Optimization of ISSR-PCR Reaction System in a Rare Medicinal Plant Rhodobryum ontariense(Bryaceae)

To seek a standardizing program of ISSR technique for the genetic diversity analysis of a rare medicinal plant Rhodobryum ontariense,a single-factor experiment was designed to optimize ISSR-PCR amplification system.A suitable ISSR reaction system was obtained,that is:20 μL reaction system containing 6 ng DNA template,0.4 μmol/L primer,2.25 mmol/L Mg²⁺,0.6 U Taq DNA polymerase,0.4 mmol/L dNTPs.The profile of ISSR amplification was 4 min at 94℃,followed by 40 cycles of denaturation for 1 min at 94℃,annealing for 1 min at 48~50℃(according to specific primer),and extension for 1 min at 72℃,and ending with a final extension for 7 min at 72℃,stored at 4℃.