High-Resolution Genotyping of Expressed Equine MHC Reveals a Highly Complex MHC Structure

Abstract

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals (n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses.

Keywords: Major Histocompatibility Complex (MHC); breed diversity; equine; high-throughput sequencing; polymorphism.

Figure 1

Schematic workflow of equine MHC genotyping using a MiSeq High-throughput sequencing approach. An overview of the bespoke bioinformatics pipeline used to analyze the equine MHCI and MHCII data generated using a MiSeq platform, starting with the raw MiSeq data to a final set of MHC alleles and haplotypes.

Figure 2

Read frequency of the MHCI alleles identified in the cohort of Thoroughbred horses. For each allele (horizontal axis), the percentage of sequencing reads (vertical axis) at which it was identified in the For1/Rev2.2 and For 3.2/Rev1 amplicons in each individual is represented by a blue and red colored dot, as described in the legend. The percentage of sequencing reads is shown on a logarithmic scale. The 0.2% cut-off threshold is shown as a horizontal bar.

Figure 3

Read frequency of the (i) DQA, (ii) DQB, and (ii) DRB loci in the Thoroughbred cohort. The relative frequencies of reads representing alleles for DQA1/DQA2, DQB1/DQB2, and DRB1/DRB2/DRB3 loci are shown. For each gene, the horizontal central line in the box plot represents the median value, the box represents the inter-quartile range, and the vertical lines extending from the box represent 1.5x the inter-quartile range. Individual outliers are represented by dots.

Figure 4

Association between expressed MHCI and MHCII haplotypes in the (A) Thoroughbred and (B) Icelandic Norwegian Fjord Horse cohorts. In the alluvial plot, the width of the lines linking MHCI and MHCII haplotypes represents the frequency at which these MHCI/MHCII are observed to co-segregate in the cohort.

Figure 5

Read frequency of the MHCI alleles identified in the cohort of Icelandic and Norwegian Fjord Horses. For each allele (horizontal axis), the percentage of sequencing reads (vertical axis) at which it was identified in the For1/Rev2.2 and For 3.2/Rev1 amplicons in each individual is represented by a blue and red colored dot as described in the legend. The percentage of sequencing reads is shown on a logarithmic scale. The 0.2% cut-off threshold is shown as a horizontal bar.