The Efficient Extraction System of Alfalfa Mitochondrial and Chloroplast DNA

The cytoplasmic male sterility (CMS) plays an important role in the utilization of heterosis of crops. To separate mitochondria and chloroplast genomes of CMS lines is a necessary step in investigating the funtional mechanisms of cytoplasmic male sterility. In this study, alfalfa (Medicago sativa L.) cytoplasmic male sterile line CMS1 and its wild type WT were selected as research materials to extract mitochondria DNA (mtDNA) and chloroplast DNA (cpDNA). The fresh leaves were gathered and then frozen homogenized, centrifuged to chloroplasts and mitochondria by differential centrifugation. Then nucleus DNA was degraded by DNase I, and chloroplasts and mitochondria were cracked by proteinase K. After proteins were removed with phenol-chloroform-isoamyl alcohol, the mtDNA and cpDNA were isolated by ethanol precipitation. The main results are as below. (1) The average number of chloroplasts in extracting solution was more than 1 500 per milliliter, and mitochondria observed with Jannus Green B dyeing showed a large number with intact structure.(2) When 4 g alfalfa leaves were extracted, the average cpDNA productions of CMS1 (WT) were 0.41 (0.47) g/g, and the average mtDNA yield of CMS1 (WT) were 1.15 (0.83) g/g. As the extracted leaves of alfalfa increased to 10 g, both of average cpDNA and mtDNA yield increased to 2.17 g/g and 2.22 g/g, respectively, and the quality of them was also good (the values of OD260/OD280 of cpDNA and mtDNA samples were around 2.0). Extension of the proteinase K digestion time (2 h, 4 h, 6h) could improve the productive rate of cpDNA and mtDNA: proteinase K incubation for 4 h gave the highest concentration of mtDNA (59.9 ng/L), and the highest yield of cpDNA up to 24.3 ng/L under 6 h incubation. (3) The agarose gel electrophoresis of alfalfa cytoplasmic genomes showed that, all the bands of these cytoplasmic genomes showed no railing phenomenon, which identified high-purity DNA without pollution of RNA furthermore. All in all, the methods above to extract and purity alfalfa samples for cytoplasmic genomes were feasible.