黄绿蜜环菌SSR-PCR反应体系的优化及引物筛选  

Establishment and Primer Screening of the SSR-PCR Reaction System for Armillaria luteo-virens

作　　者：李颖[1] 谢占玲[1,2] 田飞[1] 贾贤卿 方慧[1] 夏明哲[1] 

机构地区：[1]青海大学生态环境工程学院,青海西宁810016 [2]青海省高原作物种质资源创新与利用重点实验室,青海西宁810016

出　　处：《北方园艺》2013年第16期114-119,共6页Northern Horticulture

基　　金：国家自然科学基金资助项目(31260021);青海省自然科学基金资助项目(2011-z-724);国家重点实验室培育基地青海省高原作物种质资源创新与利用重点实验室开放课题资助项目(2011-05)

摘　　要：以CTAB法提取的黄绿蜜环菌(Armillaria luteo-virens)DNA为模板,应用L16(45)正交实验设计,对Mg2+、dNTPs、引物和Taq DNA聚合酶、模板浓度5种因素进行SSR-PCR反应体系优化,建立了适合黄绿蜜环菌SSR-PCR反应的最佳体系并对退火温度进行检测。结果表明:在15μL反应体系中包括:1×PCR Buffer,100ng模板DNA,dNTPs 217μmol/L,引物0.5μmol/L,Taq DNA聚合酶0.75U,Mg2+1.3mmol/L。稳定性检测证明,该反应体系具有较高的稳定性和重复性,并从34对引物中共筛选出扩增条带清晰、多态性丰富的SSR引物8对。该体系的建立为今后利用SSR标记对黄绿蜜环菌遗传多样性研究、亲缘关系分析及种质资源鉴定等研究提供了依据。Taking DNA extracted from Armillaria luteo-virens by CTAB method as template,L16（45） orthogonal design was used to optimize the main factors affecting the SSR-PCR system,which were Mg2＋,dNTPs,primers,Taq DNA polymerase and template DNA concentration.It established the optimum SSR-PCR reaction amplification system of Armillaria luteo-virens,and based on the optimum SSR-PCR reaction amplification system annealing temperature was selected.The results showed that a 15 μL reaction system contained 1×PCR Buffer,100 ng template DNA,dNTPs 217 μmol/L,primer 0.5 μmol/L,Taq DNA polymerase 0.75 U,Mg2＋1.3 mmol/L.The reaction system was proved in good stability and reproducibility.At the same time,8 primer combinations were selected with the optimized system among 34 primer combinations,which had abundant polymorphism bands.This study could be used in analysis of genetic diversity,genetic relationship and fingerprint in Armillaria luteo-virens.

关 键 词：黄绿蜜环菌 SSR-PCR 体系优化 正交设计 引物 

分 类 号：S503.53[农业科学—作物学]