Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia

Abstract

The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes.

Figure 1.

Genetic relationships and population structure of the 60 dogs based on all autosomal SNPs. (A) Principal component plot. The first (PC1) and second component (PC2) are shown in the main figure, while the third (PC3) and fourth component (PC4) are shown in the inset. The percentages indicate the proportion of variance explained by each component. (B) Unrooted neighbor-joining tree. The evolutionary distance is measured by the number of net nucleotide substitutions between individuals. (C) Population structures with the number of ancestral clusters K from 2 to 6. Each color represents one ancestral cluster and each vertical bar represents one dog. The length of colored segments represents corresponding ancestry attributions.

Figure 2.

Selective sweep analysis of the breeds from different altitude levels. (A) Manhattan plot of F_ST among the high- (TM, DQ), middle- (LJ), and low- (YJ, KM, GS) altitude breeds. The F_ST was calculated for each 100-kb autosomal window. The dashed line denotes a threshold of Z(F_ST) = 5. (B) Diversity π and iHH around the EPAS1 loci. The region with Z(F_ST) > 5 is shaded. π and iHH were calculated for each 10-kb window and Z-transformed on the genome scale. The values were smoothed by lowess regression. (C) π and iHH around the beta hemoglobin loci.

Figure 3.

EPAS1 mutations in the coding region. (A) Structural and evolutionary analysis of the four amino acid variants. The protein coordinate is based on Ensembl ID ENSCAFP00000003819. The coordinate of NCBI RefSeq XP_531807.2 is slightly different but the variants remain the same. The upper panel shows the Pfam domains of the protein. The orthologous protein sequences from 17 vertebrates are aligned with the mutant residues shown in red. The NJ tree derived from the multiple alignment is shown in the left panel. The red stars indicate two hydroxylation sites (Pro405 and Pro531), which are essential for oxygen sensing (Patel and Simon 2008). (PAS) Per-Arnt-Sim; (HIF) hypoxia-inducible factor; (CTAD) C-terminal transactivation domain. (B) NJ tree of the 8-kb haplotypes comprising the four nonsynonymous mutations. The evolutionary distance is defined as the number of different nucleotides between haplotypes. The haplotypes from high- and low-altitude breeds were grouped into two distinct clades. (C) Percentages of homozygotes and heterozygotes in the six breeds. The mutant and reference alleles are represented by “+” and “−”, respectively. Besides the 60 dogs sequenced with the Illumina technology, additional samples were genotyped with Sanger sequencing. The sample size is shown beside the bars. (D) Association between EPAS1 genotypes (mutant allele, A; reference allele, G) and blood flow resistance in DQ. The blood flow resistance was measured at different shear rates. In the boxplot, the line within the box defines the median; the ends of the boxes define the 25th and 75th percentiles; and the error bars define the 10th and 90th percentiles. The ANOVA F-test was performed, taking the age as covariant.