Optimization of ISSR-PCR Reaction System and Primers Screening in Schisandra sphenanthera

The main influential factors of ISSR-PCR reaction system in Schisandra sphenanthera Rehd.et Wils.were systematically studied,optimization of ISSR-PCR system was established,and 12 effective ISSR primers were selected.Single-factor experimental results showed that the suitable concentration of composition of ISSR-PCR systen was 1.50~3.50 mmol·L-1 of Mg2+,0.10~0.35 mmol·L-1 of dNTPs,0.25~0.60 μmol·L-1 of primer and 0.50~1.50 U of Taq DNA polymerase,respectively.The optimal ISSR-PCR reaction system was defined by orthogonal experiment with four-factor of three-level,i.e.20 μL reaction system contained 2.50 mmol·L-1 of Mg2+,0.20 mmol·L-1 of dNTPs,0.25 μmol·L-1 of primer,1.50 U of Taq DNA polymerase,60 ng of DNA template,2.50 μL of 10×PCR Buffer.The present study could be used in the research for evaluation of germplasm resources and analysis of genetic diversity in S.sphenanthera Rehd.et Wils..