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

## TL;DR

This study identifies a male-specific genetic system in tire track eels and creates tools for sex identification to improve aquaculture.

## Contribution

Development of two PCR-based sex markers and evidence for an XY system in Mastacembelus favus.

## Key findings

- Two PCR-based markers (W5 and W14) reliably distinguish male and female tire track eels.
- Evidence supports an XY sex determination system in Mastacembelus favus.
- Male-specific sequences show high conservation in related species despite non-transferable markers.

## Abstract

This study developed a rapid, accurate, and cost-effective genetic sex identification method for the tire track eel (Mastacembelus favus). Whole-genome resequencing identified 69 male-specific genomic regions, from which two PCR-based markers (W5 and W14) were designed. These markers reliably distinguished males and females in genetic sex identification, indicating an XY sex determination system in M. favus. Sequence conservation was further examined by BLAST in the related species Mastacembelus armatus. This work provides practical tools for monosex breeding and advances the understanding of sex determination in Mastacembelus species.

The tire track eel (Mastacembelus favus) 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 M. favus. Although the markers were not transferable to the closely related species Mastacembelus armatus, the corresponding male-specific sequences exhibited high genomic conservation. In conclusion, this study provides reliable molecular tools for genetic sex identification in M. favus. 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.