| Half-smooth tongue sole(Cynoglossus semilaevis) is a traditional economic flatfish in China. It embraces both nutrient and economical value, but studies on its genome and sex chromosomes are rarely reported and its gene functions are yet to be revealed. A 2-step-3D-PCR screening system was successfully constructed based upon the female half-smooth tongue sole fosmid library. By screening half-smooth tongue sole's W-chromosome library, one pair of female-specific primers was obtained. Using the female-specific primers, a W-chromosome DNA fragment was located in the fosmid library and assigned to W-chromosome using florescence in situ hybridization (FISH). The major results are as follows:1. Construction and verification of the fosmid library screening systemA 3D pooling scheme, which includes super pools, row pools, column pools and plate pools, is essential to the fosmid library screening system. Use every 20 96-well microtiter plates to construct 1 super pool, 8 row pools, 7 column pools and 20 plate pools. Every super pool contains 1920 fosmid clones. Serial experiments were then carried out to determine the efficiency of the PCR screening system, using 7 known genes'primers. The results showed that at least 2 clones could be located in the fosmid library for each gene, indicating the PCR screening system's qualification for gene screening and other related researches on fosmid library.2. Screening and FISH locating of the female-specific fosmid cloneBy priming and screening the half-smooth tongue sole W-chromosome library, one pair of female-specific primers were obtained and 10 male and 10 female genome DNA were then used as templates to verify the primers'exclusivity to W-chromosome by PCR. The female-specific primers were then used to screen the fosmid library and one positive clone containing W-chromosome fragment was located in fosmid library. The W-chromosome-specific fosmid clone were then extracted, cultured and assigned to C. semilaevis metaphase chromosomes using florescence in situ hybridization. The result showed that the FISH signals could only be found in the middle of W-chromosome, thus proved the the exclusivity of the primer and added an additional conformation on the efficiency of the 2-step-3D-PCR screening system.The W-chromosome-specific primers can be used in half-smooth tongue sole's pseudo-male identification and be of great help in industrial cultivation. Also the W-chromosome-specific fosmid clone can be used for further W-chromosome researches and genetic analysis in this species and will be important for a better understanding of the C. semilaevis W chromosome. |
