叶用莴苣TRAP反应体系的建立

Establishment of TRAP Reaction System for Lactuca sativa L.

以叶用莴苣为试材,采用正交设计和单因素试验2种方法研究叶用莴苣TRAP反应体系中Mg^2＋、Taq DNA聚合酶、dNTPs、引物等4个因素的浓度变化对扩增结果的影响,建立最佳反应体系。结果表明：TRAP-PCR反应最优体系是在20μL反应体系中含DNA模板60～100 ng、10×PCR buffer（Mg^2＋free）2μL、Mg^2＋终浓度2.0 mmol/L、Taq DNA聚合酶含量1.0 U、dNTPs终浓度0.2 mmol/L、引物终浓度0.75μmol/L。该体系对叶用莴苣种质的扩增结果稳定,条带清晰度高且多态性丰富,可用于对叶用莴苣种质资源的遗传多样性分析和亲缘关系鉴定。

The orthogonal design and single factor test were used to optimize and study the suitable system in TRAP reaction of Lactuca sativa L. There were four factors which influenced the effects of TRAP-PCR including Mg^2＋, Taq DNA polymerase, dNTPs and primers. The optimal reaction system for TRAP reactions in leaf lettuce was established, which was in 20 μL reaction volume containing 60-100 ng DNA, 2 μL 10×PCR Buffer, 2.0 mmo/L Mg^2＋, 1 U Taq DNA polymerase, 0.2 mmol/L dNTPs and 0.75 μmol/L primers. The result of amplified production was stable, high definition and polymorphic, indicating that the reaction system was the best to analyze genetic diversity and relationship in leaf lettuce by TRAP.