Molecular Identification Of NSa Cytoplasm In Brassica Napus L.

Cytoplasmic male sterility (CMS) is a maternally inherited character of failure to produce viable pollens. CMS is a main pollination control system for oilseed rape hybrids production in the application of heterosis. While the use of a single cytoplasm is in favor of disease spreading, exploiting and establishing novel CMS lines are of major importance for enrich the cytoplasmic genetic diversity of oilseed rape hybrids production. In OCRI-CAAS, NSa cytoplasm male sterile obtained by somatic hybridization is a potential cytoplasmic male sterility system for the breeding of hybrid .The sterility of NSa CMS is stable. Study on maintaining and restoring relationship of NSa CMS showed that NSa CMS is different from Polima CMS. In order to further identify the cytoplasm origin of NSa CMS, find the mitochondrial genes associated with male sterile cytoplasm of NSa CMS and to apply it in breeding and production, a series of studies was conducted and the main results are as following:1. Establish an efficient mitochondrial DNA (mtDNA) extraction method, which is the foundation of mtDNA sequencing and mechanism research of cytoplasmic male sterility. Using etiolated seedlings of Brassica napus, a protocol for the extraction of pure mtDNA was set up by differential centrifugation combined with sucrose padding followed by DNAase digestion, SDS lysis and extraction. The mtDNA showed fairly good integrality on the agarose gel with a sharp band without visible degradation. PCR detection using gene specific primers did not find any contamination of nuclear DNA. Compared with density gradient centrifugation method, this protocol is superior with higher efficiency, low requirement of equipment and good purity of the product.2. RFLP identifies the origin of cytoplasm of NSa CMS is from Sinapis arvensis. Southern blot with EcoRI/atp9 in mitochondrial DNA of NSa sterile line, NSA restorer, somatic hybridization parent Zhongshuang 4 and Sinapis arvensis. The blot bands of NSa CMS are same with Sinapis arvensis while are different from parent Zhongshuang 4. This result shows that the atp9 gene of NSa CMS is from Sinapis arvensis.3. Identify the expression difference of some fertility related mitochondrial genes in NSa sterile and NSa maintainer,and a differential expression mitochondrial gene is obtained preliminary. Reverse dot blot with 15 mitochondrial genes in NSa sterile cDNA and NSa maintainer cDNA. Use GeneTools to analysis the hybridization result. The result shows that the expression of mitochondrial gene nad2 in NSa sterile line is lower than in NSa maintainer, that is the expression of nad2 is inhibited in NSa CMS. So we suggest the nad2 gene may relate with NSa CMS.