基于HRM技术的大白菜InDel标记高效检测技术构建及其应用  

Construction and Usage of High Efficient InDel Marker Detecting Method of Chinese Cabbage Based on High-Resolution Melting (HRM)

作　　者：张志刚 司立英 赵智中 王荣花 王立华 李巧云 刘栓桃 Zhang Zhigang;Si Liying;Zhao Zhizhong;Wang Ronghua;Wang Lihua;Li Qiaoyun;Liu Shuantao(institute of Vegetables and Flowers,Shandong Academy of＇Agricultural Scie~wes/Shandong Bratu~.h of National Center jot Vegetables hnprovemer,t/SharMong Key Laboratory jbr Biology of Greenhouse Vegetables,Jinan 250100,CTdna;Liaocheng Dongchangju Agricultural Bureau,Liaocher~g 252000,China)

机构地区：[1]山东省农业科学院蔬菜花卉研究所/国家蔬菜改良中心山东分中心/山东省设施蔬菜生物学重点实验室,山东济南250100 [2]聊城市东昌府区农业局,山东聊城252000

出　　处：《山东农业科学》2018年第10期128-133,共6页Shandong Agricultural Sciences

基　　金：山东省自然科学基金项目(ZR2015YL073);山东省重点研发社会公益类项目(2017GNC10124);山东省农业科学院农业科技创新工程项目(CXGC2016A11);山东省泰山学者计划项目(2016-2020)

摘　　要：高分辨率熔解曲线(high-resolution melting,HRM)分析技术是基于核酸小片段之间的GC含量差异而导致双链核酸解链温度差异的一门检测技术,更多地被用于特定类型的单碱基突变筛查,很少见用于插入/缺失突变的检测。本研究从前期鉴定的可以有效区分大白菜自交系He102与06-247的593对多态性InDel引物中筛选到271对特异性好、呈共显性分布的InDel引物,采用软件TMUtility_1.5预测了差异目标片段的熔解温度(Tm)值,发现63对标记的Tm差值满足HRM分型的灵敏度(差值绝对值≥0.2℃)要求,进一步采用梯度PCR技术对上述63对引物进行PCR扩增和HRM分型,从中鉴定出20对适宜进行HRM分型的InDel标记。构建了适宜LightScanner-96高分辨率熔解曲线分析仪分析的高效检测体系,并在大白菜种质基因型精准鉴定、杂交种纯度高效鉴定方面得到成功应用。本研究开发的适宜HRM分型的InDel标记还可以作为一种标记储备用于构建遗传连锁图谱和DNA指纹图谱、鉴定杂交种纯度和真实性、关联分析、分子标记辅助育种等。基于此构建的高通量检测方法也为其它动植物材料InDel标记的高通量检测和应用提供借鉴。High -resolution mehing （HRM） curve analysis assay has been investigated for rapid detec- tion of sequence variants based on differences in GC content and the mehing profile. It was commonly used for special kind of single nucleotide mutation scanning, while less commonly used for detection of InDel muta- tions. At present study, 271 co -dominant InDel markers with good specificity were screened out from 593 fomlerly verified polymolphic InDel markers that could effectively distinguish Chinese cabbage inbred lines Hel02 and 06 -247. The mehing temperature （Tin） of all different target fragments was predicted by software TMUtilityl. 5. Only 63 pairs of codominant markers, whose Tm difference （absolute value ≥0.2℃ ） waspotentially suitable for detection sensitivity of HRM analysis, were screened out. A gradient PCR strategy was further used to test the best annealing temperature and HRM genotyping effect. As a result, 20 pairs of codominant markers were shown to be practically feasible for genotyping by HRM analysis. A set of high efficient detection method was constructed for LightScanner -96 instrument and used for Chinese cabbage geureplasm accurate identification and hybrid purity detection. The identified InDel markers suitable for HRM based geno- typing analysis could also be used as a kind of marker reserve for constructing genetic linkage map, DNA fin- gerprint, identifying the purity and authenticity of hybrid, COxTelation analysis, and molecular marker assisted selection bleeding and so on. The practiced high efficient InDel marker detection method would provide references for the high - throughput detection and application of other animal and plant materials InDel markers.

关 键 词：大白菜 插入/缺失标记 高分辨率熔解曲线分析 高效检测技术 

分 类 号：S634.1[农业科学—蔬菜学]