Establishment and optimization of SRAP-PCR system in Melia azedarach

Objective In order to study the genetic diversity and evaluate the germplasm resources of Melia azedarach using SRAP markers, the concentration of DNA template, dNTPs, Mg²⁺, primer and Taq polymerase were tested to determine their optimal levels and establish the optimal SRAP reaction system in M. azedarach.

Method Five factors, each with 8 concentration levels, were conducted to screen their feasible concentration range using single factor experiment, and then the orthogonal experiment L16(4⁵) was carried out and the optimized system was determined.

Result and conclusion The concentration range of DNA template was wider in SRAP system in M. azedarach. When the concentration of dNTPs ranged from 0.1 to 0.2 mmol·L^-1, the bands were steady; when the concentration of Mg²⁺ was around 2.0 mmol·L^-1, more bands were amplified and clear. The band type was consistent and clear when the primer concentration ranged from 0.48 to 0.64 μmol·L^-1. When the Taq polymerase ranged from 0.50 to 2.00 U, more bands were amplified and clear. The optimum SRAP-PCR system was established on the basis of orthogonal experiment, which was DNA template 30 ng, dNTPs 0.125 mmol·L^-1, Mg²⁺ 2.25 mmol·L^-1, primers 0.48 μmol·L^-1, and Taq DNA polymerase 0.75 U in the 25 μL reaction system.