Abstract: In order to establish and optimize the SSR-PCR amplification system for pear total DNA, an orthogonal design experiment of 5 elements (Taq enzyme, Mg²⁺, template DNA, dNTP, primers)with 4 levels L₁₆(4⁵) was conducted. The PCR result was analyzed by the software DPS to select the best levels of responsive factors and established the optimal SSR reaction system. The optimal SSR-PCR system of 10 μL volumes consists of template DNA 30.0 ng, Taq DNA polymerase 1.0 U, each primer 0.2 μmol/L, Mg²⁺ 2.0 mmol/L, and dNTPs 0.4 mmol/L. PCR amplification procedures was：pre-denaturation for 4 min at 94℃, followed by 30 cycles of denaturation for 45 s at 94℃, anneal for 30 s at 48℃, extension for 30 s at 72℃, the amplification was completed after extension for 10 min at 72℃, then stored at 4℃. Total DNA of 24 pear varieties was tested to amplify SSR markers, which verified the robustness of the system.

Key words: Pear, SSR, Orthogonal design, PCR