Analysis Of Molecular Mechanisms Of High-altitude Adaptation In Tibetan Pig

Tibetan pig is a local pig breed in Qinghai-Tibetan plateau and has evolved excelent adaptation to the harsh envrioment in this plateau. The adaptation in Tibet, a complex trait with a polygenic basis, is shaped by pressures from both natural selction and domesticaiton. In this study, we genotyped Tibetan pig living at high altitude(3000 m), Dahe pig that is distributed in moderate alittude(2000 m) and Wuzhishan pig that is a low-land pig breed(200 m) using Porcine SNP60 chip array. We employed a population-genomics approach to identify highly differentiated genomic regions between high- /moderate- and low-altitude pigs, copy number variation regions, as well as gene-gene interactions which have association with high altitude adaptation. Meanwhile, we used RNA-seq to compare the gene expression of lung between Tibetan and Min pig, to systemically uncover the molecular mechanisms of high-altitude adaptation in Tibetan pig.The results of identifing highly differentiated genomic regions between high- /moderate- and low-altitude pigs revealed a total of 12 specific selective genes(CCBE1, F2RL1, AGGF1, ZFPM2, IL2, FGF5, PLA2G4 A, ADAMTS9, NRBF2, JMJD1 C, VEGFC and ADAM19) for Tibetan pig and six(OGG1, FOXM, FLT3, RTEL1, CRELD1 and RHOG) for Dahe pig. In addition, six adaptive genes(VPS13A, GNA14, GDAP1, PARP8, FGF10 and ADAMTS16) were shared by the two pig breeds. Gene function analysis suggested that Tibetan pig-unique candidates have particular roles in cardiovascular conditioning, and Dahe pig-unique candidates are mainly related to DNA repair for oxidative damage. Wherease, those shared condidates are associated wth blood circulation, DNA repair and eneger metabolism.The results of copy number varation(CNV) analysis revealed 5 CNV regions contating 5 genes related to homeostasis process have potentail assocation with high altitude adaptation in plateau pigs. These genes have functions related to temperature adaptation, energy metabolism, response to hypxia, lung function and cardiovascular system.We also characterized gene-gene interactions in Tibetan pig by using a gamete-based logistic regression, with Wuzhishan pig as the low-land reference population. We totally identified 5,834 significant gene pairs(FDR <0.001). Most of these genes have small/moderate differentiation between Tibetan and Wuzhishan pig based on FST anlaysis. We then constructed an epistasis network using these gene pairs and anlayzed the network topological properties. The results suggested two typical features of most biological networks, small world and scale free. Based on poisson distribution, we determined a total of 87 significant hubs genes. Among them, 13(ADRB1, RFC3, CDK7, NRG1, ISL1, RAG2, FLT1, NOX4, NDUFA12, the homolog of human SCNN1 B, TGFBI, MYC and CDCP) were highlighted as they have some biological functions relevant to high altitude adaptation. More interestingly, eight of them(NOX4, TGFB1, CDCP1, FLT1, MYC, CDK7, RFC3, and NGR1) were further found to act in an oxygen-related signaling network with the hypoxia-inducible factor 1(HIF1) complex.The comparison of gene expression of lung between Tibetan and Min pig reveald 281 significantly differentially expressed genes. Among them, 191(67.97%) were found to be down-regulated in lung of Tibetan pig. Wherase only 90(32.08%) were up-regulated in lung of Tibetan pig. The function enrichment analysis showd that these up-regulated genes in Tibetan were mainly associated with lung strucutre, cardiovascular systerm and inflammation, indicating that the down- and up-regulation of these genes maintaine the balance between injury and repair of lung/blood vessels. Meanwhile, we compared these significantly differentially expressed genes with maps for postive selection in Tibetan pig. We identified only six common genes, including RGS2, SCD, TLE2, SLCO4C1, CXCL13 and PRCP gene. This finding suggested that most of the gene expression changes were not individually driven by positive selection at cis-regulatory sites. The expression changes my be caused by genetic differences in trans, neutral differences in cis, and/or physiological correlates of adaptation. Our results also showed that the expression changes for positively selcted genes in Tibetan pig were overall small in magnitude, which may be attributed to the minor effects of many genes underlying the selective targets.In summary, the molecular mechanisms of high-altitude adaptation is complex, involving a large number of genes. The effects of these responsible genes are often minor. Those highly differentiated genes between Tibetan and low-land pig may act by chaning the protein structure or trans-regulatory roles. Meanwhile, some other genes with modest/small differentiation with low-land pigs, such as, genes involving in HIF pathway, may exert their effects by interacting with other genes. And some genes, such as genes associated with homeostasis process, may contribute to high altitude adaptation by changing copy numbers. The expression of most genes of lung in Tibetan pig are inhibited. Wherease, parts of genes associated with lung structure, cardiovascular systerm and inflammation are up-regulated. The down- and up-regulation of these genes maintain the balance between injury and repair of lung/blood vesseles under the extreme enviroments of Tibetan pleteau.