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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (10): 173-178.doi: 10.11924/j.issn.1000-6850.2011-2748

• 林学 园艺 园林 • 上一篇    下一篇

光萼荷属植物SRAP-PCR反应体系建立与引物筛选

张飞 王炜勇 张智 葛亚英 沈福泉 郁永明   

  • 收稿日期:2011-09-20 修回日期:2012-01-11 出版日期:2012-04-05 发布日期:2012-04-05
  • 基金资助:

    石羊河下游荒漠绿洲过渡带固沙植被退化与土壤演变的数量关系研究;浙江省自然科学基金项目;浙江省重大科技专项;浙江省公益性技术应用研究计划;杭州市种子种苗专项;浙江省农科院创新能力提升工程项目

Establishment of SRAP-PCR reaction system of Aechmea species and screening for polymorphic primers

  • Received:2011-09-20 Revised:2012-01-11 Online:2012-04-05 Published:2012-04-05

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摘要：

为了建立光萼荷属植物(Aechmea) SRAP-PCR反应体系，为今后光萼荷属植物种质资源研究提供技术支持，本研究通过L16(45)正交试验设计，对光萼荷属植物SRAP反应体系中的Mg2+、dNTPs、Taq DNA聚合酶、引物和模板DNA浓度等5个因素进行优化实验，并筛选多态性SRAP引物组合。结果表明，光萼荷属植物的最佳SRAP反应体系为1.50 mmol/L Mg2+、400 μmol/L dNTPs、1.5 U Taq DNA聚合酶、15 μmol/L引物、30 ng模板DNA及1×PCR buffer。各因素对SRAP-PCR扩增反应结果影响的差异较大，依次为模板DNA>Taq DNA聚合酶>dNTPs>引物>Mg2+。从56对SRAP引物组合中筛选出51对扩增条带清晰、多态性丰富的SRAP引物组合，多态性引物比率达90%以上。通过不同光萼荷属植物和不同引物组合对该反应体系进行验证，均获得了多态性丰富、条带清晰的扩增图谱，表明本研究建立的光萼荷属植物SRAP-PCR反应体系稳定可靠。

关键词: RAPD, RAPD

Abstract:

The aim was to establish SRAP-PCR reaction system and provide a new tool for future research on Aechmea germplasm. A orthogonal design of L16(45) was used to optimize SRAP-PCR reaction system for Aechmea species with five factors, namely Mg2+, dNTPs, primers, Taq DNA polymerase and template DNA, and SRAP primer combinations were screened for polymorphism. The result showed that a suitable SRAP-PCR reaction system for Aechmea species was 1.50 mmol/L Mg2+, 400 μmol?/L dNTPs, 1.5 U Taq DNA polymerase, 15 μmol/L primer combination, 30 ng template DNA and 1× PCR buffer. In addition, each factor in SRAP-PCR reaction system had different effects on the amplified patterns and the descending order was DNA > Taq DNA polymerase > dNTPs > primer > Mg2+. A total of 51 polymorphic SRAP primer combinations were screened out from 56 SRAP primer combinations, with the polymorphic primer ratio more than 90%. The optimized SRAP-PCR reaction system was identified by different genomic DNAs of Aechmea species and different SRAP primer combinations; as a result, the amplified pattern with rich polymorphism and clear bands was obtained. It proved that the established SRAP-PCR reaction system for Aechmea species was steady and reliable.

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