The Establishment And Application Of Four Kinds Of Molecular Marker Technologys In Lyophyllumkarst

In this study Lyophyllum Karst. are used as material,prioritization the culture medium,and buile three molecule markers, TRAP,CoRAP and RSAP, prioritize the PCR system,cluster analysis were used to analyse the esterase zymogram polymorphism,TRAP,CoRAP and RSAP’s genetic relationships.The main results of this study were as follows:1、The ingredient of culture medium that suit most of the Lyophyllum Karst.contain potato200g,hydrolysis starch20g,KH2PO41g, K2HPO41g,MgSO41g,VB110mg,Agar18g,add the25%water of pine at the same time can accelerate the speed of Lyophyllum Karst. And become more vigorous.2、The result of Esterase isozyme analysis show that.the difference between te45Lyophyllum Karst. Strains are evident,and there are much inheritance diversity in it, this are in agreement of the message about the strains,for example,the strain38and stiain39,their bodys,morphology,esterase isozyme are totally the same,they may be the same race’s different individual. Esterase isozyme analysis can use to study the partition of Lyophyllum Karst.3、A new marker, target region amplified polymorphism (TRAP) was developed for the first time in Lyophyllum Karst. Experiment the best PCR-system, The15μl PCR reaction of TRAP includes40-60ng DNA templates,2.5m mol/L of Mg2+,0.25m mol/L of dNTP,1.5U of Taq DNA polymerase,600nmol/L of each primer. PCR amplification is run first degeneration at94℃for5minutes then the first5cycles with an annealing temperature of35℃, followed by35cycles with an annealing temperature of50℃. Amplified DNA fragments can be separated by denaturing polyacrylamide gels and detected by Sanger silver staining.4、A new marker Conserved Region Amplification Polymorphism (CoRAP) was developed for the first time in Lyophyllum Karst. Experiment the best PCR-system, The15μLPCR reaction of TRAP includes50ng DNA templates,2.5m mol/L of Mg2+,0.25m mol/L of dNTP,1.5U of Taq DNA polymerase,500nmol/L of each primer. PCR amplification is run first degeneration at94℃for5minutes then at an unify annealing temperature of52℃. Amplified DNA fragments can be separated by denaturing polyacrylamide gels and detected by improvement Sanger silver staining.5、A new marker, Restriction site amplified poly-morphism, RSAP was developed for the first time in Lyophyllum Karst. Experiment the best PCR-system, The20μl PCR reaction of TRAP includes25～75ng DNA templates,2.5m mol/L of Mg2+,0.2mmol/L of dNTP,1U of Taq DNA polymerase,800nmol/L of each primer. PCR amplification is run first degeneration at94℃for5minutes then the first5cycles with an annealing temperature of35℃, followed by35cycles with an annealing temperature of50℃. Amplified DNA fragments can be separated by denaturing polyacrylamide gels.6、The result of esterase zymogram polymorphism、TRAP、CoRAP and RSAP are totally the same.