Ma Hong-Yu, Chen Song-Lin, Li Jing, Tian Yong-Sheng, Ji Xiang-Shan, Zhang Li-Jing
Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research Insti-tute, Chinese Academy of Fishery Sciences, Qingdao 266071, China.
Yi Chuan. 2009 Jan;31(1):88-94. doi: 10.3724/sp.j.1005.2009.00088.
Molecular sex identification is important in studying sex control, sex determination, and all-female breeding in half-smooth tongue sole (Cynoglossus semilaevis). In the present study, a female-specific AFLP marker was isolated from Cynoglossus semilaevis by AFLP technique using the selective primer combination E-ACT/M-CAA. This marker was re-amplified, recovered from the agarose gels, cloned and sequenced. Bioinformatic analysis indicated that the length of the product was 791 bp, and the sequence showed no similarity to any known sequences deposited in the GenBank database using BLASTn. According to the DNA sequence of the female-specific AFLP marker, specific PCR primers were designed and PCR amplification was performed on 100 sex-known individuals of C. semilaevis (50 females and 50 males each). A specific band 324 bp in length was present in all females but absent in all males (except for one male), indicating that the female-specific AFLP marker was successfully converted into female-specific SCAR (sequence characterized amplified regions) marker. The sex analysis of 3-day-old C. semilaevis individuals using this female-specific SCAR marker indicated that the female ratio was 41.7%. The female-specific SCAR marker developed in this study allowed simple, reliable, and rapid molecular sex identification using small amounts of fin tissue without sacrifice of C. semilaevis especially at early stage of development.
分子性别鉴定在半滑舌鳎（Cynoglossus semilaevis）的性别控制、性别决定和全雌育种研究中具有重要意义。在本研究中，利用AFLP技术，采用选择性引物组合E-ACT/M-CAA，从半滑舌鳎中分离出一个雌性特异性AFLP标记。对该标记进行重新扩增，从琼脂糖凝胶中回收，克隆并测序。生物信息学分析表明，产物长度为791 bp，使用BLASTn程序在GenBank数据库中未发现该序列与任何已知序列相似。根据雌性特异性AFLP标记的DNA序列，设计了特异性PCR引物，并对100尾已知性别的半滑舌鳎个体（雌雄各50尾）进行PCR扩增。所有雌性个体均出现一条长度为324 bp的特异性条带，所有雄性个体（除一尾雄性外）均未出现该条带，表明该雌性特异性AFLP标记已成功转化为雌性特异性SCAR（序列特征性扩增区域）标记。利用该雌性特异性SCAR标记对3日龄半滑舌鳎个体进行性别分析，结果显示雌性比例为41.7%。本研究开发的雌性特异性SCAR标记可利用少量鳍组织进行简单、可靠、快速的分子性别鉴定，且无需牺牲半滑舌鳎，尤其是在其发育早期。
