新疆哈密瓜SRAP反应体系建立和优化

[目的]研究新疆哈密瓜SRAP反应的最佳体系,为进一步研究新疆哈密瓜分子标记辅助育种提供基础.[方法]采用均匀实验设计法对新疆哈密瓜SRAP - PCR反应体系的5个主要因子进行优化.[结果]优化后的SRAP - PCR反应体系扩增多态性高、带型清晰、稳定性好,是新疆哈密瓜SRAP扩增的最佳条件.利用该优化体系,对12个新疆哈密瓜地方品种基因组DNA进行SRAP扩增,大部分材料扩增出来的带清晰可辨、条带丰富、背景无干扰,满足分子标记应用的要求.[结论]20 μL的反应体系中各成分用量或浓度为:模板DNA 36 ng、Taq DNA聚合酶2.5U、dNTPs2.2 5 mmol/L、引物0.6μmol/L、Mg2+ 1.75 mmol/L和2μL10×PCR buffer.

Optimization of SRAP-PCR Reaction System in Hami Melon

【Objective】 The purpose of this program was to study the best system of Xinjiang Hami melon SRAP reaction,and provide a foundation for further studying of marker-assisted breeding of Xinjiang Hami melon.【Method】In this paper,the uniform design was used to optimize the sequence-related amplified polymorphism(SRAP) PCR reaction system for Xinjiang Hami melon,which involved 5 factors.【Result】 Application of this expansion of results are clear and reliable,which is the best choice of melon SRAP amplification conditions.By using this optimized system,SRAP-PCR reactions were performed on 12 Melon local varieties,most of which were amplified with a clear,legible,rich background without any interference,fitting for application of molecular markers.【Conclusion】20 μL reaction system components to the appropriate concentration or amount contain: template DNA 36 ng,Taq DNA polymeras 2.5 U,dNTPs 2.25 mmol/L,primers 0.6 μmol/L,Mg2+1.95 mmol/L and 10×PCR buffer 2 μL.