Integrated multi-omic and functional evidence uncover substantial strain-specific divergence in zebrafish

The zebrafish (Danio rerio) is a foundational vertebrate model in biomedical and developmental research, yet genetic divergence among laboratory strains remains an underappreciated source of biological and experimental variation. Among these, the AB and Tübingen (TU) strains are used globally, forming the basis for most zebrafish-based discoveries. Here, we integrated long- and short-read sequencing, ATAC-seq, Hi-C, transcriptomics, and functional assays to generate a comprehensive multi-omic comparison of these two strains. Our analyses revealed substantial genomic and regulatory divergence, including a 62-bp insertion in the asmt promoter that elevates transcription and alters melatonin biosynthesis, driving strain-specific circadian phenotypes. We also identify a non-synonymous variant (mybbp1aG2612C) associated with heightened immune responsiveness. Together, these findings construct the first genome-wide atlas of functional variation between zebrafish strains, illuminating how hidden genetic differences can shape phenotype, regulatory architecture, and experimental reproducibility in this essential vertebrate model.