茶树TRAP反应体系的建立及正交设计优化

TRAP标记技术因操作简单、重复性好、效率高等特点得到广泛应用，但该技术在茶树中的研究报道较少，且茶树TRAP-PCR体系优化尚未见报道。笔者采用正交设计的方法，对茶树TRAP-PCR反应中Mg2+、Taq酶、dNTP、固定引物、随机引物5个因素进行了优化研究，建立了茶树TRAP-PCR最佳反应体系。该20 μL反应体系含有2 μL 10×PCR Buffer (Mg2+ free)，50 ng模板DNA，1.75 mmo/L Mg2+，0.50 U Taq酶，0.20 mmol/L dNTP，0.20 mmol/L随机引物和0.20 mmol/L固定引物。根据优化结果，利用最佳反应体系，选用16个茶树品种对TRAP标记多态性进行了初步研究。

Establishment of TRAP Reaction System for Camellia sinensis and Optimization Using Orthogonal Design

TRAP markers is widely used for its simplicity, repeatability and high efficiency, however, the technology is scarcely used in the study of tea plant, and tea tree TRAP-PCR system optimization has not been reported. Orthogonal design was applied for optimizing 5 factors （Mg2＋, Taq DNA polymerase, dNTP, fixed primer, arbitrary primer） in the TRAP-PCR amplification system at 4 levels respectively. The best level of each factor were selected, a suitable TRAP-PCR reaction system was established, namely 20 p.L reaction system containing 2μL 10× PCR Buffer （Mg2＋ free）, 50 ng DNA, 1.75 mmo/L Mg2＋, 0.50 U Taq DNA polymerase, 0.20 mmol/L dNTP, 0.20 mmol/L arbitrary primer and 0.2 mmol/L fixed primer. TRAP polymorphism was studied using 16 tea germplasm.