The Development of Genetic Sex Identification Markers and Evidence of a Male Heterogametic Sex Determination System in Red Shiner

Abstract

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.

The effects of estradiol‐17β on the sex reversal, survival, and growth of Red Shiner and its use in the development of YY individuals

“…This female was heterozygous (A/G) for the sex‐linked marker XY_170 , which is typical of phenotypic males (Teal et al. 2023). The remaining 80 fish sampled from M XY × F YY crosses were males.…”

“…Red Shiner possess an XY‐male/XX‐female sex determination system (Teal et al. 2023); however, high temperatures can act as an aromatase inhibitor in some fishes that possess sex chromosomes, thus preventing production of estrogen and causing male sexual characteristics to develop (Piferrer et al. 1994).…”

“…Individuals who were heterozygous (A/G) for the male sex‐linked SNP with the sex identification marker XY_170 (Teal et al. 2023) were broodstock candidates for the next phase of spawning. All fish from the E2 treatments that were homozygous (A/A) for the female SNP were euthanized and discarded from the rest of study.…”

“…For species in which males are the heterogametic sex, such as the Red Shiner (Teal et al. 2023), the development of YY fish is often accomplished by selectively breeding feminized males (neofemales) with wild‐type males (Beardmore et al. 2001; Cotton and Wedekind 2007; Senior et al.…”

“…Although typically pursued to enhance aquaculture performance (Beardmore et al 2001;Piferrer 2001;Arslan and Phelps 2004;Wang et al 2008) or to obtain all-male cohorts for the ornamental fish industry (Pandian and Sheela 1995;Piferrer and Lim 1997), development of YY individuals for commercially unimportant taxa has gained more interest due to the possible implications for their use in invasive population control (Cotton and Wedekind 2007). For species in which males are the heterogametic sex, such as the Red Shiner (Teal et al 2023), the development of YY fish is often accomplished by selectively breeding feminized males (neofemales) with wild-type males (Beardmore et al 2001;Cotton and Wedekind 2007;Senior et al 2013;Schill et al 2016). Progeny from this cross will feature three genotypes: XX females, XY males, and YY males.…”