restriction fragment length polymorphism(PCR-RFLP) with a total of 222 individual tobacco plants,indicating that all the mitochondrial atp6 gene fragments of maintainer lines plants could be entirely digested by Bst1107 I and showed 2 bands in the electrophoresis pattern of atp6 gene,while these of 96% CMS plants displayed 3 bands in the electrophoresis pattern of atp6 gene could be partly digested by Bst1107 I due to the mutation of A→C at 59.Therefore,the A-59C SNP site in atp6 gene exhibited a significant correlation with the tobacco CMS.