Lecture 29 - Immunogenetics Case Study Pt 2 - Wild Peccaries

MHC Evolution and Diversity in Wild Pigs and Peccaries

Outline

• Recommended readings on the topic.
• Context regarding the Major Histocompatibility Complex (MHC).
• Background on wild pigs and peccaries.
• Characterizing the MHC in wild pigs and peccaries using a DNA capture approach combined with Next Generation Sequencing (NGS).
• Investigating MHC diversity in Malagasy wild bushpigs and Australian feral pigs using deep amplicon sequencing, NGS, and Sanger sequencing.

Recommended Readings

• Lee et al. (2020). "Genetic origins and diversity of bushpigs from Madagascar." Scientific Reports, 10(1), 1-18.
• Lee et al. (2018). "Inferring the evolution of the major histocompatibility complex of wild pigs and peccaries using hybridisation DNA capture-based sequencing." Immunogenetics, 70(6), 401-417.
• Lunney et al. (2009). "Molecular genetics of the swine major histocompatibility complex, the SLA complex." Dev Comp Immunol 33:362–374. DOI: 10.1016/j.dci.2008.07.002

The MHC

• MHC genes are expressed in all nucleated cells.
• They primarily present endogenous peptides for recognition by CD8 T lymphocytes.
• The MHC region is highly polymorphic.
• Key role in:
  • Response to diseases.
  • Protection against diseases.
  • Self-identification.
• Class I MHC:
  • Deals with viruses.
• Class II MHC:
  • Deals with bacteria.
• PBR (Peptide Binding Region)
  • In Class I: α1 and α2 domains.
  • In Class II: β1 and α1 domains.

Why Study the MHC?

• Key role in response and protection against diseases and self-identification.
• Model region for understanding the evolution of gene families and host-pathogen co-evolution.
• Highly polymorphic gene region.
• MHC evolutionary studies have mostly focused on species from major lineages.
• Within Suidae (pig family), the MHC of the domestic pig (Sus scrofa) known as SLA (Swine Leucocyte Antigen) has been extensively sequenced and annotated with approximately 150 genes.
• Located on SSC7 (chromosome 7) region II near the centromere.

Why Study Suidae and Tayassuidae (Pigs and Peccaries)?

• They play a role in zoonotic and emerging diseases.
• Potential models to understand the association between immune response and local adaptation to infectious diseases.

Evolutionary History

• Suidae and Tayassuidae diverged in Southeast Asia approximately 35 million years ago (Ma).
• Suidae:
  • Sus (e.g., S. scrofa)
  • Babyrousa
  • Porcula
  • Hylochoerus
  • Phacochoerus
  • Potamochoerus
• Tayassuidae:
  • Pecari
  • Tayassu
  • Catagonus
• Evolutionary lineages:
  • Sub-Saharan Africa lineages
  • Eurasian lineages

Rationale for Approach

• For species like wild pigs and peccaries where MHC region information is limited:
  • Use DNA capture to characterize the MHC across wild pig and peccary species.
  • Use MHC data obtained through DNA capture to:
    • Design primers for deep amplicon sequencing to assess MHC diversity (Antigen Binding Site) across various genes and species, particularly focusing on Australian feral pigs.
    • Design primers for PCR and Sanger sequencing to assess MHC diversity (Antigen Binding Site) of Malagasy bushpigs.

Samples Used for DNA Capture

• 9 species of Suidae (n = 69) from Eurasia and Africa.
• 3 species of Tayassuidae (n = 19) from the Americas.

Pipeline for Heterologous Capture Approach

• Sample Preparation:
  • Start with 1.5 \, \mu \text{g} of genomic DNA.
  • Fragment DNA using Covaris.
  • Perform end repair using a TruSeq kit.
  • A-tailing using a TruSeq kit.
  • Adapter ligation using a TruSeq kit.
  • PCR enrichment using a TruSeq kit.
  • Quality control (QC) using Bioanalyzer and Qubit.
• Targeted Enrichment and Capture:
  • Pool 12 samples (12-plex pooling).
  • Solid-phase array-based hybridization (using a 385K array from NimbleGen, designed based on the S. scrofa Hp1a.0 haplotype).
  • Elution (NimbleGen).
  • PCR enrichment (TruSeq kit).
  • QC using Bioanalyzer.
• Sequencing:
  • Paired-end sequencing (PE) using HiSeq.
• Bioinformatic Analysis:
  • QC of Illumina data.
  • Read mapping to reference genome (BWA).
  • Read pairing (BWA).
  • Create BAM file, perform QC, and filtering.
• Data Analysis:
  • Data integration.
  • Variant calling (SAMtools).
  • Variant filtering.
  • Hierarchical clustering.
  • Multiway ANOVA.
• Result Interpretation.

How the DNA Capture Approach Works

• Start with the MHC region.
• Prepare the genomic DNA library:
  • Quantification.
  • Fragmentation.
  • Blunt-ended repair.
  • A-tailing.
  • Adaptor ligation (12).
  • PCR (10 cycles).
  • Pooling (12 per pool).
• Hybridization using an array of 385K oligos covering 12.5X of the domestic pig (S. scrofa) MHC region (from 2.4 to 1.8 Mb).
• Hybridization cocktail.
• Discard non-target DNA.
• Array washing and elution of captured MHC sequences.
• PCR (10-18 cycles) on captured (target) sequences.
• Next-generation sequencing.
• Cost: approximately \$90 per sample.

Overall Results of MHC Capture

• Average coverage and specificity:
  • Suidae: 86X coverage and 32\% specificity.
  • Tayassuidae: 5X coverage and 20\%$ specificity.
• Total genes captured:
  • Class I: 60/62
  • Class III: 58/61
  • Class II: 30/31

Results Regarding Antigen-Peptide Binding Region of Class I SLA Genes

• Key regions:
  • Exon 1 (Leader sequence)
  • Exons 2 and 3 (Antigen-recognition site, α1 and α2 domains, ABS, CD8+ binding site)
  • Exon 4
  • Exon 5 (Transmembrane domain)
  • Exon 6 (α3 domain)
  • Exons 7 and 8 (Cytoplasmic domain)

Results: Evolutionary History of SLA Genes

• Classical and non-classical SLA genes are present in both Suidae and Tayassuidae, contradicting previous hypotheses.
• Classical Ia genes (SLA-1-5 & 9) seem to have undergone rapid differentiation.
• Non-classical Ib genes (SLA-6, 7 & 8) emerged after the classical Ia genes (1-5 & 9).

Genetic Patterns of Differentiation

• Differentiation observed for most SLA genes between Eurasian and sub-Saharan Suidae.
• Example: SLA-6 gene differentiation among various Sus species, Potamochoerus larvatus, Hylochoerus meinertzhageni, and peccaries (Pecari tajacu, Tayassu pecari).

Results: Signatures of Evolution

• MHC class Ib-7 exon 2 (SLA-7 like).
• High levels of sequence conservation (92-99\% ) among species.
• Six additional nucleotides in exon 2 of this gene for Tayassuidae and some Suidae species (except Sus), similar to that found in some other mammals.
• Deletion within Sus after divergence from other suids.
• Comparison of the MHC related class I cell surface protein gene (MIC2).

Investigating MHC Diversity Of

• Australian feral pigs (Sus scrofa).
• Malagasy wild bushpigs (Potamochoerus larvatus).

Approach for Diversity Investigation

• Used DNA capture-NGS data to inform a deep amplicon sequencing approach.
• Deep amplicon sequencing (multiple loci with adapters and high throughput sequencing) to study feral pigs.

Results: MHC Diversity of Australian Feral Pigs

• Australian feral pigs (Sus scrofa) across different locations have a higher diversity in general than other species of wild pigs and peccaries.
• Comparison includes:
  • All Suidae
  • All Sus species
  • S. scrofa (Australian feral pigs)
  • Southeast Asian pigs (S. cebifrons, S. celebensis, S. barbatus, and B. babyrussa)
  • Sub-Saharan African suids (P. africanus, Potamochoerus spp., and H. meinertzhageni)
• Analysis of number of variable sites and mean genetic diversity (nucleotide and amino acid diversity) for SLA-1, SLA-2, SLA-3, SLA-6, SLA-7 and SLA-8.

History and Context

• Despite their relatively short time in Australia, feral pigs show high MHC diversity.
• S. scrofa was domesticated in East Asian and Anatolian populations around 9000 years ago, with subsequent dispersal through Europe and China, leading to admixture with local wild boar populations.
• Australian feral pigs are descended from European and Asian domestic animals, with some introgression from Southeast Asian Sus species.
• Admixture of diverse genetic origins during domestication may have contributed to the accumulation of MHC class II diversity in Australian feral pigs.
• This diversity may contribute to their ecological success in challenging environments in Australia.
• The higher level of genetic diversity in Australian feral pigs may also allow them an evolutionary advantage to combat pathogens and become successful in adapting to the Australian environment.

Context: MHC Diversity of Malagasy Bushpigs

• Analysis of Malagasy bushpigs (n = 45$$, including samples from mainland Africa).
• It is unclear when bushpigs were introduced to Madagascar, but it was likely prior to the 9th century.
• mtDNA suggests a small founder population was introduced.
• Bushpigs are carriers of African Swine Fever Virus but do not show symptoms.
• Sanger sequencing was used to investigate the diversity of MHC class II - DQB1 ABS.

Results: MHC Diversity of Bushpigs from Madagascar

• Malagasy bushpigs have maintained similar (and unique) levels of MHC class II SLA-DQB1 ABS diversity compared to mainland animals.
• This suggests that genetic drift and founder population size had minimal effect on SLA-DQB1 ABS diversity.
• Adaptation to environmental challenges (novel parasites) after introduction to Madagascar may have driven the rise of new alleles and maintenance of genetic diversity.
• Retention of ancestral SLA-DQB1 ABS amongst Malagasy and mainland bushpigs may indicate the necessity to combat common pathogens.

Conclusions

• Heterologous capture is efficient for investigating the MHC of the family Suidae.
• This study refines the understanding of the evolutionary history of the MHC in these families, including that of the domestic pig.
• Feral pigs and Malagasy bushpigs show relatively high levels of MHC diversity, providing evolutionary potential to face and adapt to environmental challenges.
• MHC assists in understanding the adaptive diversity of introduced and wild species.