罗非鱼TRAP分子标记反应体系优化设计方案的比较  

Comparison between single factor design and orthogonal design to optimize reaction system for TRAP markers in tilapia

作　　者：马庆男[1] 董在杰[1,2] 朱文彬[2] 苏胜彦[2] 张宁[1] 袁新华[1,2] 杨弘[1,2] 

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心农业部淡水渔业和种质资源利用重点实验室,江苏无锡214081

出　　处：《南方水产科学》2013年第1期28-34,共7页South China Fisheries Science

基　　金：现代农业产业技术体系建设专项资金项目(CARS-49);现代农业人才支撑计划项目(2130106)

摘　　要：靶位区域扩增多态性(target region amplification polymorphism,TRAP)是一种新型功能性分子标记,比较了L16(45)正交试验和单因素方法优化罗非鱼(Oreochromis spp.)TRAP反应体系。2种分析方法结果中dNTPs浓度、随机引物浓度、DNA浓度相同而镁离子(Mg2+)浓度和TaqDNA聚合酶浓度不同,不同因素间存在一定的交互作用。正交设计方法比单因素法更简单、科学、合理。该试验获得罗非鱼15μL TRAP最优反应体系为DNA模板浓度为60 ng、Mg2+浓度为1.5 mmol.L-1、dNTPs浓度为0.3 mmol.L-1、TaqDNA聚合酶0.5 U、随机引物浓度为7 pmol.L-1和固定引物浓度为10 pmol.L-1。该反应体系在4个罗非鱼群体中验证,表现出良好的稳定性和重复性,该反应体系的建立为TRAP标记应用于罗非鱼遗传多样性评价、种质鉴定、分子标记辅助育种等研究提供了新的手段。The target region amplification polymorphism （TRAP） marker is a novel functional molecular marker. In present study, we compared the optimization of the reaction system for TRAP markers in tilapia （Oreochromis spp. ） between L16 （45） orthogonal design and single-factor design. The resuhs of the two approaches showed that the concentrations of dNTPs, random primer and DNA were the same, while those of Mg2＋ and TaqDNA polymerase dosage varied, and interaction among different factors were observed. The orthog- onal design is simpler, more scientific and reasonable than single factor design. The optimal TRAP reaction system for tilapia includes 60 ng DNA template, 1.5 mmol.L-1 Mg2＋ , 0. 3 mmol-L-1 dNTPs, 0. 5 U TaqDNA polymerase, 7 pmol-L-1 random primers and 10 pmol-L-1 fixed primer. The stability and repeatability of the reaction system had been verified in 4 tilapia populations. The TRAP marker provides new insights into evaluation of genetic diversity, germplasm identification, molecular marker-assisted breeding and other related researches of tilapia.

关 键 词：罗非鱼 TRAP 正交设计 单因素试验 优化 

分 类 号：Q953.3[生物学—动物学]