适于SSR-PCR的农作物基因组总DNA高通量提取方法的建立

[目的]建立一种新的、通用、快速的农作物基因组总DNA提取方法,为应用分子标记技术研究遗传多样性及变异提供方便.[方法]分别从甘蔗、水稻、玉米、花生和大豆5种作物中提取基因组DNA,用琼脂糖凝胶电泳对所提取的DNA质量进行检测,然后每种作物分别选取两对SSR引物进行PCR扩增,用聚丙烯酰胺凝胶电泳检测扩增结果.[结果]采用该提取方法,不同作物产生的纯DNA在100.0~160.0 g/g,提取DNA的A260/A280比率在1.80~1.95.此外,电泳凝胶未出现可见的RNA污染,每个作物基因组DNA单一条带非常清晰,DNA结构完整,质量较高,适用于分子标记的研究.应用SSR分子标记进行PCR扩增,重复性、稳定性好,经琼脂糖凝胶电泳检测,条带清晰且分辨率较高.[结论]建立的DNA提取方法为直接将叶片剪碎后装入EP管中,加入提取缓冲液进行DNA提取,省去了用液氮研磨的过程.与传统的DNA分离方法相比,新建立的DNA提取方法具有高通量、简单、快速、经济效益高等优点.

A method for high throughput DNA extraction from crop materials for SSR-PCR amplification

【Objective】A novel， rapid， and universal method for DNA extraction from crops was developed to accelerate the studies on the genetic diversities and variations using various molecular markers. 【Method】 The DNA isolated from five crop varieties， viz.， sugarcane， rice， maize， peanut， and soybean using the method this paper account， and the total DNA was detected by agarose gel electrophoresis. Then PCR was performed using 2 pairs of SSR primer for each crop and the products were visualized on polyacrylamide gel electrophoresis. 【Result】The presented method yielded pure DNA ranged from 100.0-160.0  g per gram of material depending upon the plant type. DNA samples with A260/A280 ratio ranged from 1.80 to 1.95. Furthermore， there was no visible RNA contamination in electrophoresed gels and the single bands of genomic DNA isolated from each crop were highly distinct. The DNA extracted from different crops showed high quality and reliability， therefore the DNA was ideal for marker applications. The PCR reactions using the simple sequence repeat （SSR） markers showed high repeatability， stability and the amplified bands were clear and distinct on the agarose gel. 【Conclusion】For this DNA isolated method， the leaf pieces were put into PCR tubes followed by the addition of DNA extraction buffer， saved the process of grind using liquid nitrogen. Compared to other traditional DNA extraction methods and DNA isolation kits， this method is high throughput， simple， rapid， and very economic.