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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。
Aiming at the main aquaculture species of Cyprinus carpio, a target region amplified polymorphism (TRAP) molecular marker reaction system was designed. The parameters influencing the TRAP reaction system including Mg2 +, dNTPs, Taq DNA polymerase, template DNA and primer concentration were optimized, and a stable and reproducible TRAP-PCR reaction system was established. In 15μlPCR reaction system, the concentration of Mg2 + was 1.5mmol / L, the concentration of dNTPs was 0.35mmol / L, the concentration of two random primers was 3 pmol / L, the concentration of primer was 10 pmol / L, the DNA template was 60ng, Taq DNA polymerase 1.0 U. The TRAP reaction program of common carp was: 1 cycle at 94 ° C for 4min; 5 cycles of 94 ° C for 45s, 35 ° C for 45s and 72 ° C for 1 min; 35 cycles of 94 ° C for 45s, 53 ° C for 45s and 72 ° C for 1 min; , 1 cycle. The establishment of this optimization system provides a new molecular marker for the future research on genetic diversity, germplasm identification, genetic linkage map and genetic relationship among common carp populations.