摘要
限制性酶切位点关联DNA测序技术(restriction-site-associated DNA sequencing,RAD-seq)是在二代测序技术基础上发展起来的一种简化基因组技术(reduced-representation genome sequencing,RRGS).RAD-seq利用限性核酸内切酶使基因组片段化,经过修饰后连接含标记的接头构建文库并进行测序.因其具有操作简单、实验成本低、通量高等优点,在分子生态学、进化基因组学、保护遗传学等领域得到应用.目前发展起来的RAD-seq技术主要包括利用稀有酶和物理打断方式进行基因组片段化的mbRAD、利用稀有酶和常见酶组合酶切的方式进行基因组片段化的ddRAD、利用IIB型限制性内切酶得到短片段文库的2bRAD、利用同裂酶和Illumina试剂盒建库的ezRAD和完全利用Nextera试剂盒建库的nextRAD.本文对每种RAD-seq技术的特点及其在昆虫种群遗传学、群落生态学、物种界定、系统发育和遗传连锁图谱构建等研究领域的应用进行了综述.
Abstract
Restriction-site-associated DNA sequencing (RAD-seq) is a technology of reducedrepresentation genome sequencing (RRGS),which is developed with next-generation sequencing.In terms of RAD-seq,genome is digested by restriction endonuclease.The digested fragments are ligated with barcoded adapters,and used to construct library for sequencing.With the advantages of simple operation,low experimental cost,and high throughput,RAD-seq is widely used in molecular ecology,evolutionary genomics and conservation genetics.Recently,five different RAD-seq methods including mbRAD,ddRAD,2bRAD,ezRAD and nextRAD have been developed.Of them,mbRAD uses a low frequency cutter and physical shearing to make genome into fragments.ddRAD uses a low frequency cutter and a high frequency cutter to digest genome.2bRAD relies on ⅡB-type restriction endonuclease to excise genomic sequences into short fragments.ezRAD uses isochimozer enzymes and Illumina library preparation kit.nextRAD entirely uses Nextera DNA library preparation kit.In this article,we reviewed the characteristics of each RAD-seq method and its application in entomology,such as population genetics,community ecology,species delimitation,phylogenetic reconstruction and construction of genetic linkage map.
基金项目
北京市自然科学基金(6162010)
国家自然科学基金(31472025)
国家重点基础研究发展计划项目(2013CB127600)
万方数据