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本研究利用Me2Em4正反向引物组合对影响梾木属SRAP-PCR反应体系的Mg2+、Taq聚合酶、dNTP和引物浓度4个因素进行L9(34)正交试验,采用正交设计直观分析及方差分析对影响SRAP反应的4个因素进行分析,并对模板DNA浓度进行了单因素试验分析。结果表明,梾木属SRAP-PCR 20μL反应体系的最佳组合为:Taq聚合酶2.0 U、Mg2+浓度1.5 mmol/L、dNTP浓度0.15 mmol/L、Primer浓度0.30 mmol/L、模板DNA浓度5.5 mg/L以及含有2μL不含Mg2+的10倍稀释缓冲液。各因素对梾木SRAP-PCR反应的影响大小依次为:dNTP、Taq聚合酶、Mg2+和Primer。最后运用优化体系从100对SRAP引物组合中筛选出26个多态性SRAP标记,可用于梾木属不同种间亲缘关系、系统进化和遗传多样性等方面的研究。
In this study, four factors, Mg2 +, Taq polymerase, dNTP and primer concentration affecting the Alnus sibirica SRAP-PCR reaction system were investigated by Me2Em4 forward and reverse primer combinations. L9 (34) orthogonal test was carried out using orthogonal design and variance Analysis of the impact of SRAP response of the four factors were analyzed, and the template DNA concentration was a single factor test analysis. The results showed that the optimal combination of 20μL reaction system of Alnus was 12 U of Taq polymerase, 1.5 mmol / L of Mg2 +, 0.15 mmol / L of dNTP, 0.30 mmol / L of Primer and 5.5 mg of template DNA / L and containing 2 [mu] L 10x dilution buffer without Mg2 +. The factors affecting the Alnus cremorium SRAP-PCR reaction size: dNTP, Taq polymerase, Mg2 + and Primer. Finally, 26 polymorphic SRAP markers were screened from 100 pairs of SRAP primer combinations using optimization system, which could be used to study the genetic relationship, phylogenetic and genetic diversity among different species of Alnus.