In Some Areas Of Qinghai Cordyceps Sinensis RAPD Analysis Of Genetic Diversity

Cordyceps sinensis (Berk.) Sacc is a Chinese tranditional medicinal fungus. The Cordyceps sinensis resources in Qinghai Province's distribution area are mainly south the plateau Yushu, Guoluo two states, also has the distribution middle Qinghai province. RAPD technique is used in analyzing the genetic diversity of 20 samples from five different regions of Qinghai province, the results would reveal the genetic background of Cordyceps sinensis of Qinghai Province and provide the basic information for protection and evaluation, use of germplasm resources. The major findings are as follows:1. Two methods--SDS method and improved CTAB method were used to extract genomic DNA from Cordyceps sinensis. The DNA samples of two methods were tested by agarose gelelectropHoresis and spectropHotometer. The result was that the two methods could be used for extracting genomic DNA from Cordyceps sinensis, and that the improved CTAB method was the best ones through which the production had the highest purity. The OD260/OD280 was better by improved CTAB method, Between in 1.6-2.0. so, we choice the improved CTAB method for extract genomic DNA.2. In the experiment, five essential factors which may affect the result of RAPD were optimized by orthogonal experiment and two single factor experiments of annealing temperature and cycle times. The result showed that the optimal conditions of RAPD-PCR system and reaction parameters were 20μl Total reacting system: 35ng template DNA, 2.0 U Taq enzyme, 0.4μmol/L random primer, 0.2mmol/L dNTPs, 1.5 mmol/L Mg²⁺, The reaction procedure was 95℃in 3 min, then 40 cycle times with 94℃in 60 s, 37℃in 1 min, 72℃in 2 min, lastly 72℃in 5 min, and stop reaction at 4℃.3. There were 40 random primers used for the PCR amplification of 20 genomic DNA fragment. Nine of the primers could amplify polymorpHic bands, Altogether 65 were amplified, Every primer could amplify an average of 7.2 bands, And in there bands Include 57 bands, PolymorpHism position spot ratio accounts for 87.69%. Every primer could amplify an average of 6.3 polymorpHic bands, Nine of the primers could amplify PolymorpHism position spot ratio of 75%-100%.4. According to random primer pairs of 20 samples of Cordyceps sinensis results build 0/1 matrix and use the software NTSYS to calculate similarity coefficients between all varieties which determines the genetic relationship between the two species, the greater the similarity coefficient, the closer genetic relationship. The value of the similarity coefficients of 20 samples were 0.1818 to 0.9876. The similarity coefficient between num 18 and num 20 is the largest of all that means the closest relationship between the two. Cluster analysis showed that: five plots of 20 samples, each sample plot of the samples were gathered in a class, indicating similarity coefficient is closely related to the sample location.