A rapid method of sex-specific marker discovery based on NGS and determination of the XX / XY sex-determination system in Channa maculata

Abstract The discovery of sex-specific markers is of great significance for the study of unisexual breeding and sex-determination mechanisms of fish. The all-male hybrid snakehead is a freshwater aquaculture species with high economic value. The breeding of all-male hybrid snakeheads depends on the discovery of sex-specific markers of Taiwan snakeheads. In this study, 24 females and 24 males of Taiwan snakeheads were sequenced by NGS, and then a set of biological information analysis process was developed to speculate that Taiwan snakeheads were XX / XY sex-determining system, and 1250 male-related contigs were assembled; In combination with indels and SNP sites analysis, Chelex 100 DNA extraction and an HRM scan, two molecular markers (CM286 and CM1053) were selected from 29 male-related contigs, which could clearly distinguish XX females, XY males, and YY supermales; Then, it was confirmed by Sanger sequencing that Taiwan snakeheads had a male heterogamete type, and the ratio of females to males in three full-sib families was all close to 1:1 (P > .05). Therefore, Taiwan snakeheads were determined to be the XX / XY sex-determining system. The results showed that the effective sex-specific markers, combined with the Chelex 100 DNA extraction and HRM scan technology, could realize high-throughput and rapid detection of the three sexes (XX, XY, and YY) of Taiwan snakeheads, and could be directly applied to the breeding of the all-male hybrid snakehead. This process of bioinformatic analysis for sex-specific marker discovery is simple and efficient, and can also be applied to the study of other fish sex-specific marker discovery and sex-determination mechanisms.