Sex-Specific Marker Development and Identification of an XY Sex Determination System in the Tire Track Eel (Mastacembelus favus) via Whole-Genome Resequencing

The tire track eel (<i>Mastacembelus favus</i>) is a freshwater fish with high economic value and aquaculture potential. However, its sex determination mechanism remains unclear, which limits the development of monosex culture and sex-controlled breeding. To address this, preliminary female and male reference genomes were generated using second-generation sequencing, followed by whole-genome resequencing of four females and four males. Comparative analyses identified 69 male-specific sequences, with a total length of approximately 44.5 kb. Based on these sequences, two PCR-based sex-specific markers (W5 and W14) were developed. Both markers showed complete concordance with phenotypic sex in parental and offspring from controlled crosses populations, providing strong evidence for a male heterogametic (XY) sex determination system in <i>M. favus</i>. Although the markers were not transferable to the closely related species <i>Mastacembelus armatus</i>, the corresponding male-specific sequences exhibited high genomic conservation. In conclusion, this study provides reliable molecular tools for genetic sex identification in <i>M. favus.</i> These tools will support monosex aquaculture and sex-controlled breeding programs, while also offering insights into sex determination and sex chromosome evolution in the genus Mastacembelus.