In Four Regions Of Qinghai Fritillaria.Unibracteata Hsiao Et K．C．Hsia RAPD Analysis Of Genetic Diversity

Fritillaria. unibracteata Hsiao et K.C.Hsia is an important medicinal plant belonging to Liliaceae, Fritillaria. RAPD technique is used in analyzing the genetic diversity of 23 samples from four different regions of Qinghai Province, the results would reveal the genetic background of Fritillaria of Qinghai Province and provide the basic information for breeding varieties and protection, evaluation, use of germplasm resources. The major findings are as follows.1. 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 25ng template DNA, 0.8μmol/L random primer, 0.20mmol/L dNTPs, 1.5 mmol/L Mg2+,0.8 U Taq enzyme. The reaction procedure was 95℃in 5 min, then 40 cycle times with 94℃in 45 s,37℃in 1 min,72℃in 2 min, lastly 72℃in 5 min, and stop reaction at 4℃.2. There were 40 random primers used for the PCR amplification of genomic DNA fragment. ten of the primers could amplify polymorphic bands,67 of 95 amplified bands were polymorphic, accounts for 70.5%.Every primer could amplify 5 to 12 polymorphic bands, with an average of 6.7 bands.3. According to S23 primer pairs of 23 samples of Fritillaria amplification results build 0 / 1 matrix and use the software NTsys2.10 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 23 samples were 0.3333 to 0.9167 and the average is 0.625.The similarity coefficient between num 4 and num 6 from Jiuzhi County is the largest of all that means the closest relationship between the two. Cluster analysis showed that: four plots of 23 samples, each sample plot of the samples were gathered in a class, indicating similarity coefficient is closely related to the sample location.