|
|
|
| A Rapid Method for Genetic Sexuality Identification in Cynoglossus semilaevis |
|
|
|
|
Abstract 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 400 μL 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 10×Buffer, 4.0 μL 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 min; 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.
|
|
Received: 03 December 2015
Published: 01 April 2016
|
|
|
|
| 陈松林. 2013. 鱼类性别控制与细胞工程育种[M]. 科学出版社, 北京. pp 2-3. (Chen S L. 2013. Fish sex control and cell engineering breeding[M]. Science Press, Beijing, pp. 2-3)陈松林, 李仰真, 张静等. 2013. 半滑舌鳎快速生长及高雌性家系的筛选. 水产学报, 37(4): 481-488. (Chen S L, Li Y Z, Zhang J, et al. 2005. Selection of the families with high growth rate and high female proportion in half-smooth tongue sole (Cynoglossus semilaevis)[J]. Journal of Fisheries of China, 37(4): 481-488.)季相山, 陈松林, 马洪雨等. 2010. 半滑舌鳎养殖群体中自然性逆转伪雄鱼的发现. 水产学报, 34(2): 322-327. (Ji X S，Chen S L，Ma H Y，et al. 2010. Natural sex reversal of female Cynoglossus semilaevis in rearing populations[J]. Journal of Fisheries of China, 34(2): 322-327.)刘洋, 陈松林, 高峰涛等. 2014. 半滑舌鳎性别特异微卫星标记的SCAR转化及其应用. 农业生物技术学报, 6(6): 787-792. (Liu Y, Chen S L, Gao F T, et al. 2014. SCAR-transformation of Sex-specific SSR Marker and Its Application in Half-smooth Tongue Sole (Cynoglossus semiliaevis)[J]. Journal of Agricultural Biotechnology, 6(6): 787-792.)柳学周, 庄志猛, 马爱军等. 2005. 半滑舌鳎繁殖生物学及繁育技术研究. 海洋水产研究, 5: 7-14. (Liu X Z, Zhuang Z M, Ma A J, et al. 2005. Reproductive biology and breeding technology of Cynoglossus semilaevis Gunther[J]. Marine Fisheries Research, 5:7-14)孟庆闻, 苏绵祥, 缪学祖. 1995. 鱼类分类学[M]. 中国农业出版社, 北京，pp 979-981. (Meng Q W, Su M X, Miao X Z. 1995. Fish taxonomy[M]. China Agriculture Press, Beijing, pp. 979-981.)周丽青, 杨爱国, 柳学周等. 2005. 半滑舌鳎染色体核型分析. 水产学报, 03:417-419. (Zhou L Q, Yang A G, Liu X Z, et al. 2005. The karyotype of the tonguefish Cynoglossus semilaevis[J]. Journal of Fisheries of China, 03:417-419.)Chen S L, Ji X S, Shao C W, et al. 2011. Induction of mitogynogenetic diploids and identification of WW super-female using sex-specific SSR markers in half-smooth tongue sole (Cynoglossus semilaevis)[J]. Marine Biotechnology, 14(1): 120-128. Chen S L, Li J, Deng S P, et al. 2007. Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (Cynoglossus semilaevis)[J]. Marine Biotechnology, 9(2): 273-280.Chen S L, Zhang G J, Shao C W, et al. 2014. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle[J]. Nature Genetics, 46(3): 253-260. Devlin R H. Mcneil B K., Groves T D, et al. 1991. Isolation of a Y-Chromosomal DNA Probe Capable of Determining Genetic Sex in Chinook Salmon (Oncorhynchus tshawytscha)[J]. Canadian Journal of Fisheries & Aquatic Sciences, 48(9): 1606-1612. Devlin R H, Nagahama Y. 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences[J]. Aquaculture, 208(3): 191–364.Felip A, Young W P, Wheeler P A, et al. 2005. An AFLP-based approach for the identification of sex-linked markers in rainbow trout (Oncorhynchus mykiss)[J]. Aquaculture, 247: 35–43. Lee B Y, Penman D J, Kocher T D. 2003. Identification of a sex-determining region in Nile tilapia (Oreochromis niloticus) using bulked segregant analysis[J]. Animal Genetics, 34(5): 379–383. Majumdar K C, Mcandrew B J. 1986. Relative DNA content of somatic nuclei and chromosomal studies in three genera,Tilapia, Sarotherodon, andOreochromis of the tribe Tilapiini (Pisces, Cichlidae)[J]. Genetica, 68(3): 175-188. Matsuda M, Kusama T, Oshiro T, et al. 1997. Isolation of a sex chromosome-specific DNA sequence in the medaka, Oryzias latipes[J]. Genes & Genetic Systems, 72(5): 263-268. Meeker N D, Hutchinson S A, Ho L, et al. 2007. Method for isolation of PCR-ready genomic DNA from zebrafish tissues[J]. Biotechniques, 43(5): 610-614. Patil J G, Hinze S J. 2008. Simplex PCR Assay for Positive Identification of Genetic Sex in the Japanese Medaka[J]. Marine Biotechnology, 10(6): 641-644.Shao C W, Li Q Y, Chen S L, et al. 2014. Epigenetic modification and inheritance in sexual reversal of fish[J]. Genome Research, 24(4): 604-615. Wang D, Mao H L, Chen H X, et al. 2009. Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populations[J]. Animal Genetics, 40(6): 978-981. Yano A, Guyomard R, Nicol B, et al. 2012. An immune-related gene evolved into the master sex-determining gene in rainbow trout, Oncorhynchus mykiss[J]. Current Biology Cb, 22(15): 1423-1428. Zhuang Z M, Wu D, Zhang S C, et al. 2006. G‐banding patterns of the chromosomes of tonguefish Cynoglossus semilaevis Günther, 1873[J]. Journal of Applied Ichthyology, 22(5): 437-440. |
|
|
|
