MAP-Based Cloning Of Fertility Restoring Gene Of CMS In Cotton

Plant cytoplasmic male sterility (CMS) is a maternally inherited trait that is unable to pruduce viable pollens, which plays an important role in utilization of hybrid vigor. Comparing with hand-emasculation and pollination, and genetic male sterile lines, utilization of CMS lines is much more effective and economical in producing commercially hybrid seeds. Currently, corn, rice, onion, castor, sorghum, canola and sugar beets have used three lines to produce hybrid seeds. Cotton as an important economic crop has a very obvious heterosis, which can effectively increase the fiber production, improve quality and increase the biotic and abiotic resistance. Because of the weak restoring force the cotton restorer line, the application of the cotton three lines has been greatly restricted in agricultural production. Thus, cloning cotton CMS fertility-restoring gene for getting better restorer lines has important theoretical and practical significance.Currently, all the fertility restorer(Rf) genes reported whether or not belong to PPR genes, have the mitochondrial targeting sequence (MTS). Therefore, containing mitochondrial targeting sequence may be the feature of Rf. Tetraploid cotton restorer gene is from wild cotton, but the maintain line and sterile line corresponding to restorer line do not contain Rf gene. In this study, our laboratory constructed the genetic and physical map of the restorer gene and screened the 0-613-2R BAC clones on the physical map of Rfl locus combined with the homology cloning method. Finally, we got a positive BAC clone. This positive BAC clone was sequenced and using online software to predict ORF (open reading frame). According to the characteristics of the target gene, we selected the target gene combined with Q-PCR, VIGS (virus-induced gene silencing). Materials used in this experiment are sporophyte sterility system containing 0-613-2R (restorer line), Simian 3 maintain line, Simian 3 sterile line, sterile Gharknessii and gametophyte sterility system containing 8518R (restorer line),8518B (maintain line),8518A (sterile line), Gtrilobum;We got 26 contigs after BAC clone was sequenced, only four PPR genes contained the MTS. One of these four gene only contained 6 PPR motifs, further analysis revealed that this was an incomplete gene may be due to an incomplete BAC clone sequencing. The remaining three genes were named ORF2,ORF3 and ORF18.Three paris of primers were designed and amplified genes in 0-613-2R, Simian 3 maintain line, Simian 3 sterile line, sterile Gharknessii and G. raimondii. As a result, the ORF18 in the maintain line, sterile line and sterile G.harknessii were different with the sequence of the restorer line, which contained lots of SNPs. According to Yang(2009), ORF3 only be amplified in 0-613-2R and Gharknessii. In addition to the 0-613-2R,ORF2 could also be amplified in sterile line and G Raimondii(the maintain line), so the ORF2 might not be the candidate gene. There was other sequence amplified in 0-613-2R, the sequence of ORF2 had more than 105 bp, named ORF2-2, which only be in 0-613-2R.Using the homologous cloning method, amplified sequence in 8518R,8518A,8518B and Gtrilobum. As a result, the one was obtained in the ORF2 and two sequences were obtained in the ORF3, named ORF3-1 and the ORF3-2. Since the genes belong to the PPR family, designed SNP primers for four candidate genes (ORF2, ORF2-2, ORF3, ORF18) in 0-613-2R and three candidate genes (ORF2, ORF3-1, ORF3-2) in 8518R to do Q-PCR detection. Only ORF2-2 was expressed in roots, stems, leaves and anthers of 0-613-2R and (Simian Ⅲ X 0-613-2R) F1 and ORF2 was expressed in 8518R and (8518A X 8518R) F1.To further verify whether the two candidate genes were or not Rf gene, we used VIGS technology to silence the expression of the two candidate genes to verify the fertility of the pollens. The sterile pollens were observed in a 0-613-2R plant that injected Agrobacterium. We extracted RNA of the plant leaf to verify the relative expression level of the ORF2-2, it’s releative expression was 19% of the control’s. There was another plant whose anther in early development state was shrivel and the releative expression of the leaf from the plant only was 39% of the control’s. In addition, Q-PCR result showed that VIGS began to lose effect when 5 months after injection at about 20℃. Since 8518R plants grew slowly, so we had not yet got the result.