Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for...Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.展开更多
The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detect...The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.展开更多
基金Supported by Funding of Utilization of Heterosis and Breeding of New Variety in Brassicaceous Vegetable(2012BAD02B01)
文摘Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.
文摘The patterns of cleavage of mtDNA by restriction endonucleases was analysed for six fowl breeds and of. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White, Abor Acres and mtDNA polymorphisms were detected in the restriction patterns with the following eight enzymes, Ava Ⅰ, Ava Ⅱ, EcoR Ⅰ, Hind Ⅲ, Bam HI, Pvu Ⅱ, Pst Ⅰ, Hinc Ⅱ. The restriction cleavage patterns were identical among these breeds. Hyline White, Hyline Brown, ISA Brown, Hisex Brown, Lohmann White—A type. The patterns of Abor Acres were B type. Based on their mtDNA restriction types, all the breeds were classified into two groups. Genetic distances among these groups were calculated in order to define their phylogenetic relationships. The relationship among five egg line breeds is close, while Abor Acres (Broiler fowl) is relatively far from them. The results suggest that the difference of mtDNA could result from the different origins. The polymorphic sites in mtDNA of Hyline White has been located on a restriction map.
