半滑舌鳎雌性特异扩增片段长度多态性标记的筛选与应用

半滑舌鳎(Cynoglossus semilaevisGünther)为东北亚特有的名贵冷温性比目鱼类,为我国养殖业的新宠。半滑舌鳎雌鱼生长速度是雄性的2~3倍,若能实现单雌化养殖将大大提高养殖业的经济效益。本研究利用扩增片段长度多态性(AFLP)技术,应用64个引物组合,检测了半滑舌鳎(Cynoglossus semilaevisGünther)雌雄基因组DNA的多态性,筛选与半滑舌鳎性别相关的AFLP分子标记。实验经过3轮筛选和验证,4个引物组合扩增出7个雌性个体出现频率为100%的DNA片段,我们认为这7个标记是半滑舌鳎雌性特异的AFLP标记,分别命名为CseF382、CseF575、CseF783、CseF464、CseF136、CseF618和CseF305。同时,将标记CseF382成功转化为SCAR标记,测定了该标记的DNA序列,建立了半滑舌鳎遗传性别鉴定的PCR技术,为半滑舌鳎性别决定机制的研究和性别控制奠定了重要基础。

Isolation and application of female specific amplified fragment length polymorphism markers in Cynoglossus semilaevis

Halfsmooth tongue sole (Cynoglossus semilaevis Günther) is a cultured marine fish exploited recently in China. The female individuals of tongue sole grow 1-2 times faster than male individuals. Thus, the development of all female stock would be of significant benefit for aquaculture. Sex related molecular marker is a useful tool for studying sex determination mechnism and controling fish sex. In order to screen sex specific molecular markers, AFLP analysis technique was firstly developed in half smooth tongue sole. DNA extraction from liver tissues was carried out according to stardand procedures. Phenotypic sex of the fish was determined by histological sectioning and staining. 64 AFLP primer combinations were used to screen the genome DNA of half smooth tongue soles. 4 primer combinations amplified 7 female specific markers that were female specific in half smooth tongue sole. The 7 female specific markers were named CseF382, CseF575, CseF783, CseF464, CseF136, CseF618 and CseF305, respectively. One female specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced. This female specific AFLP marker was converted into single locus PCR marker of a sequence characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half smooth tongue sole. This PCR method will be more efficient and less expensive than with AFLP markers. The isolation of sex specific molecular markers lays a basis for elucidation of sex determination mechanism, and provides a tool for sex control in half smooth tongue sole.