Optimization of ISSR-PCR Reaction System and Primer Selection for Olea euyopaea

摘要

[Objective] This study aimed to optimize the ISSR-PCR reaction system and select polymorphic ISSR primers for Olea euyopaea. [Method] O. euyopaea genomic DNA was extracted from leaves as the template for optimization of ISSR-PCR reaction system by single-factor experiments on the main factors including Mg2+, dNTPs, primer concentration and template amount. [Result] The optimal ISSR-PCR reaction system for O. euyopaea was obtained, with a total system volume of 20 μl containing 1 × Taq buffer, 3.5 mmol/L Mg2+, 0.4 mmol/L dNTPs, 1.0 μmol/L primers, 1.0 U of Taq DNA polymerase and 20 ng of DNA template. The optimal ISSR-PCR reaction program was started with predenaturation at 94 ℃ for 5 min, followed by 40 cycles of denaturation at 94 ℃ for 30 s, annealing at 52-55 ℃ for 30 s, and extension at 72 ℃ for 2 min; the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min to allow complete extension of PCR products. PCR products were stored at 4 ℃. Based on the above conditions, 11 primers with high polymorphism, clear amplified bands and good repeatability were selected. [Conclusion] This study laid the foundation for further diversity research and species identification of O. euyopaea germplasm resources.