The Tibetan Plateau is the largest and highest plateau in the world, with an average elevation exceeding 4000 m. This extreme environment is characterized by low oxygen levels, low atmospheric pressure, low temperatures, and intense ultraviolet (UV) radiation (1). High-altitude regions are significant socioeconomic habitats, particularly for ruminant livestock, which serve as a primary source of food and income for local communities. However, because of harsh environmental conditions and limited forage resources, ruminants face severe survival challenges (2). Nevertheless, some ruminant species, such as goats (3, 4), sheep (5), and yaks (6, 7), demonstrate remarkable adaptive capabilities, enabling them to thrive in these extreme environments. One important ruminant of this landscape, the Tibetan sheep (Ovis aries), has evolved a hypoxia-adaptive mechanism that deserves special attention.

As one of the three major coarse-wool sheep breeds in China, Tibetan sheep primarily inhabit plateau regions characterized by extreme conditions, such as severe cold, hypoxia, and intense UV radiation. They survive in harsh environments with remarkable adaptability, providing essential resources (e.g., meat, wool, and leather) that play a vital role in the development of local animal husbandry practices (8). Through long-term natural selection and artificial breeding, Tibetan sheep have developed unique anatomical and physiological traits to thrive in this habitat, making them an ideal animal model for studying high-altitude hypoxia adaptation (9).

In recent years, rapid advances in high-throughput sequencing technology have spurred the widespread use of whole-genome resequencing. Specifically, detecting whole-genome selection signals has become a crucial research area, precisely revealing which genes and their genomic regions have been significantly influenced by natural selection or artificial breeding. In-depth analysis of selective signals facilitates a comprehensive understanding of the genetic traits that species develop during adaptation to natural environments. It also allows for the precise identification of genetic factors closely related to production traits, which can be used in animal genetic breeding programs to enhance production performance (10). Selection signal analysis has become an important research method for unraveling the genetic mechanisms underlying important economic traits in Tibetan livestock, such as sheep, goats, and horses. For instance, it has led to the identification of multiple genes associated with high-altitude adaptation, including EPAS1 in Tibetan horses (11), FGF10, MMP14, SLC25A51, ALAS1, PRMT1, HMOX2, and HIF1AN in Tibetan sheep (5), and FGF2, EGFR, AKT1, PTEN, MITF, ENPEP, SIRT6, KDR, and CDC42 in Tibetan goats (4). Additionally, EPAS2, which is associated with plateau adaptation, and IRF2 and EXOC2, which are related to coat color, have been identified in goat breeds (12), further deepening our understanding of their adaptation mechanisms.

Livestock play a crucial role in human society. These domesticated animals were originally domesticated from wild animals. Through long-term natural selection and artificial breeding, they gradually evolved into diverse breeds that are adapted to local environments and meet human needs. During this process, distinct selection signatures have been accumulated in their genomes (13). By analyzing these selective signals, functional genes related to important traits can be identified, providing a theoretical basis for breed improvement. Tibetan sheep exhibit remarkable adaptability to extremely high-altitude environments, demonstrating excellent survival and reproduction performance. In this study, we investigated three populations of Tibetan sheep from different altitudes: Tao (TS, 2887 m), Tianjun White Tibetan (WT, 3331 m), and Huoerba (HB, 4614 m). Whole-genome resequencing was used to assess population differentiation via population differentiation index (Fst) values and nucleotide diversity (θπ) ratios, enabling selection signal analysis. The results of this study provide a scientific basis for sheep breeding and husbandry strategies that aim to improve breed adaptation to different altitudes, thereby enhancing production efficiency and economic benefits.

Extreme environments marked by hypoxia, low temperatures, and UV radiation pose significant survival challenges for plateau-dwelling organisms, limiting the development of animal husbandry in these regions. In response to these environmental pressures, animals on the Tibetan Plateau have evolved unique morphological and physiological characteristics. Tibetan sheep are an important source of germplasm on the Tibetan Plateau, featuring strong adaptability to cold and hypoxic environments and serving as an important economic asset for farmers and herders in the region. Native animals, such as Tibetan sheep, serve as excellent models for studying the molecular regulatory mechanisms involved in adaptation to high-altitude environments. Therefore, to reveal genome-wide selection signals related to altitude adaptation, we conducted whole-genome resequencing of three Tibetan sheep populations from different altitudes. A sequencing depth of 6.2 × enhanced the reliability of variant annotation, mutation site identification, and exploration of genome structure, while the TS/TV ratio of 2.01 indicated a well-balanced and relatively conserved genome structure (25), providing a reliable dataset for future studies on population structure and selection signals.

For this study, we analyzed the population structure and genetic background of three Tibetan sheep populations from different altitudes using PCA, an N-J tree, and ADMIXTURE analysis. Firstly, PCA was used to investigate the clustering of the selected samples. The three populations were relatively close in genetic space, with the branches of the WT and HB populations being close, while the TS population was farther away, which is consistent with the results of the N-J tree. Because PCA and N-J tree are often used in conjunction with model-based clustering methods, we also employed ADMIXTURE analysis on the selected samples. Cross-validation error rates were then compared across different K values, revealing a K value of 2 as the optimal modeling choice. The TS population exhibited separation from the other two, with a predominantly dark blue background. As the K value was increased, the population structure exhibited a mixed state with a diverse genetic composition and relatively complex origins. Our findings point to obvious gene flow among the different Tibetan sheep populations, which is consistent with their geographical locations. The results obtained through mutual verification of the three methods were consistent, indicating the reliability of the data.

Altitude adaptation is a complex process resulting from the combined action of multiple genes and pathways (26). When individuals live at high-altitudes for an extended period, their bodies undergo a series of adaptive changes to maintain normal physiological functions and stabilize the internal environment. These changes involve multiple mechanisms such as energy metabolism, hypoxic response, and cardiovascular function (27). In this study, altitude adaptation-related genes were found to be mainly concentrated in metabolic pathways (GSTA1, ALAS1, HMOX2, SCD, ME1, ACSL6, PIK3C2G), melanogenesis (MITF, EP300), and the HIF-1 signaling pathway (HIF1A, ERBB2, RELA). The hypoxic conditions of high-altitude environments can lead to mitochondrial dysfunction and increased oxidative stress (28, 29). GSTA1 (Glutathione S-transferase alpha 1) is a widely expressed enzyme that plays a central role in alleviating oxidative stress and detoxification, particularly in the process of scavenging hydrogen peroxide (30, 31). The gene expression and activity of GSTA1 are modulated under hypoxic conditions, potentially helping cells adapt to low-oxygen environments through its antioxidant and detoxification functions. Heme is a cofactor for various proteins (e.g., hemoglobin) and plays an important role in multiple processes, including oxygen transport, electron transfer, and detoxification. The activity of the rate-limiting enzyme in heme synthesis, ALAS1 (5-aminolevulinate synthase 1), directly affects heme production, which is crucial for oxygen transport and uptake. HMOX2 (Heme oxygenase 2) plays an important role in heme metabolism, catalyzing the decomposition of heme into carbon monoxide (CO), a crucial gaseous signaling molecule. CO plays a significant role in regulating vascular relaxation and oxidative stress, thereby influencing oxygen utilization and cellular metabolism (32). Additionally, HMOX2 also plays a key role in angiogenesis regulation, particularly in maintaining endothelial cell function and vascular stability (33). The enzyme encoded by the SCD (Stearoyl-CoA desaturase) gene catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids. In the plateau lizard Phrynocephalus vlangalii (34), upregulated expression of SCD promotes fatty acid synthesis and storage, helping larvae adapt to high-altitude environments. ME1 (Malic enzyme 1) is a key metabolic enzyme that catalyzes the conversion of malate to pyruvate while generating NADPH (35), an important cellular reducing agent that participates in various biosynthetic and antioxidant reactions. In high-altitude, low-oxygen environments, the activity of ME1 is thought to affect the cellular redox state and metabolic pathways by regulating the NADPH/NADP+ ratio (36, 37). ACSL6 (Acyl-CoA synthetase long chain family member 6), a key enzyme for the uptake and transport of free fatty acids, regulates the expression of a series of downstream genes involved in lipid metabolism (38). ACSL6 has been associated with lipid metabolism under hypoxic stress (39), suggesting that adaptation to low-oxygen environments may depend on enhanced fatty acid oxidation. Additionally, a study compared copy number variations between high-altitude and low-altitude cattle breeds in Ethiopia. It was found that the PIK3C2G (Phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 gamma) gene exhibited significant differentiation in high-altitude cattle breeds (40). This finding in cattle, further supported by similar observations in pigs (41), suggests that PIK3C2G may be associated with high-altitude adaptation, and indicates its possible importance in Tibetan sheep adaptation to high-altitude, low-oxygen environments. Collectively, these genes appear to play crucial roles in enabling organisms to effectively cope with the challenges posed by low-oxygen environments through the regulation of various adaptive mechanisms.

Generally, UV radiation is stronger at high-altitudes, potentially leading to DNA damage in skin tissues. In this study, we found that the MITF (Microphthalmia-associated transcription factor) gene is significantly enriched in the melanogenesis pathway, which is related to UV resistance (42), with similar research conducted in Tibetan goats (4). Hu et al. (2) found that MITF may be a candidate gene for high-altitude adaptation in sheep and plays a significant role in the adaptive gradual infiltration from argali to Tibetan sheep (43). Alternatively, populations residing at high altitudes have been shown to exhibit strong positive selection signals for the EP300(E1A binding protein p300) gene, which plays a crucial role in the regulation of sensory cell damage (44). Furthermore, there is a significant correlation between the expression level of EP300 and the content of nitric oxide (NO) in the blood. Specifically, EP300 may enhance the adaptability of Tibetan populations to hypoxic environments by increasing the level of NO (44). In summary, MITF and EP300 may contribute to the body’s response to hypoxic challenges in high-altitude environments.

Hypoxia-inducible factor-1 (HIF-1) can be activated in hypoxic environments to influence the expression of many genes, playing a crucial role in regulating the stability of oxygen levels in the body (45). In this study, we identified enrichment of key genes in the HIF-1 signaling pathway that may play pivotal roles in high-altitude adaptation, namely HIF1A, ERBB2, and RELA. HIF1A, the core regulatory factor of the HIF-1 signaling pathway, is transcribed at low levels under normal oxygen conditions, but significantly increases in hypoxic states, which affects erythrocyte production, influencing processes such as cell proliferation and death, and leading to hypoxic adaptive responses (46). The ERBB2 (Tyrosine kinase receptor-2) gene plays a key regulatory role in the growth and drug resistance of breast cancer. Studies have shown that ERBB2 relies on HIF-1 to promote breast cancer growth in vivo, enhancing the adaptability of tumor cells to hypoxic environments through HIF-1-mediated signaling pathways, thereby generating oxygen resistance (47). Cyclooxygenase-2 (COX-2) is induced by hypoxic environments in vascular endothelial cells, with the induction process mediated by RELA (V-rel reticuloendotheliosis viral oncogene homolog A) (48). The interactions among these genes and their synergistic effects in the HIF-1 signaling pathway provide new insights into the molecular mechanisms of high-altitude hypoxic adaptation. However, their specific functions and regulatory networks in high-altitude adaptation require verification through further experimental studies.

This study reports genome-wide selection signals in Tibetan sheep populations at high, medium, and low altitudes using comprehensive Fst and θπ ratio analyses. Adaptation to high altitudes was found to involve genes associated with metabolic pathways (GSTA1, ALAS1, HMOX2, SCD, ME1, ACSL6, PIK3C2G), melanogenesis (MITF, EP300), and the HIF-1 signaling pathway (HIF1A, ERBB2, RELA). These genes are primarily linked to energy metabolism, angiogenesis, the hypoxic response, and UV protection. Our findings provide data for further exploration of the hypoxic adaptation mechanisms of Tibetan sheep, and serve as a reference for the prevention and treatment of high-altitude illnesses in humans.