Limited evidence for extensive genetic differentiation between X and Y chromosomes in Hybognathus amarus (Cypriniformes: Leuciscidae).

This article focuses on identifying and validating sex-linked genetic markers to infer the sex determination system in the endangered Rio Grande silvery minnow (Hybognathus amarus), a species with previously unknown sex determination mechanisms. Using a combination of Nextera-tagmented reductively amplified DNA sequencing (nextRAD-seq) from individuals of known sex and whole-genome sequencing (WGS) of male and female genomes, the study identified five male-associated markers and two single-nucleotide polymorphisms (SNPs) consistent with an XY sex determination system. PCR validation confirmed one marker (HAM3) as sex-associated but not strictly male-specific, showing higher copy number in males, while the SNP-containing locus (HAM6) exhibited allele patterns supporting males as the heterogametic sex. The findings suggest limited differentiation between sex chromosomes in this species and highlight the utility of integrating reduced representation sequencing with WGS for sex marker discovery in nonmodel organisms, with implications for conservation and captive breeding programs.