Identification of two novel female-specific DNA sequences in half-smooth tongue sole, Cynoglossus semilaevis

Abstract Half-smooth tongue sole ( Cynoglossus semilaevis ) is a kind of cultured teleost fish with larger size of females than males. In order to produce all-female progeny, it is necessary to distinguish the genetic females and males precisely. In this study, a W chromosome library, which had been constructed by laser microdissection of W chromosomes, was screened. Among four hundreds and twelve pairs of designed PCR primers, two pairs (W045 and W131) could produce female-specific fragments in fifty individuals. To verify the primers and optimize the method, another one hundred individuals were tested by multiplex PCR with both W045 and a pair of positive control primers. Furthermore, two sex-reversed individuals were also identified correctly. Fluorescent in situ hybridization (FISH) further confirmed that fragments form W045 and W131 primers only localized to the W chromosome in female individuals, and the FISH technique with the two female-specific fragments could help to isolate super-female individuals. The identification of two novel female-specific DNA sequences and the techniques used here may be applied to distinguish the genetic gender of half-smooth tongue sole precisely. Besides, these also help us to obtain a better understanding of fish sex determination.