一种快速鉴定半滑舌鳎遗传性别的方法  

A Rapid Method for Genetic Sexuality Identification in Cynoglossus semilaevis

作　　者：董忠典[1,2,3] 张宁[1,2,3] 徐文腾 刘洋[2,3] 邵长伟 温海深[1] 陈松林[2,3] 

机构地区：[1]中国海洋大学,青岛266003 [2]中国水产科学研究院黄海水产研究所/农业部海洋渔业可持续发展重点实验室,青岛266071 [3]青岛海洋科学与技术国家实验室/海洋渔业科学与食物产出过程功能实验室,青岛266237

出　　处：《农业生物技术学报》2016年第5期764-769,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金重点项目(No.31130057);国家自然科学基金面上项目(No.31472269);山东省泰山学者攀登计划专项资助

摘　　要：半滑舌鳎(Cynoglossus semiliaevis)遗传性别快速准确鉴定的方法是性别控制与高雌苗种培育的基础,其性别决定机制为ZW型,遗传雌鱼为异型染色体ZW,遗传雄鱼为同型染色体ZZ。本研究在半滑舌鳎全基因组测序结果的基础上,通过分析Z染色体和W染色体差异序列,设计一对跨Z/W染色体同源差异DNA片段的引物CS-SEX-F和CS-SEX-R,取半滑舌鳎部分鳍条通过碱煮沸方法获得的DNA为模板,采用PCR扩增,琼脂糖凝胶电泳方法鉴定半滑舌鳎遗传性别。通过该方法在遗传雄鱼(ZZ)个体中可以扩增出366 bp的DNA条带,遗传雌鱼(ZW)个体中可以扩增出366和253 bp两种DNA条带。本研究建立了一种新的成本低廉、操作快速、结果准确可靠的鉴定半滑舌鳎遗传性别的方法,该方法与已有方法相比,操作简便省时,为半滑舌鳎遗传学研究与性别控制育种相关研究提供了可靠分子标记,在半滑舌鳎遗传性别鉴定和养殖场所内伪雄鱼快速检测方面具有广阔的应用前景。Establishment of a rapid and accurate method for genetic sexuality identification in half-smooth tongue sole （Cynoglossus semilaevis） is the basis of sex control technology and high-female breeding of half- smooth tongue sole. This fish harbors ZZ/ZW sex determination system, in which female contains ZW heteromorphic chromosomes compared to male （ZZ）. In this method, a pair of primers （CS-SEX-F and CS- SEX-R）, which were located in the different regions between Z and W chromosome, was firstly designed according to the whole genome sequencing data. Then genomic DNA templates were extracted from the fin by boiling method as follows： 30-50 mg of fin samples were picked into 1.5 mL eppendorf tube and 400μL NaOH （50 mmol/L） was added, then they were mixed thoroughly and centrifuged at 12 000 r/min for 10 min. The supernatant was used as template in PCR reaction system, which contained 5.0 μL 10xBuffer, 4.0μL dNTP （2.5 mmol/L）, 1.0 μL CS-SEX-F/CS-SEX-R （10 μmol/L）, 0.5 μL rTaq （5 U/μL）, 5.0 μL DNA supernatant. The program was 95℃ 10 rain; 95℃30 s, 60℃ 30 s, 72℃ 30 s for 35 cycles; 72℃ 10 min. The PCR products were analyzed by electrophoresis in agarose gel at 150 V and 20 min. Genetic sexuality could be distinguished via different band patterns, where ZZ male exhibited single band （366 bp）, and ZW female harbored double bands （366 and 253 bp）. The advantages of this newly established method lie in its simplicity and time-effective compared to the previous ones, so it has tremendous potential in the future for application to accurate identification of genetic sexuality and efficient screening of pseudo-male fish in aqua farm.

关 键 词：半滑舌鳎 DNA提取 引物开发 性别鉴定 

分 类 号：S917[农业科学—水产科学]