Population Genomics Analysis Revealed Origin and High-altitude Adaptation of Tibetan Pigs

Abstract

Tibetan pig is native to the Qinghai-Tibet Plateau and has adapted to the high-altitude environmental condition such as hypoxia. However, its origin and genetic mechanisms underlying high-altitude adaptation still remain controversial and enigmatic. Herein, we analyze 229 genomes of wild and domestic pigs from Eurasia, including 63 Tibetan pigs, and detect 49.6 million high-quality variants. Phylogenomic and structure analyses show that Tibetan pigs have a close relationship with low-land domestic pigs in China, implying a common domestication origin. Positively selected genes in Tibetan pigs involved in high-altitude physiology, such as hypoxia, cardiovascular systems, UV damage, DNA repair. Three of loci with strong signals of selection are associated with EPAS1, CYP4F2, and THSD7A genes, related to hypoxia and circulation. We validated four non-coding mutations nearby EPAS1 and CYP4F2 showing reduced transcriptional activity in Tibetan pigs. A high-frequency missense mutation is found in THSD7A (Lys561Arg) in Tibetan pigs. The selective sweeps in Tibetan pigs was found in association with selection against non-coding variants, indicating an important role of regulatory mutations in Tibetan pig evolution. This study is important in understanding the evolution of Tibetan pigs and advancing our knowledge on animal adaptation to high-altitude environments.

Figure 1

Geographic distribution and population genetic analysis of the Tibetan pigs and other Chinese pigs. (A) Sites origin for the different Chinese breeds and wild boars. Sampling sites of the wild boar only include newly sequenced samples. (B) Whole-genome Neighbor-Joining tree of pigs in this study. The branch length of OG artificially shortened and is shown as a dashed line. (C) Principal component analysis. PC1 is the first principal component. PC2 is the second principal component. (D) Population structure analysis with K from 2 to 5. Abbreviations (B,D) defined as follow. OG: outgroup (Sumatra wild boar).

Figure 2

Selection signals on the autosomes of the Tibetan pigs. (A) Genome-wide F_ST values in the Tibetan pigs. The black dotted line represents top 99% threshold of F_ST at whole-genome level. (B) XP-EHH values of the autosomes in the Tibetan pigs. Each dot represents the average value of a10-kb sliding window. The red dotted line represents top 99% threshold of XP-EHH at whole-genome level.

Figure 3

Selective signals and haplotypes of EPAS1. (A) F_ST values of each SNP between the Tibetan and control populations. The x axis is the physical position on chromosome 3 (Sus scrofa 10.2 build). The region between the two green dashed lines is the candidate sweep. The red box defines the SNPs with the largest genetic differentiation, used to analyze haplotypes. (B) Haplotype pattern of highly differentiated SNPs between the Tibetan and control pig populations using 227 pigs from East Asia and Europe. Each column is a polymorphic genomic location and each row is a phased haplotype. The blue cell represents the ancestor allele and the red cell represents the derived allele. OG: outgroup, TP: Tibetan pigs, CDP: Chinese domestic pig, CWB: Chinese wild boar, EDP: European domestic pig, EWB: European wild boar. (C) The Median-Joining network of haplotypes within EPAS1. Only haplotypes with frequency more than 1 were used to draw the network.

Figure 4

Enrichment of variants under different divergence levels and transcription activity assay of alleles in predicted motifs near EPAS1. (A) Enrichment pattern of SNPs at whole genome level. SNPs were divided to different bins by F_ST between Tibetan and lowland pigs. (B) Enrichment pattern of SNPs from selected sweep regions in Tibetan pigs. (C) Selective signals of EPAS1. The black vertical bar in downstream of EPAS1 indicated differentiated SNP positions between Tibetan and lowland pigs. The red dotted line represents top 99% threshold of F_ST at whole-genome level. (D) Predicted motifs with differentiated SNPs in downstream of EPAS1. (E) Transcription activity assay of different alleles within predicted motifs near EPAS1 in pig lung fibroblast cell. AA names of different pGLS3 vectors means ancestral allele and DA means derived allele. (F) Transcription activity assay of different alleles within predicted motifs near EPAS1 in BEAS-2B cell. The two-tailed t test was used for statistical assessment of transcription activity change.