Comparative Whole Genome Analysis and Targeted Validation of Variants in Three Greek Indigenous Sheep Breeds

Indigenous sheep breeds represent valuable reservoirs of genetic diversity shaped by long-term adaptation to local environments and management systems. Greek autochthonous sheep breeds remain underrepresented in genomic and functional studies. The objective of this study was to characterize and compare coding sequence variation in three indigenous Greek sheep breeds-Lesvos (LES), Serres (SER), and Thrace (THR)-and to identify shared and breed-associated functional patterns. The study was designed using a two-stage approach, comprising a discovery (exploratory) phase and a validation phase. In the discovery phase, whole genome sequencing data (one animal per breed; total n = 3; mean sequencing depth ~36.9×) were analyzed to identify protein-altering exonic variants, focusing on missense single-nucleotide polymorphisms (SNPs) and exonic insertions/deletions (indels). Variants were examined at breed-specific and comparative levels, followed by functional enrichment analyses using Gene Ontology (GO) and KEGG pathways. Normalized variant density metrics identified genes with elevated polymorphism levels. In the validation phase, a subset of prioritized missense SNPs was genotyped in an independent cohort of 54 animals (18 per breed) using MassARRAY genotyping. Genes harboring prioritized missense SNPs showed a conserved enrichment profile across breeds, dominated by genome maintenance, DNA repair, cytoskeletal organization, and core regulatory functions. Distinct breed-associated patterns were also observed. LES showed enrichment in metabolic, biosynthetic, and sensory-related processes, SER in regulatory and signaling functions, and THR in cytoskeletal, extracellular matrix, and organelle-associated pathways. Polymorphism density analyses highlighted highly variable genes across breeds, including olfactory receptor (OR) gene families, keratin-associated protein genes (KRTAPs), and loci involved in immune and regulatory functions (e.g., PRKDC, CDH15). The validation phase confirmed the expected allele frequency patterns for most prioritized SNPs, supporting the robustness of the approach. This study identifies functionally relevant coding variation across Greek indigenous sheep breeds, revealing conserved genomic patterns and breed-associated signatures linked to metabolic, structural, and regulatory processes.