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.
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