Artificial propagation and wild release may influence the genetic integrity of wild populations. This practice has been prevalent in fisheries for millennia and is often termed “stocking”. In the Laurentian Great Lakes, walleye populations faced declines from the 1950s to the 1970s, prompting extensive stocking efforts for restoration. By the mid-2010s, walleye populations showed signs of recovery, but the genetic legacy of stocking on population structure at the genomic level remains unclear. Using a dataset of 45,600 genome-aligned SNP loci genotyped in 1,075 walleye individuals, we investigated the genetic impacts of over 50 years of stocking across the Great Lakes. Natural geographic barriers shaped walleye population structure, but pairwise comparisons revealed changes in genetic structure due to stocking from non-native sources also significantly contribute to population structure. Admixture between Lake Erie walleye and walleye from the re-populated Tittabawassee River indicate that stocking may have re-distributed putatively adaptive alleles around the Great Lakes. Genome scans identified FST outliers and evidence of selective sweeps, indicating local adaptation of spawning populations is likely. Notably, one genomic region showed strong differentiation between Muskegon River and walleye from the Tittabawassee River which was re-populated by Muskegon Strain walleye, suggesting admixture and selection both impact the observed genetic diversity. Overall, our study underscores how artificial propagation and translocations can significantly alter the evolutionary trajectory of populations. The findings highlight the complex interplay between stocking practices and genetic diversity, emphasizing the need for careful management strategies to preserve the genetic integrity of wild populations amidst conservation efforts.

Conservation and management professionals often works across jurisdictional boundaries to identify broad ecological patterns. These collaborations help to protect populations whose distributions span political borders. One common limitation to multijurisdictional collaboration is consistency in data recording and reporting. This limitation can impact genetic research which relies on data about specific markers in an organism’s genome. Incomplete overlap of markers between separate studies can prevent direct comparisons. Standardized marker panels can reduce the impact this issue and provide a common starting place for new research. Genotyping-in-thousands (GTSeq) is one approach used to create standardized marker panels for non-model organisms. Here we describe the development, optimization, and early assessments of a new GTSeq panel for use with walleye (Sander vitreus) from the Great Lakes region of North America. High genome-coverage sequencing conducted using RAD-capture provided genotypes for thousands of single nucleotide polymorphisms (SNPs). From these markers, SNP and microhaplotype makers were chosen that were informative for genetic stock identification (GSI) and kinship analysis. The final GTSeq panel contained 500 markers, including 197 microhaplotypes and 303 SNPs. Leave-one-out GSI simulations indicated that GSI accuracy should be greater than 80% in most jurisdictions. The false-positive rates of parent-offspring and full-sibling kinship identification was found to be low. Finally, genotypes could be consistently scored among separate sequencing runs >94% of the time. Results indicate that the GTSeq panel we developed should perform well for multijurisdictional research throughout the Great Lakes region.