Unveiling potential sex-determining genes and sex-specific markers in autotetraploid Carassius auratus

Autotetraploid Carassius auratus is a stable hereditary autotetraploid fish resulting from the hybridization of Carassius auratus red var. (RCC, ♀) × Megalobrama amblycephala (BSB, ♂), containing four sets of RCC chromosomes. However, the molecular mechanism underlying the determination of sex in this species remains largely unknown. Currently, there lacks a full understanding of the molecular mechanisms governing sex determination and specific molecular markers to differentiate sex in this species. In this study, 25,801,677 SNPs (Single-nucleotide polymorphism) and 6,210,306 Indels (insertion-deletion) were obtained from whole-genome resequencing of 100 individuals (including 50 female and 50 male). Further identification confirmed the candidate chromosomes as Chr46B, with the sex-determining region located at Chr46B: 22,500,000–22,800,000 bp. Based on the male-specific insertion (26 bp) within the candidate sex-determining region, a pair of sex-specific molecular markers has been identified. In addition, based on the screening of candidate sex-determining region genes and RT-qPCR validation analysis, ADAM10, AQP9 and tc1a were identified as candidate sex-determining genes. These findings provide a robust foundation for investigating sex determination mechanisms in fish, the evolution of sex chromosomes, and the development of monosex populations.