[目的]建立并优化老芒麦ISSR - PCR反应体系和扩增程序,以期为探讨老芒麦种质资源的遗传多样性提供科学依据.[方法]采用正交设计试验和单因素试验相结合的方法对老芒麦的ISSR - PCR的反应体系进行构建和优化,对影响ISSR - PCR扩增效果的Taq酶、模板DNA的浓度、Mg2+、dNTP和引物浓度等因素进行优化,同时对退火温度、循环次数和延伸时间进行筛选.[结果]在25μl体系中各反应物的最适含量为:0.2 mmol/L dNTP,0.2 μmol/L ISSR引物,1.50 U Taq DNA聚合酶,2.5μl 10×PCR Buffer,1.5 mmol/L MgCl2 和40 ng模板DNA;循环次数和延伸时间分别是35次和90 s.[结论]该研究建立并优化了老芒麦ISSR - PCR反应体系,通过2份老芒麦种质对所优化体系得验证,结果显示体系稳定,可用于老芒麦种质资源遗传分析.%[Objective] This study aimed to establish and optimize the ISSR-PCR reaction system and amplification process for Elymus sibirkus L. , to provide scientific basis for exploring the genetic diversity of E. sibiricus germplasm resources. [Method] Orthogonal design and single factor test were applied to establish the ISSR-PCR reaction system of E. sibiricus, optimize the affecting factors including Taq DNA polymer-ase, template DNA concentration, Mg2+ , dNTP, primer concentration, and screen the annealing temperature, number of cycles and extension time. [ Result] The optimal reaction system for ISSR analysis contains 0. 2 mmol/L dNTPs ,0.2 μmol/L ISSR primers, 1. 5 U of Taq DNA polymerase, 2.5 μl of 10 X PCR Buffer ,1.5 mmol/L MgCl2 and 40 ng of template DNA in 25 Μl total volume; the amplification was conducted with 35 cycles and extension time of 90 s. [Conclusion] ISSR-PCR reaction system for E. sibiricus L. was established and optimized in this study, which was verified using two E. sibiricus germplasms, results show that the ISSR-PCR reaction system is stable and can be used for the genetic analysis of E. sibirkus.
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