摘要
以蒙古冰草(Agropyron mongolicum var.mongolicum)DNA为模板,利用单因素与正交试验设计相结合的方法,对影响冰草SSR-PCR体系的dNTP浓度、Mg2+浓度、引物浓度、模板DNA浓度和Taq DNA聚合酶用量5个因素进行优化。结果表明,冰草SSR-PCR的最佳体系为:dNTP浓度250μmol·L^–1、Mg^2+浓度2.25 mmol·L^–1、引物浓度0.60μmol·L^–1、模板DNA浓度40 ng·μL^–1和Taq DNA聚合酶用量0.75 U,其余为ddH2O,反应总体积为20μL。本研究筛选出适于冰草SSR-PCR的最佳反应体系,可提高冰草SSR扩增条带的清晰度和可靠性,这为后续深入开展冰草的遗传作图、重要性状QTL定位及分子标记辅助育种等研究奠定了基础。
Using wheatgrass DNA as a template,both single-factor and orthogonal-design methods were used to optimize the concentrations of five components(dNTPs,Mg2+,DNA template,primers,and Taq DNA polymerase)that influence marker amplification in simple sequence repeat(SSR)-PCR systems.The optimal SSR-PCR system for wheatgrass included 250μmol·L^–1 dNTP,2.25 mmol·L^–1 Mg^2+,0.60 mmol·L^–1 for each primer,40 ng·μL^–1 DNA,and 0.75 U Taq DNA polymerase,with a total reaction volume of 20μL.This optimization,which could improve the definition and reliability of SSR bands,establishes a foundation for genetic mapping in wheatgrass,as well as for the localization of important QTLs and,thus,molecular marker-assisted breeding.
作者
于肖夏
杨东升
于卓
卢倩倩
吴国芳
李景伟
张明飞
YU Xiaoxia;YANG Dongsheng;YU Zhuo;LU Qianqian;WU Guofang;LI Jingwei;ZHANG Mingfei(Agronomy College,Inner Mongolia Agricultural University,Hohhot 010019,Inner Mongolia,China)
出处
《草业科学》
CAS
CSCD
2020年第9期1760-1769,共10页
Pratacultural Science
基金
国家自然科学基金项目(31660681)。
