Whole genome detection of sequence and structural polymorphism in six diverse horses

Abstract

The domesticated horse has played a unique role in human history, serving not just as a source of animal protein, but also as a catalyst for long-distance migration and military conquest. As a result, the horse developed unique physiological adaptations to meet the demands of both their climatic environment and their relationship with man. Completed in 2009, the first domesticated horse reference genome assembly (EquCab 2.0) produced most of the publicly available genetic variations annotations in this species. Yet, there are around 400 geographically and physiologically diverse breeds of horse. To enrich the current collection of genetic variants in the horse, we sequenced whole genomes from six horses of six different breeds: an American Miniature, a Percheron, an Arabian, a Mangalarga Marchador, a Native Mongolian Chakouyi, and a Tennessee Walking Horse, and mapped them to EquCab3.0 genome. Aside from extreme contrasts in body size, these breeds originate from diverse global locations and each possess unique adaptive physiology. A total of 1.3 billion reads were generated for the six horses with coverage between 15x to 24x per horse. After applying rigorous filtration, we identified and functionally annotated 17,514,723 Single Nucleotide Polymorphisms (SNPs), and 1,923,693 Insertions/Deletions (INDELs), as well as an average of 1,540 Copy Number Variations (CNVs) and 3,321 Structural Variations (SVs) per horse. Our results revealed putative functional variants including genes associated with size variation like LCORL gene (found in all horses), ZFAT in the Arabian, American Miniature and Percheron horses and ANKRD1 in the Native Mongolian Chakouyi horse. We detected a copy number variation in the Latherin gene that may be the result of evolutionary selection impacting thermoregulation by sweating, an important component of athleticism and heat tolerance. The newly discovered variants were formatted into user-friendly browser tracks and will provide a foundational database for future studies of the genetic underpinnings of diverse phenotypes within the horse.

Fig 1. Circos plot summarizing the genetic variants detected in each horse.

The pattern of variation across the genomes of the six horses reveals structurally diverse regions important for immunity and olfactory reception at chromosomes 12 and 20 in all six horses. From the inside out, each plot shows two endpoints of the inter- (orange) and intra- (blue) chromosomal translocations. Intrachromosomal translocations > 5 MB are in dark blue. The yellow ring shows the copy number variations (green = normal, blue = loss, red = gain). The histogram (in orange) shows the density of SNPs detected using 1MB windows.

Fig 2. RT-qPCR results of the LATH CNV region for seven horses belonging to different breeds, a thoroughbred (TB), a Percheron (PER) and an American Miniature (AMH), and an Arabian (ARB), a Tennessee Walking Horse (TWH), a Mangalarga Marchador (MM) and a Native Mongolian Chakouyi (CH).

The Y axis represents the copy number and the X axis, represents horses from different breeds. The results are shown for different primers in the order they appear in in the genome are shown, starting with BPIFB4 to BPIFA1. The results show statistically significant difference in copy number variation between horses for BPIFB4 (a) and BPIFA1 (d) two genes that flank LATH (c) in the EquCab 2.0 assembly.