Establishment of SRAP-PCR Reaction System on Docynia delavayi (Franch.) Schneid. and Selection of Primers

Purpose To establish the optimal SRAP-PCR reaction system in Docynia delavayi and selection of SRAP-PCR polymorphism primer combinations.

Method The improved CTAB method was used to extract DNA from young leaves of D. delavayi from three natural populations of Muli, Lancang and Yingjiang. In order to establish the SRAP-PCR reaction system applied to D. delavayi, the orthogonal design L₁₆(4⁵) was used to optimize five important influencing factors such as Taq DNA polymerase, Mg²⁺, dNTPs, template DNA and primers. And the primer combinations with high SRAP-PCR polymorphism were screened from 100 primer combinations.

Result The optimal reaction system for the SRAP-PCR of D. delavayi as (25 μL): Taq DNA polymerase 0.5 U, Mg²⁺ 2.5 mmol/L, dNTPs 0.25 mmol/L, template DNA 80 ng, primers 0.8 µmol/L. In addition, the effect degrees of the factors on the SRAP-PCR reaction were in the order of dNTPs<template DNA<Taq DNA polymerase<Mg²⁺<primers. Using the established and optimized SRAP-PCR system, 20 pairs of primers were used to amplify DNA ofD. delavayi, and a total of 212 amplified bands were obtained, the polymorphism sites was 187, the percentage was 88.21%.

Conclusion A stable and reliable SRAP-PCR system was established to support the subsequent study of genetic diversity in D. delavayi.