Abstract：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 and singletons. In addition, 19 contigs were simultaneously located with 2 or more markers. The physical location of these sex-linked markers can promote accurate assembly of sex chromosomes sequences of C. semilaevis.
