A Y-specific amhr2 duplication (amhr2Y) is the candidate master sex determining gene in rabbitfish (Siganus canaliculatus).

The rabbitfish (Siganus canaliculatus), a member of the family Siganidae, is an economically important species in China's marine aquaculture industry. However, the genetic basis of its sex determination remains largely unknown, limiting its application in artificial breeding. In this study, we constructed haplotype-resolved genome assemblies and performed whole-genome re-sequencing to characterize the sex determination system and identify the candidate sex-determining gene. We found that S. canaliculatus possesses a male-heterogametic (XX/XY) system, with chromosome 23 acting as the sex chromosome. A small ~400-kb sex-determining region was identified on the Y chromosome, with no homologous sequence detected on the X chromosome. Within this region, a Y-linked duplicate of the autosomal amhr2 (amhr2Y) was consistently present in all examined males, strongly supporting its role as a candidate male sex-determining gene. Sequence analyses suggest that amhr2Y originated from an ancient duplication of amhr2 and was subsequently translocated to its current location relatively recently. Based on the sequence divergence between the X and Y chromosomes, we developed and validated sex-linked markers in two captive stocks derived from wild populations, enabling rapid and accurate genetic sex identification diagnosis. Together, these findings provide valuable genomic resources and practical tools for elucidating the sex determination mechanism and for accelerating broodstock sexing and selective breeding programs in rabbitfish.