Gene-set enrichment analysis to identify genomic regions associated with environmental adaptation in some Iranian sheep breeds

Extended Abstract
Introduction: Animal breeding in sheep, is significantly influenced by environmental adaptations that allow different breeds to thrive in diverse ecological conditions. Understanding the genetic basis for such adaptations can greatly enhance breeding programs aimed at improving resilience and productivity. Various indigenous sheep breeds exhibit unique traits suited for their respective habitats in Iran. This study aims to identify genomic loci associated with environmental adaptation through genome-wide association studies (GWAS) based on gene-set enrichment (GSE) analysis. By leveraging high-density SNP arrays, we explore the genetic mechanisms underlying thermotolerance, altitude adaptation, and coat type differentiation, which are critical for sustaining sheep production under Iran’s heterogeneous climates. These adaptations are particularly relevant given Iran’s diverse topography, where temperatures range from -20°C in mountainous regions to +50°C in deserts, creating strong selective pressures on local breeds.
Materials and Methods: The Illumina HD ovine SNP600K BeadChip genomic arrays were utilized to analyze 139 animals from nine Iranian sheep breeds, namely Kermani (n=15), Sanjabi (n=14), Lori-Bakhtiari (n=15), Qezel (n=15), Gharagol (n=15), SiahKabod (n=15), GrayShiraz (n=9), Afshari (n=14), Shal (n=15) and Baluchi (n=12). Animals were grouped based on three adaptive traits: (1) tropical vs. cold climate resilience, (2) high vs. low altitude distribution, and (3) wool vs. skin coat type. Quality control filters included minor allele frequency (MAF) > 0.05, SNP call rate > 95%, and removal of markers with unknown genomic positions. To assess the relationship between specific traits and SNP variations across the genome, GWAS was conducted based on logistic regression models using PLINK software (v 1.9), with principal component analysis (PCA) to correct for population stratification. SNPs were assigned to respective genes based on their locations within the genomic sequence or within a flanking region of 50 kb upstream and downstream of each significant SNPs. Following this analysis, we employed various bioinformatics databases to interpret gene sets and ascertain their biological functions related to selected genomic regions.
Results and discussion: The results of the GWAS showed that 2431, 2244 and 2145 SNP markers were associated with adaptation to tropical-cold environmental conditions, distribution in areas with high-low height of sea level and wool-skin body covering in Iranian indigenous sheep, respectively (P-value ≤ 0.05). Gene-set enrichment analysis identified, the biological pathways (and candidate genes) of defense response to gram-positive bacterium (HSPA4L, DNAJB4, MSRB3), regulation of immunoglobulin mediated immune response (IL27A, TRAF3IP2, LY96), regulation of muscle system process and cell junction (BMP2, THBS1, MYH10), regulation of anatomical structure size (FGF2, ACTR3), regulation of ossification and organ growth (TMEM117, PTBP1) and skin epidermis development (KR25, KR27, KRT71). The pathways identified in the current study for adaptation traits in indigenous breeds, played an important role in the regulation of immune system, muscle structure development, osteoclast differentiation, body size, and regulation of wool growth. The insights gained from this study greatly extend our understanding of how specific genetic determinants contribute toward functional adaptations in Iranian sheep breeds under diverse environments. For example, the identification of HSPA4L in thermotolerance aligns with its known role in heat shock response, while novel candidates like TMEM117 may represent breed-specific adaptations. Some genes have also not a well-established biological role, and there may be potential mutual effects that are not yet understood in this context. Therefore, to determine the precise role of these genes, it is essential to conduct further comprehensive functional studies and biological system analyses.
Conclusion: In general, our findings provide valuable knowledge on genomic regions linked with vital adaptation traits among indigenous Iranian sheep breeds. The integration of GWAS and pathway analysis revealed not only expected candidates (e.g., KRT genes for wool) but also novel genes (e.g., LY96 for immunity) that broaden our understanding of genomic adaptability. These results may serve future research endeavors focused on dissecting genetic architectures underlying adaptability, particularly for marker-assisted selection in breeding programs. However, the study’s limitations, including sample size and the need for functional validation of candidate genes, highlight opportunities for future work. A pivotal component influencing successful breeding strategies aimed at improving these animals' resilience while considering changing global climates.
Keywords: Genome wide association analysis (GWAS), Pathway analysis, Candidate gene, Sheep
Conflicts of interest: The authors declare no conflicts of interest.
Funding: The authors received no specific funding for this project.