Validation of male-specific markers confirmed an XY/XX-type sex determination system in Chinese dark sleeper (Odontobutis sinensis).

Next generation sequencing (NGS) method has been successfully used to identify sex-specific markers in many fish species. The Chinese dark sleeper (Odontobutis sinensis) is a commercially important aquaculture species in China. Here, by conducting whole genome sequencing of three females, three males and one female mixed pool, we initially identified 658 male-specific sequences and 242 female-specific sequences of Chinese dark sleeper. Based on polymerase chain reaction (PCR) confirmation, three markers showed 100 % male-specific amplification in 16 female and 16 male individuals from the Wuhan population, and this result was further validated using an additional 16 females and 16 males from the Guangdong population, demonstrating the reliability of these markers across multiple populations. These findings suggest a male heterogametic sex determination system in Chinese dark sleeper. Moreover, male-specific genes including high mobility group box 3 (hmgxb3) and insulin-like growth factor 2b (igf2b) were identified through NR annotation, which might be candidate sex-determining genes in Chinese dark sleeper. Generally, these validated male-specific markers first demonstrated the existence of an XY-type sex determination system in Chinese dark sleeper and would make a significant contribution to the development of all-male breeding and understanding of sex determination mechanisms.