Male-specific genome sequences for non-invasive sex identification in Nishikigoi (Cyprinus carpio)

Nishikigoi, ornamental cultivars of the common carp (Cyprinus carpio), are economically valuable aquaculture teleosts in which mature females command significantly higher market prices due to superior body conformation and coloration. Consequently, early and non-invasive sex identification is highly desirable for commercial breeding. Although sex determination in common carp follows an XX/XY system, sex-chromosome-specific markers and field-applicable diagnostic methods for Nishikigoi have remained limited. In this study, we conducted sequence-based bulked segregant analysis using whole-genome shotgun sequencing of female and male pools in Kohaku, a representative Nishikigoi lineage. We identified male-specific candidate contigs and successfully developed PCR-based male-specific markers. Validation across multiple Nishikigoi varieties and wild common carp demonstrated that primary primer pairs achieved high diagnostic accuracy. Comparative genomic analysis revealed that these candidate contigs mapped to the genomic region on chromosome 25B containing the male-determining gene Pangu, with the primary marker located in immediate physical proximity to the gene. To enable field diagnostics, we combined non-invasive epidermal mucus DNA extraction, loop-mediated isothermal amplification (LAMP), and chromatography-printed array strip (C-PAS) technology, establishing a rapid on-site detection workflow. Overall, this study provides highly reliable PCR markers associated with the sex-determination locus and establishes a non-invasive, field-applicable diagnostic platform to streamline commercial Nishikigoi breeding and sex-selected stock management.