黄瓜基于扩增子测序的TILLING技术体系的建立  

Establishment of TILLING Technology Platform Based on Amplicon Sequencing in Cucumber

作　　者：厉彦钢 王怡 张孟茹 娄群峰[1] LI Yangang;WANG Yi;ZHANG Mengru;LOU Qunfeng(College of Horticulture,Nanjing Agricultural University,Nanjing,Jiangsu 210095)

机构地区：[1]南京农业大学园艺学院,江苏南京210095

出　　处：《核农学报》2024年第12期2305-2311,共7页Journal of Nuclear Agricultural Sciences

基　　金：江苏省重点研发项目(BE2021357);种业振兴“揭榜挂帅”项目(JBGS2021070)。

摘　　要：定向诱导基因组局部突变(TILLING)技术能够对突变群体中的点突变进行快速筛选,而样品混池深度是影响其效率的主要因素。为提高TILLING的突变检测效率,本研究以甲基磺酸乙酯(EMS)诱变群体构建的突变体库为基础,对其中部分M2代植株进行突变性状观察,结果表明表型突变率为10.80%。同时利用已知矮化突变体与野生型进行混池,分别构建96、192、288、384四个不同深度的测序池,使用GATK软件进行突变检测,为对假阳性位点进行有效过滤,候选突变位点分别根据突变类型及归一化深度作为阈值两次进行过滤。结果表明,96~288倍的混池深度均能稳定检出预设的已知突变位点,并与Sanger测序结果相一致。与前人相比,本研究通过优化混池深度与过滤阈值,实现了更高混池深度的突变检测,并在黄瓜中建立了基于扩增子测序的TILLING技术体系,可实现更高通量地对大规模突变群体进行目标突变的鉴定。TILLING(Targeting Induced Local Lesions in Genomes)technique enables the rapid screening of point mutations within mutant populations.However,the mixing depth of sample pools is a critical factor affecting its efficiency.To enhance the efficiency of TILLING detection,an ethyl methylsulfonate(EMS)mutagenized populations were constructed and the mutant characters of some M2 plants were investigated.The results showed that the phenotypic mutation rate was 10.80%.Then,a known dwarfing mutant was pooled with wild types to construct four sequencing pools with varying depths:96,192,288 and 384.The Genome Analysis Toolkit(GATK)was used for mutation detection.To effectively filter false positive sites,candidate mutation sites were subjected to a two-step filtering process based on mutation type and normalized depth as thresholds.The results showed that the preset known mutation sites could be reliably detected in 96 to 288-fold pools,which was consistent with the results of Sanger sequencing.Compared with previous studies,a mutation detection with a greater mixing depth was achieved by optimizing the mixing depth and filtering thresholds.Meanwhile,a TILLING technology system based on amplicon sequencing in cucumber was established,which can facilitate throughput identification of target mutations in large-scale mutant populations.

关 键 词：黄瓜 扩增子测序 TILLING 生物信息学 突变检测 

分 类 号：S642.2[农业科学—蔬菜学]