The development of genetic sex identification markers and evidence of a male heterogametic sex determination system in Red Shiner

The Red Shiner Cyprinella lutrensis is of increasing management interest as an invasive species that negatively impacts many native fishes throughout North America. Trojan sex chromosome (TSC)-carrying individuals could theoretically control invasive fish populations by skewing the sex ratio to 100% male. The efficacy of TSC-based control programs requires an understanding of a population's sex determination system, yet such information is lacking for Red Shiner. We used single-digest restriction site-associated DNA sequencing to discover sex-linked single-nucleotide polymorphisms (SNPs), and we conducted a series of breeding experiments to uncover the sex determination system. All candidate sex-linked SNPs that fit our selection criteria exhibited a pattern of male heterogamety. We developed two sex-identification (sex-ID) marker assays, XY_248 and XY_170, which showed phenotype–genotype concordance scores of 77.00% and 84.35%, respectively. These sex-ID markers exhibited relatively high phenotype–genotype concordance in females (XY_248 = 96.30%; XY_170 = 98.61%), which allowed for selective breeding of phenotypically feminized genetic males. We observed a 3:1 male : female sex ratio in spawns from feminized males crossed with wild-type males, indicative of a male heterogametic sex determination system (i.e., XY male/XX female). The discovery of a male heterogametic sex determination system, in combination with our two markers, increases the likelihood of developing an effective TSC eradication strategy for invasive Red Shiner populations.