Study On RAPD Genetic Diversity And Relationship Among The Strains Of Agaricus Bisporus

There are abundant germplasm resources in Agaricus bisporus all over the world. Because of human's culture and natural hybridization, the genetic diversity formed in Agaricus bisporus, which makes the boundary of interspecific population and intraspecific varieties to be unclear and research for genetics and variation to be difficult.Total DNAs of Agaricus bisporus were extracted by the CTAB method from 40 samples. The genetic diversity and relationship among 40 varieties of Agaricus bisporus were analyzed by RAPD results.Three methods with chloroform, chloroform-Tris and CTAB for DNA extraction were compared. The results indicated that the DNA extracted by CTAB method is suitable to RAPD-PCR analysis.The parameters of RAPD-PCR including the dosage of Taq enzyme, template DNA, magnesiumion, dNTPs and arbitrary primer were optimized one by one, to establish the optimal RAPD-PCR system, i.e. the 20μL volume of the reaction is consisted by 10×buffer 2.0μL, 1.5 unit of Taq DNA enzyme, 80 ng of template DNA, 2.0 mmol·L^-1 of MgCl₂, 0.2 mmol·L^-1 of dNTPs, 0.3μmol·L^-1 of arbitrary primer, adding pure water to 20μL of final volume. The thermal programmer, for RAPD-PCR was 5 min denaturation at 94℃, then 1 min denaturation at 94℃, 1 min annealing at 44℃, 1min 50sec polymerization at 72℃, for 40 cycles, and 7 min final polymerization at 72℃.The eletrophoresis patterns of RAPD-PCR products were clear, stable and polymorphic, were found in 71 arbitrary primers of 108 ones. According to the RAPD-PCR data coded in 0 or 1, the cluster analysis was carried out by means of the Between-groups Linkage method and the coefficient of Euclidean Distance Square (CEDS) in the software of SPSS 11.0 for Windows.The results of the cluster analysis showed that abundant genetic diversity in the 40 varieties were found. The 40 varieties could be classified into 3 variety groups at 20 in CEDS, or could be classified into 6 variety groups at 5 in CEDS. These results are rather agreement with the results based on isoenzymic datas from the researcher who provied materials in this study. Five varieties could be distinguished from 40 ones of Agaricus bisporus by the patterns of DNA fingerprint with variety-specific based on RAPD-PCR datas.