Chinese tongue sole (Cynoglossus semilaevis) males and females exhibit great differences in growth rate and appearance. The species is heterogametic (ZW/ZZ) and has sex-reversed “pseudomales” that are genetically female and physiologically male. In this study, we identified eight sex-specific single nucleotide polymorphism (SNP) markers for the sex identification of C. semilaevis by using a combination of genome-wide association study (GWAS) screening and SnaPshot validation. Candidate SNPs were screened using genotyping by sequencing to perform GWAS of the differential SNPs between the sexes of C. semilaevis. The SNP loci were amplified using a multiplex PCR system and detected via SNaPshot, which enables multiplexing of up to 30–40 SNPs in a single assay and ensures high accuracy of the results. The molecular markers detected in our study were used to successfully identify normal males and pseudomales from 45 caught and 40 cultured C. semilaevis specimens. Linkage disequilibrium analysis showed that the eight SNP loci were related to each other, with a strong linkage. Moreover, we investigated the expression of prdm6 mRNA containing a missense SNP and confirmed that the gene is differentially expressed in the gonads of the different sexes of C. semilaevis; the expression of prdm6 mRNA was significantly higher in the males than in the females and pseudomales. This means prdm6 may be related to sex differentiation in C. semilaevis.

雄性和雌性舌tongue（Cynoglossus semilaevis）的生长速度和外观差异很大。该物种是异配的（ZW / ZZ），具有与性别相反的“伪雄性”，即遗传上是雌性，生理上是雄性。在这项研究中，我们确定了性别鉴定的8性别特异性单核苷酸多态性（SNP）标记物C.鳎通过使用全基因组关联研究（GWAS）筛选和验证快照的组合。使用基因分型方法，通过测序对候选SNP进行筛选，以对半隐梭菌性别之间的差异SNP进行GWAS分析。。使用多重PCR系统扩增SNP位点，并通过SNaPshot进行检测，这使得在一次测定中最多可复用30–40个SNP，并确保结果的高精度。在我们的研究中检测到的分子标记用于成功地从45个捕获的和40个培养的C. semilaevis标本中鉴定正常雄性和假性雄性。连锁不平衡分析表明，八个SNP位点彼此相关，具有很强的连锁性。此外，我们研究的表达prdm6的mRNA含有错义SNP，并确认该基因在不同性别的性腺差异表达C.鳎; prdm6的表达男性中的mRNA显着高于女性和假性男性。这意味着prdm6可能与半叶念珠菌的性别分化有关。