<scp>GT</scp> ‐Seq Panel Development for Species Identification and Parentage Analysis of Closely Related Hybridising <i>Scaphirhynchus</i> Sturgeons

Hatchery supplementation is vital for conserving dwindling fish populations. Effective augmentation requires distinguishing hatchery-origin from wild individuals and accurately identifying species, particularly in systems where closely related species coexist. Genetic monitoring is key to quantifying genetic differences, but conventional markers do not distinguish hybrids, especially backcrosses. Misidentifying hybrids in hatchery programs compromises wild gene pools because hatchery broodstock contributes to numerous offspring being released into the wild. Here, we present a workflow for developing and evaluating the Genotyping-in-Thousands by sequencing (GT-seq) single nucleotide polymorphism (SNP) panel for North American river sturgeons (Scaphirhynchus spp.). This panel is designed to detect complex hybrid classes and to determine parent-offspring relationships. Our species identification panel (S-loci) contains 155 SNPs selected for high genetic differentiation (F_ST) between Pallid Sturgeon (S. albus) and Shovelnose Sturgeon (S. platorynchus), and the parentage assignment panel (P-loci) includes 112 SNPs with high heterozygosity within Pallid Sturgeon. Simulation analyses demonstrated that our GT-seq S-loci panel reliably classifies pure species, F1, F2 and backcross hybrids, even with up to 70% missing data. The P-loci panel achieves high-confidence parentage assignment with ≥ 80% typed loci, with performance influenced by the proportion of sampled parents. Overall, the novel Scaphirhynchus GT-seq panel developed in this study represents a robust and efficient tool for detecting hybridisation, assigning parentage and providing critical information for management decisions in ongoing Pallid Sturgeon conservation.