Novel molecular markers for high-throughput sex characterization of Cynoglossus semilaevis

Abstract

Male and female Chinese tongue soles (Cynoglossus semilaevis) exhibit a great difference in growth rates. Karyotype analysis has revealed that the species is heterogametic (ZW/ZZ), and sex-reversed “pseudo-males” exist under both natural and aquaculture conditions. In this study, we developed three female-specific molecular markers for the sex identification of C. semilaevis. The markers were an insertion-deletion (indel)-type structural variation and two single nucleotide polymorphism (SNP) loci on the ZW sex chromosome (SNP_chr_8936186_C_G and SNP_chr_8935925_C_T). The indel marker can be used for female-specific identification with traditional PCR and agarose gel electrophoresis. The detection and use of the SNPs are based on the penta-primer amplification refractory mutation system. The SNPs on the ZW chromosome were successfully identified with high-throughput quantitative PCR. The two types of molecular markers were successfully used to identify normal males and pseudo-males in a population of 60 C. semilaevis. When compared with currently used markers, the markers identified in this study have high accuracy and can help in high-throughput detection. Our findings could provide an effective method for detecting the gender of fish with the ZW karyotype.