一种适宜高通量基因型鉴定的植物组织取样法及其应用  

A Plant Tissue Sampling Method Suitable for High-throughput Genotyping and Its Application

作　　者：仲威宇 朱晓斌 夏正俊 李艳华[2] 张丽辉 ZHONG Wei-yu;ZHU Xiao-bin;XIA Zheng-jun;LI Yan-hua;ZHANG Li-hui(School of Life Sciences,Changchun Normal University,Changchun 130032,China;Northeast Institute of Geography and Agroecology,The Innovative Academy of Seed Design,Chinese Academy of Sciences,Harbin 150081,China)

机构地区：[1]长春师范大学生命科学学院,吉林长春130032 [2]东北地理与农业生态研究所中国科学院种子创新研究院,黑龙江哈尔滨150081

出　　处：《长春师范大学学报》2023年第12期102-106,共5页Journal of Changchun Normal University

摘　　要：高效快速植物基因型鉴定是现代分子育种及分子生物研究中基因定位与克隆的前提条件,因而快速且高通量的取样技术有利于建立高效快速植物基因型鉴定技术体系。随着高通量测序技术的飞速发展,可对重要遗传材料进行重测序来进行全基因组范围内基因型鉴定,但因成本及鉴定周期等限制,该技术尚难应用于大群体遗传材料的图位克隆及基于基因型鉴定的分子育种。虽然目前已有多种不同的植物叶片取样方法、DNA提取方法,但步骤繁琐,耗时较长,或所需要特殊的仪器设备,其效率并不能满足现代高通量的技术需求。本研究以外直径为3.0 mm的塑料圆管结合木质牙签为简易的取样器,将大豆叶片均匀准确地取样至384深孔微孔板,同时优化了配套的DNA提取方法与步骤,建立了一整套的高效大豆植株基因型鉴定流程。本鉴定技术体系可大大提高工作效率,缩短鉴定周期。应用该技术流程,成功地对杂合型洐生后代群体1150个植株个体进行了基因型鉴定,确定了与黄化突变相关分子标记之间的联系,为进一步精细定位与克隆控制黄化突变基因奠定了基础。本研究所建立的植物组织高效取样法及基因型鉴定流程可扩展应用于水稻、玉米等多种作物的基因克隆研究与分子育种中。Efficient and rapid identification of plant genotypes is a prerequisite for gene localization and cloning in modern molecular breeding and molecular biology research.Therefore,the development of rapid and high-throughput sampling technology is essential for establishing an efficient plant genotype identification system.Although the rapid advancement of high-throughput sequencing technology has made it possible to resequence important genetic materials for genome-wide genotype identification,applying this technology to locus cloning of large quantity of genetic materials and molecular breeding based on genotype identification remains challenging due to cost limitations and lengthy identification periods.Additionally,existing plant leaf sampling methods and DNA extraction techniques are cumbersome,time-consuming,or require specialized instrumentation and equipment.Their efficiency does not meet the demands of modern high-throughput technology.In this study,we utilized a simple sampling device consisting of a 3.0 mm diameter plastic tube and a wooden toothpick to evenly and accurately sample soybean leaves into 384-deep-well microtiter plates.Simultaneously,we optimized the corresponding DNA extraction methods and steps,thereby establishing a comprehensive set of highly efficient genotype identification processes for soybean plants.This technological system significantly improves work efficiency while reducing the time required for identification.By implementing this process,we successfully genotyped 1150 individual plants from a heterozygous population of derived progeny.Furthermore,we identified the linkage between molecular markers associated with the yellowing mutation,laying the foundation for further fine localization and cloning of genes controlling this mutation.The efficient plant tissue sampling method and genotype identification process established in this study can be extended and applied to gene cloning research and molecular breeding of various major crops,including rice and maize.

关 键 词：DNA提取 高通量取样 384微孔深孔板 基因型鉴定 

分 类 号：Q331[生物学—遗传学]