玉米花粉粒直接PCR技术研究

利用成熟花粉粒制成悬液作为玉米基因组DNA模板直接进行PCR扩散，研究了不同花粉悬液浓度、花粉上清液对RAPD-PCR的影响，结果表明：花粉悬液浓度在4μg/50μl以上，用10碱基随机引物均能扩增出较清晰的PCR条带，且与叶片按改良Guidet法提取的DNA模板扩增的RAPD带型无明显差异，利用花粉悬液能有效地进行基因组DNA变异的RAPD分析和基因SCAR标记的检测。玉米单株花粉量可用于数百次以上的PCR反应，较叶片等植物组织用常规法提取DNA快速、简便、廉价，可有效地应用于以PCR为基础的植物基因组DNA变异，农艺性状的RAPD标记和分子标记辅助育种的研究。

The Studies on Direct PCR using Maize Pollen Grain Suspensions

Direct PCR amplifications were carried out using maize pollen grain suspension as template of genomic DNA. The effect on different pollen suspension concentrations and pollensupernatant to RAPD-PCR were studied. The results show that RAPD-PCR amplification obtained using a collection of decamer primers generated clear banding patterns in pollen grain suspension concentrations ranging from 4～100μg/50μl, and the RAPD profile between pollen suspension and leaves DNA extracted with improved Guidet method have not clearly different. The RAPD analysis of genomic DNA variation and identification of SCAR marker link to opposite gene op4 were carried out efficiently using pollen suspension. The amount of pollen grains obtained from individual maize plant can be used in up to several hundred PCRs. Direct PCR using pollen grain suspension was more rapid, simple and lower cost than that using leaves DNA extraction with others methods. These results suggest that direct PCR using pollen suspensions can be applied efficient