新疆野苹果SSR-PCR 反应体系的优化

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。

Optimization of SSR reaction system in Malus sieversii

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 μL consisted of 0.5 U Taq DNA polymerase,0.2 μmol/L primers,5 ng template DNA,0.2 mmol/L dNTP and 1.0 mmol/L Mg2+.The suitable amplification temperature profiles were as following:2 min at 94℃ for pre-denaturing,followed by 4 cycles of 30 s at 94℃ for denaturing,1 min at 65℃ for anncaling,1 min at 72℃ for extending,then followed by 30 cycles of 30 s at 94℃ for denaturing,1 min at 60℃ for anncaling,1 min at 72℃ for extending,and finally kept the reaction mixture at 4℃ after a final extension step of 72℃ 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.