通过对半滑舌鳎雌鱼fosmid文库进行有序混合,成功构建了两步-三维PCR筛选体系,为后续的基因筛选奠定了基础.通过对半滑舌鳎W染色体文库序列进行引物设计,并分别在雌、雄鱼基因组DNA中筛选,获得1对雌鱼特异性引物.使用该引物对fosrnid文库进行筛选,得到1个雌鱼特异性克隆,并通过荧光原位杂交技术将克隆定位到了W性染色体上,证实了使用该体系进行文库筛选的有效性以及雌龟引物的特异性,建立了半滑舌鳎遗传性别的分子及细胞遗传学鉴定方法,为后续半滑舌鳎基因组及性染色体的研究奠定了基础.%A 2-step-3D-PCR screening system of the female half-smooth tongue sole fosmid library was successfully constructed by a orderly mixing of the fosmid clones. By priming and screening W-chromo-some library of half-smooth tongue sole, one pair of female-specific primers were 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), which proved the exclusivity of the primers and provided a conformation on the efficiency of the 2-step-3D-PCR screening system. The female-specific primers could be of good use in pseudo-male detection of half-smooth tongue sole. This study provides a base for further genetic analysis in this species and will be important for a better understanding of the C. Semilaevis W chromosome.
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