Establishment and Optimization for ISSR-PCR System of Ferula ferulaeoides

OBJECTIVE:To establish and optimize the ISSR-PCR system for Ferula ferulaeoides.METHODS:The ranges of Mg2+,dNTPs,primers,Taq DNA,polymerase and template DNA,which influenced ISSR-PCR system of F.ferulaeoides,were determined by single factor test;using them as factors and levels,the ISSR-PCR system was optimized by orthogonal experiment.ISSR-PCR result was analyzed by SPSS statistical software.RESULTS:Most of the factors at different levels had the significant effects on the result of PCR,and the most remarkable factors were the concentration of Mg2+,dNTPs and primers.The optimized ISSR-PCR system of 50 μl F.ferulaeoides as follows:10×Taq Buffer 5 μl,Mg2+2.0 mmol/L,dNTPs 0.4 mmol/L,primer 0.4 μmol/L,Taq DNA polymerase 1.75 u/50 μl,DNA template 0.25 ng/μl.15 ISSR primers with stable amplification and abundant polymorphism were selected from 100 ISSR primers.CONCLUSIONS:Established ISSR-PCR system is stable and reproducible,and provides the basis for further study on genetic diversity analysis,source identification and genetic relationship analysis of F.ferulaeoides by ISSR molecular marker technique.