摘要
[目的]建立并优化老芒麦ISSR-PCR反应体系和扩增程序,以期为探讨老芒麦种质资源的遗传多样性提供科学依据。[方法]采用正交设计试验和单因素试验相结合的方法对老芒麦的ISSR-PCR的反应体系进行构建和优化,对影响ISSR-PCR扩增效果的Taq酶、模板DNA的浓度、Mg2+、dNTP和引物浓度等因素进行优化,同时对退火温度、循环次数和延伸时间进行筛选。[结果]在25μl体系中各反应物的最适含量为:0.2 mmol/L dNTP,0.2μmol/L ISSR引物,1.50 U Taq DNA聚合酶,2.5μl 10×PCR Buffer,1.5 mmol/L MgCl2和40 ng模板DNA;循环次数和延伸时间分别是35次和90 s。[结论]该研究建立并优化了老芒麦ISSR-PCR反应体系,通过2份老芒麦种质对所优化体系得验证,结果显示体系稳定,可用于老芒麦种质资源遗传分析。
[Objective] This study aimed to establish and optimize the ISSR-PCR reaction system and amplification process for Elymus sibiricus L.,to provide scientific basis for exploring the genetic diversity of E.sibiricus germplasm resources.[Method] Orthogonal design and single factor test were applied to establish the ISSR-PCR reaction system of E.sibiricus,optimize the affecting factors including Taq DNA polymerase,template DNA concentration,Mg2＋,dNTP,primer concentration,and screen the annealing temperature,number of cycles and extension time.[Result] The optimal reaction system for ISSR analysis contains 0.2 mmol/L dNTPs,0.2 μmol/L ISSR primers,1.5 U of Taq DNA polymerase,2.5 μl of 10×PCR Buffer,1.5 mmol/L MgCl2 and 40 ng of template DNA in 25 μl total volume;the amplification was conducted with 35 cycles and extension time of 90 s.[Conclusion] ISSR-PCR reaction system for E.sibiricus L.was established and optimized in this study,which was verified using two E.sibiricus germplasms,results show that the ISSR-PCR reaction system is stable and can be used for the genetic analysis of E.sibiricus.
出处
《安徽农业科学》
CAS
2012年第28期13709-13712,13719,共5页
Journal of Anhui Agricultural Sciences
基金
多年生牧草种质资源收集
编目与利用项目(NB2012-2130-135-33)
牧草种质资源保护项目
关键词
老芒麦
ISSR
正交试验
单因素试验
Elymus sibiricus L.
ISSR
Orthogonal experiment
Single factor test
