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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (7): 400-404.

• 水产 渔业 • 上一篇    下一篇

甘肃金鳟AFLP体系建立及应用

苏军虎 张艳萍 魏彦明   

  • 收稿日期:2010-09-10 修回日期:2010-10-29 出版日期:2011-04-05 发布日期:2011-04-05
  • 基金资助:

    甘肃省技术研究与开发专项计划项目

Establishment and Application of AFLP Reaction System in Gansu Rainbow Trout

  • Received:2010-09-10 Revised:2010-10-29 Online:2011-04-05 Published:2011-04-05

摘要：

甘肃金鳟是我国自主培育的虹鳟新品种，为进行其种质资源研究和遗传管理，以其尾鳍为试验材料,提取基因组DNA，对影响甘肃金鳟扩增片断长度多态性(AFLP)反应体系进行优化，包括模板DNA浓度、基因组酶切时间、选择性扩增中Mg2+、预扩增产物稀释倍数及选扩性引物M+3/E+3配比等进行比较分析，建立了适于甘肃金鳟的AFLP反应体系。即：100 ng 基因组DNA，3 U EcoR I 37℃酶切3 h，再 Mse I 65℃酶切5 h；然后用1 U的T4连接酶连接12 h, 选扩25 μl PCR反应体系中Mg2+2.0 mmol/L,预扩产物稀释30倍,选扩引物M+3/E+3配比为8∶1，所得产物经电泳和银染后可获得清晰条带，效果良好；筛选出了适宜甘肃金鳟品种分析的13对选择性引物。

关键词: 番茄, 番茄, 色差, 番茄红素, 主基因+多基因遗传

Abstract:

Oncorhynchus mykiss is an important reared freshwater species, which has been cultured for more than thirty years in Gansu Province, China. Gansu golden trout is a new breed developed in Gansu, which had pass the National Aquaculture Species Identification Committee identifying and was recommend as the species reared in the northwestern areas. In this study, the DNA was extracted from the caudal fin of Gansu rainbow trout, and the conditions for AFLP analysis were optimized, including DNA concentration, dNTPs concentration, primer concentrati- on, denaturation time and annealing temperature.Then the AFLP reaction system was established. 100 ng genomic DNA was digested for 3 h at 37℃with34 U EcoR I and then 65℃for 5 h with 4 U Mse I , respectively; the product was ligated for 12 h with 1U ligase and was pre-amplificated; the products of pre-ampification was diluted 30 times, 8∶1 (w∶w) MseⅠ/ EcoRⅠselective primers for selective amplification; the amplification patterns were obtained clearly and stably. Finally, thirteen AFLP primer combinations that are suitable for Gansu Rainbow Trout cultivar were screened.