Genomic evidence for Z-W differentiation in Pacific saury (Cololabis saira) and sex-chromosome diversity in Beloniformes

Genetic sex-determination mechanisms evolve rapidly in teleost fishes and are diverse within Beloniformes. In the present study, the sex-determination system of Pacific saury (Cololabis saira) was confirmed using double-digest, restriction-site-associated DNA sequencing (ddRAD-seq) and pooled whole-genome sequencing (Pool-seq), and a DNA marker was developed for inferring genetic sex. DNA sequence analysis detected numerous markers occurring predominantly or exclusively in phenotypic females suggesting a ZZ/ZW sex-determination system. Mapping of ddRAD-seq loci showed that these markers were concentrated within a 3-19 Mb region of chromosome 17 (Chr17). Pool-seq analyses also revealed elevated FST, sex-specific differences in sequencing depth, and nucleotide diversity within this region, supporting a ZZ/ZW sex-determination system involving Chr17. A PCR marker designated female-specific 1 (FS1), developed from a female-associated ddRAD sequence unmapped from the male-derived reference genome, was detected in all 43 phenotypic females and none of the 50 phenotypic males collected in 2025. In an independent sample collected in 2026, FS1 was detected in all 19 phenotypic females and one of the 29 phenotypic males, resulting in 97.9% concordance with phenotypic sex. The FS1-positive male may represent a genotypic-phenotypic sex mismatch or reflect recombination between FS1 and the causal sex-determining region; the linkage of FS1 to the W chromosome remains unconfirmed. Chromosome-scale synteny analysis showed that Pacific saury Chr17 and the sex chromosomes of Hyporhamphus sajori and Oryzias latipes are nonhomologous to one another. These findings reveal substantial sex-chromosome diversity among beloniform fishes and establish a basis for investigating genetic sex determination and genotypic-phenotypic sex concordance in wild Pacific saury.