| 刘遵春,苗卫东,刘大亮,陈学森.新疆野苹果SSR-PCR 反应体系的优化[J].广东农业科学,2012,39(4):97-99 |
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新疆野苹果SSR-PCR 反应体系的优化 |
| Optimization of SSR reaction system in Malus sieversii |
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| DOI： |
| 中文关键词: 新疆野苹果 SSR-PCR 优化 |
| 英文关键词: Malus sieversii SSR-PCR optimization |
| 基金项目: |
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| 摘要点击次数: 1237 |
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| 中文摘要: |
| 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（25 μL）中含Taq DNA聚合酶0.5 U、模板DNA 5.0 ng、dNTPs 0.2 mmol/L、引物0.2 μmol/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。 |
| 英文摘要: |
| 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. |
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