摘要
甘肃金鳟是中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:100ng基因组DNA,3U EcoR I37℃酶切3h,再Mse I65℃酶切5h;然后用1U的T4连接酶连接12h,选扩25μL PCR反应体系中Mg2+2.0mmol/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;最后,筛选出了适宜甘肃金鳟品种分析的13对选择性引物。
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 concentration,denaturation time and annealing temperature.Then the AFLP reaction system was established.100 ng genomic DNA was digested for 3 h at 37℃ with 34 U EcoR I and then 65℃ for 5 h with 4 U Mse I ,respectively;the product was ligated for 12 h with 1 U ligase and was pre-amplificated;the products of pre-ampification was diluted 30 times,8：1 （w：w） Mse Ⅰ/EcoR Ⅰ selective 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.
出处
《中国农学通报》
CSCD
北大核心
2011年第7期400-404,共5页
Chinese Agricultural Science Bulletin
基金
科技部成果转化项目"三倍体虹鳟网箱养殖标准化技术集成示范"(2009GB2G100373)
甘肃省自然科学基金项目"电子标签在甘肃金鳟家系选育中的应用"(096RJZA031)
