Rapid Molecular Determination of Sex for Western North American Chub (Gila spp.)

Abstract

AbstractGathering sex data is frequently a complicated matter for fish species that lack sexually dimorphic traits. Secondary sexual characteristics are cryptic or nonexistent in many minnows (Cyprinidae) during early life stages or outside of the breeding season, which may require lethal methods for determining sex. This complicates many conservation actions and population genetic studies, including (1) the development of hatchery broodstocks for threatened and endangered species, (2) sex ratio determination in wild populations, (3) the influence of sex ratio on the effective population size estimates, and (4) limitations on the implementation of genetic tagging and parentage assignment in wild populations. Here, we remedy this issue for four western North American chub species (Gila spp.) through restriction‐site‐associated DNA sequencing of Bonytail G. elegans, which identified a single locus indicating an XY sex determination system and no candidate loci consistent with a WZ system. This locus cross‐amplified in three additional Gila species (Chihuahua Chub G. nigrescens, Virgin Chub G. seminuda, and White River Chub G. jordani), although species‐specific differences necessitated the development of three novel primer/probe sets. Real‐time PCR with hydrolysis probe assays was used to genotype sex‐linked single‐nucleotide polymorphisms for each species. The measurement of endpoint fluorescence in a real‐time PCR thermal cycler allowed for rapid and accurate sex genotyping of these species, with accuracy >97% relative to phenotypic sex identification when three PCR replicates were employed. These assays provided high precision, with unanimous results across >87% of the replicates and a majority consensus genotype for 98.6% of the samples. The locus identified herein was conserved across all of the Gila species that we tested (n = 6) and other western North American cyprinids; however, sex‐linked polymorphisms were found only in the four species that are named above. Consequently, this locus has promise for the identification and development of sex‐linked assays for other Gila and closely related cyprinids.