鲤TRAP分子标记反应体系的建立与优化

Establishment and Optimization of Reaction System for TRAP Markers in Common Carp (Cyprinus carpio)

针对鲤(Cyprinus carpio)这一主要水产养殖品种设计了靶位区域扩增多态性(target region amplified polymorphism,TRAP)分子标记的反应体系,对影响TRAP反应体系的各参数,包括Mg2+、dNTPs、TaqDNA聚合酶、模板DNA和引物浓度进行了优化,建立了适合鲤的、稳定、可重复的TRAP-PCR反应体系。在15μlPCR反应体系中,Mg2+浓度为1.5mmol/L、dNTPs浓度为0.35mmol/L、两个随机引物浓度均为3pmol/L、固定引物浓度为10pmol/L、含DNA模板60ng、TaqDNA聚合酶1.0U。鲤的TRAP反应程序为:94℃4min,1个循环;94℃45s,35℃45s,72℃1min,5个循环;94℃45s,53℃45s,72℃1min,35个循环;72℃10min,1个循环。这一优化体系的建立为今后进行鲤群体遗传多样性、种质鉴定、遗传连锁图谱及亲缘关系分析等方面的研究提供了新的分子标记。

The reaction system for TRAP makers of Common Carp （Cyprinus carpio）,one of the major cultured fishes,was designed. The factors of reaction system including Mg2 ＋ ,dNTPs,Taq DNA polymerase,DNA template and primer concentrations were optimized and a stable,repeatable TRAP-PCR system for Common Carp was established. The PCR was performed with a final volume of 15 μl reaction solution containing 1. 5 mmol /L of Mg2 ＋ ,0. 35 mmol /L of dNTPs,3 mmol /L of each unlabeled 700- and 800- arbitrary primers,10 pmol /L of the fixed primer,60 ng of DNA template and 1. 0 U Taq DNA polymerase. The TRAP reaction program consisted of a pre-denaturing of template DNA at 94℃ for 4 min,followed by 5 cycles of denaturing at 94℃ for 45 s,annealing at 35℃ for 45 s,prolonging at 72℃ for 1min,35 cycles of denaturing at 94℃ for 45 s,annealing at 53℃ for 45 s,prolonging at 72℃ for 1min,and a final prolonging at 72℃ for 10min. This optimized TRAP- PCR reaction system provided a new molecular marker for the future study of genetic diversity,germplasm identification,genetic linkage map construction and relationship analysis in Common Carp.