An Effective Extraction And RAPD Analysis Of Mitochondrial DNA In Citrus

Mitochondria are an important organelle with their own genetic material(mtDNA). Mitochondrial genome has been reported to involve in chemical resistance,cell life cycle and cytoplasmic male sterility(CMS).It is foundation of studying both mitochondrial genome and molecular mechanism of CMS to extract mtDNA of high-quality.Here we reported a high-efficient extracting protocol and random ampliphied polymorphic DNA(RAPD) analysis of citrus mtDNA from embryogenic callus induced with the undeveloped ovules of Jiangxi seedless Ponkan were used as explants.The main results were as followed:1.The undeveloped ovules of Jiangxi seedless Ponkan were cultured onto 3 different media.The results showed that the regeneration rate of embryogenic callus varied on different conditions.Malt extract,GA₃ and darkness enhanced the inducement of embryogenic callus.During the subculture,additional silver nitrate(5 mg/L) enhanced the weak embryoids to form embryogenic callus.2.A method for isolating mtDNA from embryogenic callus of citrus was applied. Separating by the differential centrifugation and purifying based on sucrose shelf.The results showed that the obtained mitochondria had high quality.After treated with SDS, DNaseI,proteinase K,phenol/chloroform/isoamyl and chloroform/isoamyl,precipitated by ethanol,the mtDNA was examined by 0.7%agarose gel electrophoresis and UV-spectrophotometer.The result showed that the band of mtDNA was very regular.The mtDNA isolated by this method showed both high yield and structural integrity without impurities such as nuclear DNA,RNA and protein.Compared with other methods,this protocol cost less time and money and had higher efficiency.3.14 primers were selected from 30 RAPD primers to test 7 Citrus cultivars.Clear amplified bands were scored after the gel electrophores.The polymorphic fragments were ranged from 250 bp to 2000 bp.