Isolation of female-specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis)
Isolation of female-specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis)
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DOI：
10.1007/s10126-006-6081-x
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发表时间：
2007-03-01
影响因子：
3
通讯作者：
Xu, Jian-Yong
中科院分区：
文献类型：
--
作者：
Chen, Song-Lin;Li, Jing;Xu, Jian-Yong
性别特异分子标记是研究鱼类性别决定机制和性别控制的重要基因资源。以人工养殖的半滑舌鳎雌雄个体为材料，筛选性别特异的AFLP分子标记。采用生殖腺组织切片法对28只舌底鱼进行了性别鉴定。用64对引物组合对15个雌、雄个体进行AFLP分析，共获得4681条可标记带，其中42.11%的多态性带在雌个体中出现，43.39%的多态性带在雄个体中出现。共筛选出7个雌性特异性AFLP标记，分别命名为CseF382、CseF575、CscF783、CseF464、CseF136、CseF618和CseF305。扩增一个雌性特异性AFLP标记（CseF382），从凝胶中回收，克隆并测序（登录号DQ487760）。将该雌性特异性AFLP标记转化为具有序列特征的扩增区域的单位点聚合酶链反应（PCR）标记。利用所设计的特异性引物，建立了一种简便的半滑舌鳎遗传性别鉴定方法。PCR产物显示，最初的15个雌性个体产生约350 bp的雌性特异性条带，而最初的13个雄性个体没有产生条带。我们还调查了PCR引物在其他舌鳎个体中的适用性。在另外59个雌性个体中发现了相同的约350 bp的雌性特异性片段，而在另外58个雄性个体中没有发现。这种基于AFLP的分子性别鉴定技术在半滑舌鳎性别决定机制的阐明和性别控制方面具有很大的应用潜力。
The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382 ' CseF575, CscF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase- chain reaction (PCR) marker of a sequence-characterized amplified region OSCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.