The goal of this project is to develop a deep understanding of the evolutionary processes that generate and maintain diversity in the Major Histocompatibility Complex of the horse. Based upon our preliminary results, we postulate that recombination of genes within the MHC chromosomal region continually generates new combinations of MHC genes within horse breeds. The rate of recombination in the equine MHC that we have estimated can provide a vast resource of genetic variation within Thoroughbreds or Standardbreds. Countering this positive generation of MHC diversity is the negative effect of horse breeding strategies, where a small number of stallions produce many offspring each year. This harem mating system will decrease MHC diversity within a breed, and thus may decrease biological fitness. For our proposed project we would test the hypothesis that intra-MHC recombination is the major driver of MHC diversity in the horse. We would integrate data from three independent platforms for assessing MHC diversity: through Single Nucleotide Polymorphisms (SNPs), microsatellites, and by obtaining coding sequences of MHC class I and class II genes through targeted cDNA sequencing. Most of the samples required for this study have already been collected from other projects in our laboratory, and every sample has been tested on at least one platform. The objective of this project is to "fill in the gaps" in our dataset by testing selected horses and analyzing the resulting data.

Specific Aims: In this project we would undertake the following work:
Aim 1) Determine MHC Single Nucleotide Polymorphism (SNP) profiles of 96 selected horses using the Axiom 670k SNP genotyping array. This molecular assay determines individual DNA nucleotides at over 600,000 sites within the horse genome, including several thousand positions in the MHC region. The horses would be selected primarily from samples we have already collected from other projects and partially characterized using an alternative, but less informative, assay for MHC diversity based on microsatellites. We would determine the correlation between the SNP-based and microsatellite-determined MHC types using standard statistical methods. We expect the correlation between these two methods to be over 90%. Aim 2) Here we would determine the protein coding sequences of MHC class I and class II genes from up to 10 horses that carry two identical copies of the MHC genes (homozygotes) and link the functional MHC gene sequences with the SNP and microsatellite markers that we determined in Aim 1.