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为了为鸭茅分子标记辅助育种提供理论依据,研究采用L16(45)正交试验设计,对相关序列扩增多态性(SRAP)-PCR反应体系中的4种主要参数(Taq DNA聚合酶、Mg2+、dNTP、引物)进行了分析。结果表明:建立的鸭茅SRAP-PCR最佳反应体系为Taq DNA聚合酶1.0 U、Mg2+1.5 mmol/L、dNTP 250μmol/L、引物0.4μmol/L,总体积为25μL。最佳PCR循环条件为94℃预变性5 min;94℃变性1 min,35℃复性1 min,72℃延伸1 min,共5个循环;94℃变性1 min,52℃复性1 min,72℃延伸1 min,共35个循环;最后72℃延伸10 min;4℃保存。
In order to provide a theoretical basis for molecular marker assisted breeding of Dactylis glomerulus, four major parameters of Taq DNA polymerase, such as Taq DNA polymerase, Mg2 +, dNTPs, primers) were analyzed. The results showed that the optimal reaction system for SRAP-PCR was 1.0 U of Taq DNA polymerase, 1.5 mmol / L of Mg2 +, 250 μmol / L of dNTP and 0.4 μmol / L of primer, with a total volume of 25 μL. The optimum PCR conditions were as follows: pre-denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 1 min, denaturation at 35 ° C for 1 min, extension at 72 ° C for 1 min for 5 cycles, denaturation at 94 ° C for 1 min, 72 ℃ extension 1 min, a total of 35 cycles; the last 72 ℃ extended 10 min; 4 ℃ preservation.