珍稀濒危植物银缕梅ISSR-PCR反应体系建立及优化

目的]建立和优化银缕梅ISSR-PCR反应体系。方法]采用正交试验和单因子试验对影响PCR扩增体系中的Mg~(2+)浓度、dNTPs、模板DNA及引物浓度、Taq酶用量5个因子进行分析研究。结果]银缕梅ISSR-PCR 25μL反应体系中5个因子最优水平:DNA模板(20 ng/μL)2.50μL,引物(10μmol/L)1.00μL,Taq酶(5 U/μL)0.10μL,Mg~(2+)(25 mmol/L)3.00μL,d NTPs(2.5 mmol/L)1.50μL。结论]银缕梅ISSR-PCR反应最优体系的建立为进一步利用ISSR对其进行分子标记辅助育种、分子指纹图谱构建和遗传多样性分析等后续研究奠定了基础。

Establishment and Optimization of ISSR-PCR Reaction System for Rare and Endangered Plant Parrotia subaequalis

Objective]To establish and optimize ISSR-PCR reaction system.Method]Effect for ISSR-PCR reaction of Parrotia subaequalis on 5 factors(Mg2 +,dNTPs,DNA template,Taq polymerase and primer)were analyzed by orthogonal design and single factor test.Result]The optimal ISSR-PCR reaction system(25 μL)mixture contained DNA template(20 ng/μL)2.50 μL,primer(10 μmol/L)1.00 μL,Taq polymerase (5 U/μL)0.10 μL,Mg2 +(25 mmol/L)3.00 μL,dNTPs(2.5 mmol/L)1.50 μL.Conclusion]The establishment of ISSR-PCR reaction system for P.subaequalis laid the foundation for the further use of ISSR to study molecular marker-assisted breeding,molecular identity and genetic diver-sity.