Optimization for ISSR-PCR reaction system of Illicium difengpi by orthogonal design / 中草药

ABSTRACT

Objective: To establish a stable, reproducible, and suitable reaction system for ISSR analysis of genetic differences in Illicium difengpi. Methods: The ISSR-PCR amplification system on I. difengpi in five factors (Mg2+, dNTPs, primers, Taq DNA polymerase, and DNA template) was optimized by orthogonal design, and the PCR result was analyzed by SPSS. Then based on the optimal ISSR-PCR amplification system, the annealing temperature and cycle times in PCR were proposed by gradient determenation. Results: Most of the factors in different levels had the significant effects on the result of PCR, and the most remarkable factor was the quantity of Taq DNA polymerase. The optimized ISSR-PCR reaction system (20 μL) for I. difengpi was constructed of Mg2+ (1.60 mmol/L), dNTP (0.22 mmol/L), primer (0.90 μmol/L), Taq polymerase (0.50 U), and DNA template (70.00 ng). The optimized annealing temperature and cycle times were 51.8 °C and 40 cycles, respectively. Thirteen ISSR primers with stable amplification and abundant polymorphism were selected from 62 ISSR primers. Conclusion: The established and optimized ISSR reaction system is stable and credible according to the testing results of 16 samples of I. difengpi, and provides the basis for the genetic analysis of I. difengpi.