Identification of male-specific markers via 2b-RAD sequencing reveals an XX/XY sex determination system in Acrossocheilus longipinnis

Acrossocheilus longipinnis is a commercially significant aquaculture species native to southwestern China, highly favored by consumers for its excellent meat quality, high nutritional value, and abundance of polyunsaturated fatty acids. Although the species exhibits pronounced sexual dimorphism with females growing significantly faster than males, its sex determination system and underlying mechanisms remain unknown, hindering the development of monosex breeding technologies and the optimization of production efficiency. In this study, a total of 3 male-specific sequences were successfully identified from XY males and XX females using 2b-RAD-seq and genome assembly approaches in A. longipinnis . Synteny and coverage depth analyses localized these three sequences to a Y-specific insertion at 5,724,913–5,869,801 bp, and their male-specificity was further confirmed in 72 individuals from 2 populations by PCR amplification. Our results reveal an XX/XY sex-determination system and provide applicable male-specific markers for sex control breeding in A. longipinnis . These findings lay a solid foundation for future studies on sex chromosome identification, fine mapping of the sex-determining region (SDR), and cloning of the master sex-determining gene (SDG) in this species.