Development of a high-density sub-species-specific targeted SNP assay for Rocky Mountain bighorn sheep (Ovis canadensis canadensis)

Abstract

Due to their abundance and relative ease of genotyping, single nucleotide polymorphisms (SNPs) are a commonly used molecular marker for contemporary population genetic and genomic studies. A high-density and cost-effective way to type SNP loci is Allegro targeted genotyping (ATG), which is a form of targeted genotyping by sequencing developed and offered by Tecan genomics. One major drawback of this technology is the need for a reference genome and information on SNP loci when designing a SNP assay. However, for some non-model species genomic information from other closely related species can be used. Here we describe our process of developing an ATG assay to target 50,000 SNPs in Rocky Mountain bighorn sheep, using a reference genome from domestic sheep and SNP resources from prior bighorn sheep studies. We successfully developed a high accuracy, high-density, and relatively low-cost SNP assay for genotyping Rocky Mountain bighorn sheep that genotyped ~45,000 SNP loci. These loci were relatively evenly distributed throughout the genome. Furthermore, the assay produced genotypes at tens of thousands of SNP loci when tested on other mountain sheep species and subspecies.

Keywords: Allegro; Bighorn; Development; Genetic; Genomic; SNP; SPET; Sheep; Tecan; Ungulate.

Figure 1. Filtering steps applied to variant data sourced from the five studies.

The studies Miller et al. (2012), Miller, Hogg & Coltman (2013), Miller et al. (2015), Kardos et al. (2015), and Miller, Festa-Bianchet & Coltman (2018) are represented by M12, M13, M15, K15, and M18, respectively. Each step describes a process applied to data from the previous step, process is indicated in bold, source studies of variant data in plain text, and software and command used in italics.

Figure 2. Sampling locations of Rocky Mountain bighorn sheep genotyped on the 10 and 50k assays.

Rocky Mountain bighorn sheep ranges in Alberta is shown in light grey. Sampling location abbreviations: Cadomin Mountain (CM), Castle Yarrow (CY), Narraway (NW), Ram Mountain (RM), Stornoway (ST). Contains information licensed under the Open Government Licence—Canada and Alberta.

Figure 3. Rainfall plot characterising the position, inter-loci distance, and type of substitution for each of the 50,000 loci targeted by the 50k assay.

Density across chromosome regions shown in grey.

Figure 4. Proportion of the total Ovis aries 3.1 genome contained within each chromosome (grey) alongside the proportion of SNPs in the assay located on each chromosome (gold).

Figure 5. Distribution of inter-loci distances in base pairs between neighboring SNP loci in the 50k assay.

Main plot shows 99% of the data excluding the longest 1% of distances, insert (top right) shows all data.

Figure 6. Patterns of isolation-by-distance for Nei’s standard distance and Euclidean geographic distance between the five Rocky Mountain bighorn sheep sampling locations.

Genotypes from the 10k SNP assay (A) and 50k SNP assay (B).

Figure 7. Principal component analysis of the five Rocky Mountain bighorn sheep sampling locations.

Cadomin Mountain (CM), Castle Yarrow (CY), Narraway (NW), Ram Mountain (RM), Stornoway (ST), genotyped by the 10k SNP assay (A) and 50k SNP assay (B). As noted in the methods and results individuals were genotyped in duplicate, hence each individual successfully genotyped in duplicate will show two points on the PCA.