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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (19): 189-193.doi: 10.11924/j.issn.1000-6850.2012-0206

• 林学 园艺 园林 • 上一篇    下一篇

牡丹杂交品系SRAP-PCR反应体系优化及引物筛选

苏美和 赵兰勇   

  • 收稿日期:2012-01-29 修回日期:2012-03-07 出版日期:2012-07-05 发布日期:2012-07-05

Optimization for SRAP-PCR System in Paeonia suffruticosa Hybrids Selection of Primers

  • Received:2012-01-29 Revised:2012-03-07 Online:2012-07-05 Published:2012-07-05

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摘要：

通过研究牡丹杂交新品系的遗传多样性，解决其在牡丹品种分类体系中位置的问题。利用正交设计，从Mg2+、dNTPs、引物浓度、DNA聚合酶和不同模板DNA浓度5种因素4个水平来优化牡丹杂交品系SRAP-PCR反应体系，对引物进行筛选。建立牡丹杂交品系SRAP-PCR反应最佳体系(25 μL)为： 2.0 mmol/L Mg2+、1.5 U Taq酶、0.25 mmol/L dNTPs、2 ng/μL模板DNA、0.25 μmol/L引物；运用试验结果从100对引物中筛选出扩增条带清晰、多态性丰富的SRAP引物30对。优化体系的建立及引物的筛选，可为利用SRAP标记技术研究牡丹杂交品系的遗传多样性及亲缘关系提供技术基础和理论依据。

关键词: 贵州省, 贵州省

Abstract:

Researching genetic diversity of Paeonia suffruticosa hybrids is to ensure location of Paeonia suffruticosa hybrids in variety classification system. Paeonia suffruticosa hybrids genomic DNA from leaf tissue was isolated according to the SDS procedure. An orthogonal diagram as experimental design was applied to study the effects of four levels of Mg2+, dNTPs, primers, Taq DNA and genomic DNA polymerase respectively on the reaction system of SRAP, and the SRAP reaction system was optimized by analyzed effects of the concentrations of DNA template. The results showed that all factors had the significant effect on the result of SRAP-PCR. And a better amplification of SRAP-PCR was obtained with the reaction system for Paeonia suffruticosa containing 2.0 mmol/L Mg2+, 1.5 U Taq DNA polymerase, 0.25 mmol/L dNTPs, 2 ng/μL genomic templates DNA and 0.25 μmol/L primer in the total volume of 25 μL. At the same time, 30 primer combinations were selected with the optimized system among 100 primer combinations, which had abundant polymorphism bands. This study would be conducted to the optimized SRAP-PCR system and selection of primers which would play an important role in genetic diversity and genetic relationship in taxonomic hierarchies of Paeonia suffruticosa.