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.
出处
《广东农业科学》
CAS
CSCD
北大核心
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
