Background/Objectives: The domestication of the grey wolf (Canis lupus) and subsequent creation of modern dog breeds have significantly shaped the genetic landscape of domestic canines. This study investigates the genomic effects of hybridization and breeding management practices in two hybrid wolfdog breeds: the Czechoslovakian Wolfdog (CSW) and the Saarloos Wolfdog (SAW). Methods: We analyzed the genomes of 46 CSWs and 20 SAWs, comparing them to 12 German Shepherds (GSHs) and 20 wolves (WLFs), which served as their ancestral populations approximately 70-90 years ago. Results: Our findings highlight that hybridization can increase genetic variability and mitigate the effects of inbreeding, as evidenced by the observed heterozygosity levels in both wolfdog breeds. However, the SAW genome revealed a higher coefficient of inbreeding and longer runs of homozygosity compared to the CSW, reflecting significant inbreeding during its development. Discriminant Analysis of Principal Components and fixation index analyses demonstrate that the CSW exhibits closer genetic proximity to the GSH than the SAW, likely due to differences in the numbers of GSHs used during their creation. Maximum likelihood clustering further confirmed clear genetic differentiation between these hybrid breeds and their respective ancestors, while shared ancestral polymorphism was detectable in all populations. Conclusions: These results highlight the role of controlled hybridization with captive-bred wolves and peculiar breeding strategies in shaping the genetic structure of wolfdog breeds. To ensure the long-term genetic health of these breeds, it is recommended to promote adequate and sustainable breeding practices to maintain genetic diversity, minimize inbreeding, and incorporate the careful selection of unrelated individuals from diverse lineages, while avoiding additional, uncontrolled crossings with wild wolves.

Biotechnical Faculty University of Ljubljana Jamnikarjeva ulica 101 1000 Ljubljana Slovenia

Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia Catedràtic José Beltrán 2 46980 Paterna Spain

Department of Animal Science and Food Processing Faculty of Tropical AgriSciences Czech University of Life Sciences Prague Kamýcká 129 16500 Prague Czech Republic

Department of Biomedical Sciences Cornell University Ithaca NY 14853 USA

Department of Zoology Charles University Viničná 7 12843 Prague Czech Republic

Unit for Conservation Genetics Via Cà Fornacetta 9 40064 Ozzano dell'Emilia Italy

WWF Italy Via Po 25c 00198 Rome Italy

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