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Stocking programs have been widely implemented to re-establish extirpated fish species to their historical ranges; when employed in species with complex life histories, such management activities should include careful consideration of resulting hybridization dynamics with resident stocks and corresponding outcomes on recovery initiatives. Genetic monitoring can be instrumental for quantifying the extent of introgression over time, however conventional markers typically have limited power for the identification of advanced hybrid classes, especially at the intra-specific level. Here, we demonstrate a workflow for developing, evaluating and deploying a Genotyping-in-Thousands by Sequencing (GT-seq) SNP panel with the power to detect advanced hybrid classes to assess the extent and trajectory of intra-specific hybridization, using the sockeye salmon ( Oncorhynchus nerka ) stocking program in Skaha Lake, British Columbia as a case study. Previous analyses detected significant levels of hybridization between the anadromous (sockeye) and freshwater resident (kokanee) forms of  O. nerka , but were restricted to assigning individuals to pure-stock or “hybrid”. Simulation analyses indicated our GT-seq panel had high accuracy, efficiency and power (&gt; 94.5%) of assignment to pure-stock sockeye salmon/kokanee, F 1 , F 2 , and B 2  backcross-sockeye/kokanee. Re-analysis of 2016/2017 spawners previously analyzed using TaqMan® assays and otolith microchemistry revealed shifts in assignment of some hybrids to adjacent pure-stock or B 2  backcross classes, while new assignment of 2019 spawners revealed hybrids comprised 31% of the population, ~ 74% of which were B 2  backcross or F 2 . Overall, the GT-seq panel development workflow presented here could be applied to virtually any system where genetic stock identification and intra-specific hybridization are important management parameters.

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Genotyping-in-Thousands by sequencing panel development and application for high-resolution monitoring of introgressive hybridization within sockeye salmon