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【目的】建立适合两面针简单重复序列—聚合酶链反应(ISSR-PCR)体系和扩增程序。【方法】采用改良的高盐低pH法提取两面针基因组DNA,通过单因素试验和正交试验相结合对ISSR反应体系中的主要成分(Mg2+、Taq DNA聚合酶、引物和模板)进行筛选和优化,并考察退火温度对扩增结果的影响。【结果】最佳的PCR反应体系为:总体积为25μL,含1.50 mmol/L Mg2+、150μmol/L dNTPs、0.04 U/μL Taq DNA聚合酶、0.60μmol/L引物、2.40 ng/μL DNA模板。扩增程序为:94℃预变性5 min;94℃变性30 s,51.4℃退火45 s,72℃延伸2 min;35个循环;72℃再延伸10 min。【结论】优化了两面针ISSR-PCR反应体系和扩增程序,为两面针ISSR遗传多样性分析打下基础。
【Objective】 The objective of this study was to establish an ISSR-PCR system and an amplification program suitable for the double-sided needle. 【Method】 The genomic DNA of two-sided needle was extracted by modified high-salt and low-pH method. The main components (Mg2 +, Taq DNA polymerase, primer and template) in ISSR reaction system were screened by single factor experiment and orthogonal experiment Optimize, and examine the effect of annealing temperature on the amplification results. 【Result】 The optimal PCR reaction system was as follows: a total volume of 25 μL containing 1.50 mmol / L Mg2 +, 150 μmol / L dNTPs, 0.04 U / μL Taq DNA polymerase, 0.60 μmol / L primer and 2.40 ng / μL DNA template. The amplification program was: denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 30 s, annealing at 51.4 ° C for 45 s, extension at 72 ° C for 2 min, 35 cycles, and extension at 72 ° C for 10 min. 【Conclusion】 The two-sided ISSR-PCR reaction system and amplification program were optimized, which laid the foundation for ISSR genetic diversity analysis.