半滑舌鳎3D-BAC池构建及性别连锁标记的物理定位  

Construction of 3D-BAC Pools and Physical Location of Sex-linked Markers in Half-smooth Tongue-sole(Cynoglossus semilaevis)

作　　者：张俊杰[1,2,3] 张文婷[1] 高峰涛[1] 董忠典[1] 张丽燕[1] 刘琨[1] 邵长伟[1] 陈松林[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]新疆农业大学动物科学学院,乌鲁木齐830052

出　　处：《农业生物技术学报》2015年第2期141-150,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金重点项目(No.31130057);国家高技术研究发展计划(863)项目(No.2012AA092203和No.2012AA10A403-2);山东省泰山学者项目

摘　　要：半滑舌鳎(Cynoglossus semilaevis)属于雌配子异形染色体性别决定机制(ZW/ZZ),其W染色体包含的大量重复序列阻碍了W染色体序列的精确组装。目前只有物理图谱可以有效地解决由重复序列所造成的组装难题。本研究在已有物理图谱和遗传图谱的基础上,利用3D(three-dimensional)-BAC(bacterial artificial chromosome)池构建和PCR筛选策略进行了性别连锁标记的物理定位。首先将所选44个384孔板的BAC克隆进行接种和预培养,再接种到4个96孔深孔板中。将每两个384孔微孔板的BAC克隆分别汇集获得相应的板池、行池和列池,在提取DNA后,再将相应板池的部分DNA混合得到超级池。最终构建的3D-BAC池,共包括22个超级池和440个基池,覆盖半滑舌鳎基因组4.2倍,完成一个阳性超级池的基池筛选只需24个反应(20个BAC基池和4个对照)。然后选用159个半滑舌鳎性别连锁标记对这些克隆池进行两步PCR筛选。在对超级池的筛选中,有142个标记获得阳性扩增。对这些阳性超级池进行基池筛选后,最终有100个标记得到准确定位。定位到半滑舌鳎物理图谱的84个重叠群(contigs)和20个单个克隆(singletons)上,共计1 760个克隆,包含有21 927个共享条带,物理长度为42.4Mb,覆盖半滑舌鳎基因组约5.3%;确定一个定位关系(contig与标记之间的)平均使用了2.2个克隆。在这些标记中,有17个同时定位到了两个或两个以上的contigs上,有11个标记同时定位到contigs和singletons上。另外有19个contigs定位了2个或2个以上标记。这些性别连锁标记的物理定位将有助于半滑舌鳎性染色体基因组序列的精确组装。The chromosome sex determining mechanism of half-smooth tongue sole （Cynoglossus semilaevis） is heteromorphism in female gamete, and the large amount of repetitive sequences, W chromosome contains, impede the accurate assembly of W chromosome sequence, which can only be solved by physical map effectively, at present. Based on the available physical map and genetic map, physical location of sex linked markers was performed in this study using the strategies of 3D（three-dimensional）-BAC（bacterial artificial chromosome） pool construction and PCR screening. Firstly, the BAC clones of 44 384-well microtiter plates were inoculated and precultivated with new 384-well plates, and then inoculated with four 96 deep-well plates. The plate, row and column pools were obtained through pooling the BAC clone cultures of each two 384-well microtiter plates, and then DNA were extracted and the super pools were obtained by mixing some DNA of corresponding plate pools. Eventually, 3D-BAC pools including 22 BAC super pools and 440 corresponding matrix pools were constructed, which represented 4.2 genome coverage of C. semilaevis. Only 24 reactions （20 BAC matrix pools and 4 controls） were needed to complete the screening on matrix pools of a positive super pool. Then two-step PCR screening was performed on these pools with 159 sex-linked markers, and 142 markers obtained positive amplifications in the screening of super pools. Finally, a total of 100 markers were accurately located in the screening of matrix pools of these positive super pools, and they were located in 84 contigs and 20 singletons of the C. semilaevis physical map, which included a total of 1 760 clones and 21 927 consensus bands. Their physical length was 42.4 Mb, covering 5.3% of the genome of C. semilaevis. On average, 2.2 clones were used to determine a location relationship between a contig and a marker. Among these markers, 17 markers were simultaneously located in 2 or more than 2 contigs, and 11 markers were simultaneously located in contigs a

关 键 词：半滑舌鳎 物理图谱 3D-BAC池 性别连锁标记 物理定位 

分 类 号：S66[农业科学—果树学]