Optimization of ISSR-PCR reaction system and primer selection for Olea euyopaea.

[Objective] This study aimed to optimize the ISSR-PCR reaction system and select polymorphic ISSR primers for Olea euyopaea.[Method] Olea euyopaea genomic DNA was extracted from leaves as the template,for optimization of ISSR-PCR reaction system by single-factor experiment of the main factors including Mg2+,dNTPs,primer concentration and template amount.[Result] The optimal ISSR-PCR reaction system for Olea euyopaea was obtained,with a total system volume of 20 μl containing 1 × of Taq buffer,3.5 mmol/L of Mg2+,0.4 mmol/L of dNTPs,1.0 μmol/L of primers,1.0 U of Taq DNA polymerase,20 ng of DNA template.The optimal ISSR-PCR reaction program was started with predenaturing at 94 ℃ for 5 min,followed by 40 cycles of denaturing 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 Olea euyopaea germplasm resources.