Optimization of ISSR-PCR Reaction System in Pinellia ternata

Abstract

In order to establish and perfect ISSR-PCR reaction system of Pinellia ternata , we used P. ternata from Sichuan region as the test material in this research. Genomic DNA of whole plant of P. ternata was used as template, UBC818 primer was used in ISSR-PCR system, L 16 (4 5 ) orthogonal test and single factor experiment were used to optimize the system. Based on the visual analysis of orthogonal experiment, we found the following are the influences of various factors on the experimental results in turn: dNTPs, primer, Mg 2+, Taq DNA polymerase, DNA template. Based on single factor screening test, we found the best ISSR-PCR reaction system. The total reaction volume is 20.0 μL, containing dNTPs 0.2 mmol/L, primer 0.8 μmol/L, Mg 2+ 2.0 mmol/L, Taq DNA polymerase 0.075 U/μL, DNA 15 ng, annealing temperature 56℃. In this study, ISSR-PCR reaction system was optimized which will be applied to detect the genetic stability of P . ternata tissue culture seedlings.