高通量提取西红柿基因组DNA方法探究及其在SNP分型中的应用  

A High Throughput Tomato Genomic DNA Extraction Method and Its Application in SNP Genotyping

作　　者：王海英 韩德俊[2] 李晓蕊 WANG Haiying;HAN Dejun;LI Xiaorui(College of Horticulture,Northwest Agriculture and Forestry University,Yangling,Shaanxi 712100;College of Agronomy,Northwest Agriculture and Forestry University,Yangling,Shaanxi 712100;Agriculture,Rural and Water Administration of Xi’an Hi-tech Industries Development Zone,Xi’an 710077)

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100 [2]西北农林科技大学农学院,陕西杨凌712100 [3]西安高新区农业农村和水务局,西安710077

出　　处：《中国农学通报》2023年第9期45-51,共7页Chinese Agricultural Science Bulletin

基　　金：陕西省烟草公司项目“烟草赤星病生物防治与生态调控技术研发与应用”(KJ-2021-02)。

摘　　要：随着植物分子生物学的发展,植物分子试验对于大规模样品DNA提取的需求越来越大。本研究旨在开发快速、简便、高通量的DNA提取技术。以西红柿叶片为试验材料,用96孔盒对西红柿叶片进行冷冻干燥及粉碎,基于改良的SDS提取DNA方法,采取排枪“两步加样一步抽提”,简化提取步骤,实现DNA的高通量提取。该方法提取DNA样品的平均浓度在138.6 ng/µL左右,满足一般性的PCR扩增要求;琼脂糖凝胶电泳结果显示绝大部分样品泳道可见清晰完整的DNA条带,无明显降解,并伴有少量的RNA污染。利用3个等位基因特异的定量PCR基因分型系统(AQP)分子标记对192个样本进行检测,结果显示3个标记的整体检测结果良好,均能有效区分样品间的不同等位基因位点。样品的有效荧光值占比在97%~99%之间,可以用于分子标记检测或遗传群体的基因分型。本研究在前人基础上探讨了一种简便、快速、高通量的西红柿基因组DNA提取方法,对于提取其他植物叶片组织DNA同样适用,并且该方法能够成功应用于AQP等其他类似的荧光分子标记检测,为大规模分子标记检测等实验DNA样品准备提供了借鉴。With the development of plant molecular biology,there is a growing demand for DNA extraction from large-scale samples in plant molecular experiments.In the study,we aim to develop a rapid,simple and high-throughput DNA extraction method.Tomato leaves as test materials were freeze-dried and crushed in a 96-hole box.To simplify the extraction steps and achieve high-throughput DNA extraction,we used the improved SDS-DNA extraction method by“two-step sampling and one-step extraction”.The average concentration of DNA sample extracted by this method was about 138.6 ng/mL,which met the need of PCR amplification.Agarose gel electrophoresis showed that most of the samples had clear and complete DNA bands without obvious degradation,but accompanied by some RNA.A total of 192 samples were detected by using 3 allele-specific quantitative PCR(AQP)markers.The results showed that the overall detection results of the 3 markers were good,and all of them could effectively distinguish different allelic loci between the samples.The effective fluorescence value of samples detected ranged from 97%to 99%,such DNA samples could satisfy molecular marker detection and genetic population genotyping.In this study,a simple,rapid and high throughput method for DNA extraction from tomato was developed,which was also suitable for DNA extraction from other plant leaf tissues.This method could be successfully applied to AQP and other similar fluorescent molecular marker detection,and provide reference for large-scale molecular marker detection and other experimental DNA sample preparation.

关 键 词：西红柿 改良的SDS方法 高通量提取DNA 琼脂糖凝胶电泳 AQP分子标记检测 

分 类 号：S641.2[农业科学—蔬菜学]