菰(Zizania latifolia)ISSR反应体系的建立及优化  

Establishment and Optimization of an ISSR Reaction System for the Wild-rice Zizania latifolia Griseb(Turcz)

作　　者：吴国林[1,2,3] 王惠梅[3] 黄奇娜[3] 奉保华[3] 江绍琳[1] 吴建利[3] 黄长干[1] 江绍玫[2] 

机构地区：[1]江西农业大学,南昌330045 [2]江西财经大学,南昌330045 [3]中国水稻研究所,杭州310006

出　　处：《植物遗传资源学报》2014年第6期1395-1400,共6页Journal of Plant Genetic Resources

基　　金：国家自然科学基金(31160271);江西省科技支撑项目(20133BBF6005)

摘　　要：菰是一种稻族野生资源,水生或沼生,在我国有着广泛的分布。野生菰群体不仅是一种重要的育种基因资源,同时还有着巨大的生态作用。为建立适宜于菰的ISSR反应体系,以菰DNA基因组为模板对ISSR反应体系中的主要影响因子进行优化。采用单因素变量法在12个不同梯度水平上设计正交试验针对体系中d NTPs浓度、Mg2+浓度、Taq聚合酶浓度、引物浓度、模板DNA浓度及引物退火温度等6个因子进行优化,确定了在20μL ISSR反应体系中各组分的最适因素水平分别为:3.0 mmol Mg2+(10×Buffer),0.5 mmol d NTPs、1.0μmol引物浓度、0.24 U/μL Taq聚合酶、1.5 ng/μL DNA模板以及55℃退火温度。应用该优化体系对80个菰材料进行扩增,证实了该体系的适用性和稳定性,为菰遗传资源的鉴定、评价与利用提供了技术支撑。Wild rice Zizania latifolia Griseb（Turcz）,an aquatic/wetland genus,is a member of tribe Oryzeae widely distributed across China.Z.latifolia is not only an important genetic resource for crop improvement but also contributes significantly to the ecosystem.However,studies on Z.latifolia have been slow because of the lack of methodology and knowledge in all aspects.In this work,we first tried to develop and establish an optimized ISSRPCR reaction system for genotyping of Z.latifolia using the single factor variable method in which 6 factors（d NTPs,Mg^2＋,Taq polymerase,primer,template DNA and primer annealing temperature） at 12 different concentrations or gradients were tested.We finally set up an optimized 20 μL reaction system as follows：0.5 mmol d NTPs,3.0 mmol Mg^2 ＋（10 × Buffer）,0.24 U/μL Taq polymerase,1.0 μmol primer,1.5 ng/μL DNA template and annealing temperature at 55 ℃.The optimized ISSR system was successfully applied in the amplification of 80 Z.latifolia individual samples,thus,the adaptability and stability of the system were testified.

关 键 词：菰 ISSR 居群 

分 类 号：S511.9[农业科学—作物学]