利用差减链式反应构建白菜基因组代表性文库  

Construction of Brassica campestris Representative Library Using Differential Subtractive Chain

作　　者：武剑[1] 冯艳丽[1] 和江明[2] 王晓武[1] 

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081 [2]云南省农业科学院园艺作物研究所,昆明650205

出　　处：《农业生物技术学报》2009年第2期307-311,共5页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究规划(973)项目(No.2006CB101606);中国农业科学院作物科学研究所院所基金基本科研业务费专项(No.082060302-09)资助

摘　　要：构建多态性比例高的基因组代表性文库是提高芯片和测序基础上的标记技术效率的关键。通过差减链式(differen-tial subtractive chain,DSC)反应,去除同一物种内的不同栽培种群间不具多态性的片段,能够提高基因组代表性文库中多态性片段的比例。研究利用白菜类作物(Brassica campestris)8个栽培种群的材料构建testerDNA样品,利用水菜(B.campestris ssp. nipposinica)DNA作为driver样品。这两个DNA样品经EcoRⅠ/MseⅠ酶切,酶切产物连接不同的接头。3遍DSC反应后,将反应产物连接到pDONR201载体中,构建基因组复杂性降低文库。分2批随机取其中20个单克隆和95个单克隆,分别进行Southern斑点杂交,结果分别显示9个和98个多态性片段,其比例分别为45%和61%,较之前关于多态性芯片技术的相关研究中15%～17%的多态性克隆比例大大提高。High polymorphism genome representative library is the key for an efficient molecular marker technique based on mieroarray or sequencing. A genome representative libray with relatively high polymorphism ratio was developed by differential substractive chain （DSC）. Nine accessions belonging to different Brassica campestris cultivar groups were used, eight of the nine accessions were mixed as tester DNA, while one accession mizuna （B.campestris ssp. nipposinica） was used as driver. The two DNA samples were both digested by EcoR Ⅰ/Mse Ⅰ before ligated to different adapters. Three rounds DSC were performed before the product was ligated to the Gateway^TM vector pDONR201. Southern Dot blots were proformed by tester or driver probes using two randomly selected batches of clones （20 and 95 clones）, respectively. The results showed 9 clones in the first batch and 58 clones in the second batch were polymorphic, with polymorphism rate of 45% and 61% respectively, indicating that the constructed library has a significantly higher polymorphism rate than those in previous reported research （15%- 17%） by diversity array technology （DART）.

关 键 词：分子标记 差减链式反应 基因组复杂性降低文库 多态性比例 

分 类 号：S188[农业科学—农业基础科学]