新疆野生欧洲李ISSR-PCR反应体系的建立与优化

为建立适合新疆野生欧洲李的ISSR-PCR反应体系,本研究以野生欧洲李为供试材料,采用L₁₆(4⁵)正交试验设计和单因素试验设计相结合的方法,对影响野生欧洲李ISSR-PCR反应体系的5个因素(DNA模板,Taq酶,dNTPs,引物,Mg²⁺)进行筛选与优化,对16条引物的退火温度进行筛选。结果表明,Taq酶对PCR扩增反应的影响最大,野生欧洲李ISSR-PCR最佳反应体系为:总体积20μL,5 U Taq酶0.10μL,10 mmol/L引物1.00μL,2.5 mmol/L dNTPs 1.50μL,10×Buffer(含Mg²⁺)2.00μL,50 ng/μL DNA模板1.00μL,双蒸水14.40μL。建立的ISSR-PCR反应体系经过22份野生欧洲李样品验证,表明反应体系稳定可靠,可用于后续遗传多样性研究,为野生欧洲李种质资源的保护和利用提供理论参考。

Establishment and Optimization of ISSR-PCR Reaction System in Xinjiang Prunus domestica L.

In order to establish an ISSR-PCR reaction system suitable for Xinjiang Prunus domestica L.,the Prunus domestica was used as the test material.In this research,the ISSR-PCR reaction system for Prunus domestica was optimized by using the L₁₆(4⁵)orthogonal experimental design combined with signal factor analysis to screen five main factors(DNA template,Taq polymerase,dNTPs,primer,Mg²⁺)of PCR reaction.Based on the optimal ISSR-PCR reaction system of Prunus domestica,the optimal annealing temperature of 16 primers was determined.The results showed that Taq polymerase had the greatest influence on PCR amplification reaction.The optimal reaction system of Prunus domestica ISSR-PCR was:total volume 20μL,5 U Taq polymerase 0.10μL,10 mmol/L primer 1.00μL,2.5 mmol/L dNTPs 1.50μL,10×Buffer(containing Mg²⁺)2.00μL,50 ng/μL DNA template 1.00μL,and double distilled water 14.4μL.The established ISSR-PCR reaction system was verified by 22 Prunus domestica samples,which indicated that the reaction system was stable and reliable.The ISSR-PCR reaction system could be used for subsequent genetic diversity research,providing a theoretical reference for the protection and utilization of Prunus domestica germplasm resources.