新疆野苹果SSR-PCR反应体系的优化  

Optimization of SSR reaction system in Malus sieversii

作　　者：刘遵春[1,2] 苗卫东[2] 刘大亮[1] 陈学森[1] 

机构地区：[1]山东农业大学园艺科学与工程学院/作物生物学国家重点实验室,山东泰安271018 [2]河南科技学院园艺园林学院,河南新乡453003

出　　处：《广东农业科学》2012年第4期97-100,共4页Guangdong Agricultural Sciences

基　　金：国家自然科学基金(30871679);国家"863"计划(2006AA100108);山东省农业良种工程项目

摘　　要：以4个新疆野苹果株系为试材,利用CTAB法提取DNA,对影响SSR-PCR扩增结果的主要因子设计了多梯度的优化试验。结果表明:新疆野苹果SSR-PCR反应体系(25μL)中含Taq DNA聚合酶0.5 U、模板DNA 5.0 ng、dNTPs 0.2 mmol/L、引物0.2μmol/L、Mg2+1.0 mmol/L、退火温度为60℃时效果最佳。最佳扩增程序为:94℃预变性2 min,94℃变性30 s,65℃退火1 min,72℃延伸1 min,4个循环;94℃变性30 s,60℃退火1 min,72℃延伸1 min,30个循环;72℃延伸5 min,4℃保存。利用此反应体系对30份新疆野苹果进行SSR-PCR扩增和电泳检测,扩增谱带清晰且多态性较好,表明该体系适用于新疆野苹果的基因连锁图谱构建和QTL定位。DNA of four Malus sieversii accessions were distilled by CTAB.An optimal experimental design was employed to evaluate five factors （template DNA,Mg2＋,dNTPs, Taq DNA polymerase and primer） at five different concentrations.The results indicated that the optimal SSR-PCR reaction system of terminal volume 25 ILL consisted of 0.5 U Taq DNA polymerase,0.2 txmol/L primers,5 ng template DNA,0.2 retool/ L dNTP and 1.0 mmol/L Mg2~.The suitable amplification temperature profiles were as following：2 vain at 9422 for pre-denaturing, followed by 4 cycles of 30 s at 9422 for denaturing,1 rain at 6522 for anncaling,1 rain at 7222 for extending,then followed by 30 cycles of 30 s at 9422 for denaturing,1 min at 6022 for annealing,1 min at 7222 for extending, and finally kept the reaction mixture at 422 after a final extension step of 7222 for 5 min.Using the above optimal SSR-PCR reaction system,SSR fragments of 30 Malus sieversii accessions were obtained.The clear and abundant polymorphism indicated this reaction system was suitable for construction gene linkage map and QTL mapping.

关 键 词：新疆野苹果 SSR-PCR 优化 

分 类 号：Q78[生物学—分子生物学]