Evolution of genes and gene families

To examine the mutation rate and type of protein-coding genes that show accelerated evolution in pigs, we identified ∼9,000 as 1:1 orthologues within a group of six mammals (human, mouse, dog, horse, cow and pig). This orthologous gene set was used to identify proteins that show accelerated evolution in each of these six mammalian lineages (Supplementary Information). The observed number of synonymous substitutions per synonymous site (dS) for the pig lineage (0.160) is similar to that of the other mammals (0.138–0.201) except for the mouse (0.458), indicating similar evolutionary rates in pigs and other mammals. The observed dN/dS ratio (ratio of the rate of non-synonymous substitutions to the rate of synonymous substitutions) of 0.144 is between those of humans (0.163) and mice (0.116), indicating an intermediate level of purifying selection pressure in the pig. Genes showing increased dN/dS ratios in each lineage were analysed using DAVID8 to examine whether these rapidly evolving genes were enriched for specific biological processes. Most lineages show different fast-evolving pathways, but some pathways are shared (Fig. 1).

Figure 1: Phylogeny of the six mammals used in the dN/dS analysis. KEGG pathways with genes that show accelerated evolution for each of the six mammals used in the dN/dS analysis. The bar charts show the individual dN/dS and dS values for each of the six mammals. The dN/dS and dS values refer to the time period of each of the six individual lineages. The number of proteins that show significantly accelerated dN/dS ratios in each lineage varies from 84 in the mouse to 311 in the pig lineage. Pathways significantly (P < 0.05) enriched within this group of genes are also shown with the number of genes shown in brackets. HPI, Helicobacter pylori infection. PowerPoint slide Full size image

Immune genes are known to be actively evolving in mammals9,10. Because many immune genes were not included in the analysis of 1:1 orthologues, we examined a randomly selected subset of 158 immunity-related pig proteins for evidence of accelerated evolution (Supplementary information). Twenty-seven of these genes (17%) demonstrated accelerated evolution (Supplementary Table 8). A parallel analysis of 143 human and 145 bovine orthologues revealed very similar rates of evolution (18% in human and 12% in cattle, respectively). Using a branch-site analysis, we detected accelerated evolution of amino acids in PRSS12, CD1D and TRAF3 specific to pig (positive selection on pig branch), as well as amino acids in TREM1, IL1B and SCARA5 specific to pig and cow (positive selection on the cetartiodactyl branch).

Further analysis of porcine immune genes (Supplementary Table 5) revealed evidence for specific gene duplications and gene-family expansions (Supplementary Tables 6 and 7). The analysis of this second cetartiodactyl genome indicates that some expansions are cetartiodactyl-specific (cathelicidin) whereas others are ruminant/bovine-specific (β-defensins, C-type lyzozymes) or potentially porcine-specific (type I interferon, δ subfamily).

Pigs have at least 39 type I interferon (IFN) genes, which is twice the number identified in humans and significantly more than in mice. We also detected 16 pseudogenes in this family. Cattle have 51 type I IFNs (13 pseudogenes), indicating that both bovine and porcine type I IFN families have undergone expansion. This is particularly important for interferon subtypes δ (IFND), ω (IFNW) and τ (IFNT); pigs and cattle are evolving species-specific subtypes of IFND and IFNT, respectively. Both species are expanding the IFNW family and share many more IFNW isoforms than other species. Thus, expansion of interferon genes is not ruminant-specific as proposed earlier10, although duplication within some specific sub-families seems to be either bovine- or porcine-specific.

Within the immunity-related genes annotated, we found evidence for duplication of six immune-related genes: IL1B, CD36, CD68, CD163, CRP and IFIT1, and one non-immune gene, RDH16. The CD36 gene is also duplicated in the bovine genome, whereas the IL1B gene duplication, where evidence for a partial duplication was reported previously11, is unique in mammals. Other key immune genes in the major histocompatibility complex, immunoglobulin, T-cell-receptor and natural killer cell receptor loci have been characterized in detail12,13,14,15,16,17,18,19 (Supplementary Information).

Another significant porcine genome expansion is the olfactory receptor gene family. We identified 1,301 porcine olfactory receptor genes and 343 partial olfactory receptor genes20. The fraction of pseudogenes within these olfactory receptor sequences (14%) is the lowest observed in any species so far. This large number of functional olfactory receptor genes most probably reflects the strong reliance of pigs on their sense of smell while scavenging for food.

Conservation of synteny and evolutionary breakpoints

Alignment of the porcine genome against seven other mammalian genomes (Supplementary Information) identified homologous synteny blocks (HSBs). Using porcine HSBs and stringent filtering criteria, 192 pig-specific evolutionary breakpoint regions (EBRs) were located. The number of porcine EBRs (146, Supplementary Table 11 and Supplementary Fig. 16) is comparable to the number of bovine-lineage-specific EBRs (100) reported earlier using a slightly lower resolution (500 kilobases (kb)), indicating that both lineages evolved with an average rate of ∼2.1 large-scale rearrangements per million years after the divergence from a common cetartiodactyl ancestor ∼60 Myr ago2. This rate compares to ∼1.9 rearrangements per million years within the primate lineage (Supplementary Table 11). A total of 20 and 18 cetartiodactyl EBRs (shared by pigs and cattle) were detected using the pig and human genomes as a reference, respectively.

Pig-specific EBRs were enriched for LTR endogenous retrovirus 1 (LTR-ERV1) transposons and satellite repeats (Supplementary Table 12), indicating that these two families of repetitive sequences have contributed to chromosomal evolution in the pig lineage. Different families of transposable elements seem to have been active in the cetartiodactyl ancestor. The cetartiodactyl EBRs are enriched for LINE1 elements and tRNAGlu-derived SINEs. tRNAGlu-derived SINEs, previously found over-represented in cetartiodactyl EBRs defined in the bovine genome10, originated in the common ancestor of cetartiodactyls21. Our observation that these elements are also enriched in porcine EBRs strongly supports the hypothesis that active transposable elements promote lineage-specific genomic rearrangements.

A stringent set of porcine to human one-to-one orthologues using the MetaCore database revealed that porcine EBRs and adjacent intervals are enriched for genes involved in sensory perception of taste (P < 8.9 × 10−6; FDR <0.05), indicating that taste phenotypes may have been affected by events associated with genomic rearrangements. Pigs have a limited ability to taste NaCl22. SCNN1B, a gene encoding a sodium channel involved in the perception of salty tastes, is located in a porcine-specific EBR. Another gene, ITPR3, encoding a receptor for inositol triphosphate and a calcium channel involved in the perception of umami and sweet tastes, has been affected by the insertion of several porcine-specific SINE mobile elements into its 3′ untranslated region (3′ UTR), consistent with our observation of a higher density of transposable elements in EBRs. In addition to 8 bitter taste receptor genes annotated by Ensembl and which were used in the gene enrichment analysis, we identified 9 intact genes, to give a total number of 17 TAS2R receptors in the pig (Supplementary Table 13). This compares to 18 intact bitter taste receptors in cattle, 19 in horse, 15 in dog and 25 in humans23,24. Of the 14 bitter taste receptor genes that were mapped to a specific pig chromosome (SSC), 10 were found near 2 EBRs on SSC5 and SSC18 (Supplementary Tables 13 and 15). We also found that at least four taste receptors (TAS1R2, TAS2R1, TAS2R40 and TAS2R39) have been under relaxed selection (Supplementary Information). Pigs are not sensitive to bitter tastes and tolerate higher concentrations of bitter compounds than humans22,25. Thus, pigs can eat food that is unpalatable to humans. A review of the porcine taste transduction network (Supplementary Fig. 17) revealed additional genes affected by rearrangements that affect ‘apical and taste receptor cell’ processes. Together with the observed over-representation of genes related to ‘adrenergic receptor activity’ and ‘angiotensin and other binding’ categories in the pig EBRs (Supplementary Fig. 18), our data indicate that chromosomal rearrangements significantly contributed to adaptation in the suid lineage.