To date, there are many reports regarding CNVs of sheep using aCGH, SNP array and genome resequencing. Due to the difference in CNV detection platforms, the quantity of detected CNVs varies widely in different studies. In terms of the CNVs captured by aCGH and SNP chips, the number usually ranges from dozens to hundreds, and the average length is approximately 100-300 kb [28–32]. Using whole genome resequencing, Cheng et al [33] reported that 4301 CNVRs were identified in three Chinese breeds. A total of 7228 CNVRs were obtained, including 861 duplications, 6345 deletions, and 22 both events in Chinese indigenous fine-wool sheep [18]. Similarly, there were differences in the amounts of CNVR, and the proportion accounted for chromosomes. In the present study, we obtained 2777 CNVRs, including 1965 duplications and 812 deletions. These CNVRs accounted for ∼0.3% of the sheep reference genome. This coverage ratio is comparable with previous reports [29, 33] but lower than the 10% reported by Salehian-Dehkordi et al. [34]. There is no rule about which (duplication or deletion) happened more frequently in previous studies. In addition to the detection platform, the reason for these variations may be the differences in calling algorithms and standards. Meanwhile, the breed and sample size used in the study were other considerable factors [35].
The GO enrichment analysis showed that the detected CNVRs harboring genes shared by the three populations were significantly enriched in ion transport, sensory perception system, gas transport and pigmentation. Among them, sensory perception was also significantly enriched in yaks [36, 37] and other sheep breeds [18, 29]. The three TS populations live in alpine grasslands where the weather conditions are much harsher. As yak, TS faced the same living conditions as the lack of herbage in the cold season. The well-developed sensory perception system was important to improve the yak and sheep’s ability to acquire food and avoid noxious weeds. The gas transport term was also enriched in TS, and efficient oxygen exchange may facilitate hypoxia adaptation. In the KEGG pathway analysis, it is noteworthy that the CNVR-harboring genes shared by the three populations were significantly enriched in nutrition metabolism, ABC transporters, disease defense and hematopoietic cell lineage. The enrichment of nutrient metabolism terms, including nitrogen metabolism, citrate cycle, and bile secretion, was important for the digestion of nutrients in TS, especially in the cold season. Similar results showing the enrichment of CNVR-harboring genes in ABC transporters were also reported in humans [38, 39], cattle [10, 40, 41] and goats [42]. In mammals, ABC transporters can carry a broad array of endogenous metabolites, such as amino acids, peptides, and sugars, across lipid membranes, which facilitate the absorption and utilization of these nutrients. Hypoxia can promote hematopoiesis. Here, CNVR-harboring genes were enriched in the hematopoietic cell lineage in Tibetan sheep, which may make it suitable for their adaptation to high altitudes with low oxygen. Overall, the number of enriched CNVR overlapping genes associated with forage consumption and oxygen transport may be helpful for their adaptation to the local environment.
Notably, the olfactory transduction pathway was significantly enriched specifically in OTSs. Enrichment of the olfactory transduction pathway has been reported in cattle [11, 16, 43, 44], yaks [45], sheep [18, 28, 46] and goats [42]. It has been revealed to influence food consumption [47] and as a factor to assess feed efficiency and performance in crossbred beef cattle [48] and residual feed intake in pigs [49]. Meanwhile, more CNVR havrboring genes were enriched in amino acid and VFA metabolism pathways in the OTS. OTS has a much better meat performance than HTS and VTS. More CNVR harboring genes enriched in the olfactory transduction pathway, together with protein and energy metabolism pathways, may explain the better production performance in OTS.
QTLs, which contain genetic variants affecting the economic traits of domestic animals, can be used to select candidate CNVR-harboring genes that affect phenotypes in sheep. After integrating CNVs into QTLs, we found 188 CNVRs overlapping with 97 sheep QTL regions in this study. Many CNVs harboring genes, such as PCDH15, APP and GRID2, are located in growth and carcass QTL regions. The PCDH15 gene was identified to be associated with the concentration of the neurotransmitter glutamate (Glu) in cattle [50]. APP is one of the well-known pathogeneses in Alzheimer's disease. Zheng et al. indicated that homozygous APP deficiency leads to a 15–20% reduction in body weight [51]. An et al. reported that adipocyte-specific and mitochondria-targeted APP-overexpressing mice display increased body mass [52]. GRID2 was also identified as being associated with body weight in rats [53].
Selective sweeping can reveal putative regions that undergo environmental and artificial selection during local adaptation and domestication. To screen the critical CNVR significantly divergent between different populations, the pairwise Vst value was estimated [18, 50, 54]. Here, the five CNVR horboring genes RUNX1, LOC101104348, LOC105604082, PAG11 and LOC101111988 showed significant pairwise differentiation among HTS, OTS and VTS. It is well known that RUNX1 plays a crucial role in hematopoiesis, leukemogenesis and neural development. Lin et al. reported that GWAS hit SNPs associated with colostrum albumin concentration were enriched in RUNX1 in Chinese Holsteins, and these mutations might initiate the hyperactivation of inflammatory and innate immunity [55]. The GWAS hit SNP within RUNX1 is associated with the mean corpuscular volume level in pigs [56]. Moreover, HIF-1α facilitates RUNX1 transcriptional activity under hypoxic conditions and triggers hematopoietic stem cell differentiation, which ultimately improves oxygen transport to peripheral tissues [57]. Chaka sheep were a cultivated breed mainly with Tibetan sheep as basic ewes and adapted to a low oxygen environment for a long time. Hence, we speculated that CNVR-harboring RUNX1 underwent strict selection pressure in Tibetan and Chaka sheep, possibly helpful for their adaptation to a low oxygen environment at high altitudes. PAG11 is a pregnancy-associated glycoprotein that is expressed in the trophoblast of the ruminant placenta and influences embryo growth and survival [58]. HTS should face a shortage of forage and cold environments in their late pregnancy stage, in contrast to VTS. We thought the divergence of CNVR-harboring PAG11 between HTS and VTS may be related to their different habitat conditions, especially in the pregnancy stage. Notably, the ASIP gene, duplication of CNVR-harboring LOC101111988, located upstream of ASIP, was divergent between VTS vs OTS (Vst = 0.489) and HTS vs OTS (Vst = 0.284). The ASIP gene has been widely studied in mammals for its effect on animal coat color. Individuals with normal or duplication alleles of the ASIP gene are generally white or gray coat color, but individuals with normal or single deletion alleles in ASIP almost entirely have solid-black coat color in sheep [59–61]. In our study, the duplication of the LOC101111988 gene in VTS and HTS might account for their mainly white coat color. Meanwhile, the deletion in LOC101111988 CNV might be the basis for the primary brown covering color, especially in the neck in OTS.