Genome-Wide Computational Analysis of miRNA Regulatory Variants Associated with Thermotolerance in Dairy Cows

Heat stress poses a critical threat to dairy cattle productivity, fertility and welfare, yet the heritable genetic mechanisms underlying individual variation in thermotolerance remain poorly understood. While previous studies have focused predominantly on miRNA expression profiles or coding gene variants, genome-wide identification of regulatory variants within miRNA genes has remained largely unexplored. In this study, whole-genome sequencing was applied to thermotolerant and thermosensitive Holstein cows, phenotypically classified based on reproductive performance under summer heat stress, to identify group-specific genetic variants within miRNA genes. Thermotolerant-specific variants (n=15) were identified across 15 distinct miRNA loci, including one novel SNP. Five variants were located within mature miRNA sequences, and several were associated with predicted changes in minimum free energy (ΔG), suggesting alterations in secondary structure and stability. Two seed-region variants in bta-mir-2883 and bta-mir-2388-3p were predicted to substantially reshape miRNA–mRNA target repertoires, with gains of approximately 3,000 target genes and losses of approximately 600 per miRNA. Functional enrichment analysis revealed a coordinated redistribution of miRNA-mediated regulation: gained targets were enriched in stress-responsive signaling pathways and metabolic adaptation, while lost targets were associated with intracellular trafficking, vesicle-mediated transport, and membrane dynamics. These findings imply that thermotolerance in dairy cows involves a heritable reorganization of post-transcriptional regulation supporting both controlled stress signaling and cellular flexibility. Collectively, these heritable miRNA variants expand the current toolkit for genomic selection by introducing regulatory, non-coding loci as actionable targets for breeding heat-resilient dairy cattle.