A simple PCR-based genetic sex identification method in the blotched snakehead (Channa maculata) developed by high-throughput sequencing

Abstract Sex dimorphism is a common phenomenon in animals. Blotched snakehead (Channa maculata) is an economically important freshwater fish with a faster growth rate in males. However, existing sex-specific markers were too few and difficult to use in production practice, which hindered the development of all-male breeding. Here, we generated 15.64, 20.77 and 29.03 Gb paired-end genomic sequence reads for three male libraries, 12.82, 19.36 and 28.33 Gb for three female libraries and 30.93 Gb for a female mixed pool libraries to screen male-specific sequences. After assembly, we obtained a male reference genome with 541,932 assembled sequences and a female reference genome containing 853,706 assembled sequences. After mapping, 23 male-specific sequences were identified. Six candidate male-specific markers were successfully validated using PCR reaction. Moreover, based on homology analysis and coverage information, we found a candidate X chromosome sequence that was partially similar to the corresponding Y chromosome sequence, and two resulting primers were successfully used in the identification of female (XX), male (XY) and supermale (YY) individuals. In general, a deep genome sequencing method was first provided to effectively enrich large sex-specific segments of Blotched snakehead, which would greatly contribute to elucidation of its sex determination mechanisms and development of all-male breeding.