Development of a genotype‐by‐sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure

Abstract

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High‐throughput sequencing (HTS) technologies, such as genotype‐by‐sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization‐based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to “capture” specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300‐kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on‐target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer‐read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune‐related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.

Construction of Red Fox Chromosomal Fragments from the Short-Read Genome Assembly

“…Adaptation and response to selection in the red fox is therefore of interest not only to wildlife ecology, but also fields including behavioral and evolutionary genetics. The draft red fox genome assembly, which is currently in version 2.2 and known as vv2.2, has already proven valuable for studies of fox behavioral genetics [ 12 ], immunological adaptation [ 13 ], and population diversity [ 14 ].…”

The role of a mechanistic host in maintaining arctic rabies variant distributions: Assessment of functional genetic diversity in Alaskan red fox (Vulpes vulpes)

“…While the neutral genetic structure of host species can be used to infer relationships with disease maintenance and spread [ 37 , 51 , 52 ], understanding deeper co-evolutionary patterns between hosts and pathogens requires insight into the variation that exists in genes that interact with the reciprocal selective pressures. To this end and building on the data from Goldsmith et al [ 37 ], Donaldson et al [ 53 ] developed a GBS assay to specifically target 116 immunogenetically relevant regions of the red fox genome. Donaldson et al [ 53 ], tested the assay on a small sample size of red foxes from regions with different AR variants and regions without rabies and found 15 F ST -based outlier SNPs that divided the samples into two genetic clusters corresponding to regions with and without AR, similar to the results by Goldsmith et al [ 37 ].…”

“…To this end and building on the data from Goldsmith et al [ 37 ], Donaldson et al [ 53 ] developed a GBS assay to specifically target 116 immunogenetically relevant regions of the red fox genome. Donaldson et al [ 53 ], tested the assay on a small sample size of red foxes from regions with different AR variants and regions without rabies and found 15 F ST -based outlier SNPs that divided the samples into two genetic clusters corresponding to regions with and without AR, similar to the results by Goldsmith et al [ 37 ]. In both studies, inferences on the relationship between host genetic clustering and rabies distributions were undermined by either small sample sizes or assessments on regions of the genome unlikely to show patterns of selection from infectious diseases.…”

“…In both studies, inferences on the relationship between host genetic clustering and rabies distributions were undermined by either small sample sizes or assessments on regions of the genome unlikely to show patterns of selection from infectious diseases. However, in these studies, red fox genetic structure was more pronounced in the data from the immunogenetic assay [ 53 ] relative to the microsatellite data [ 37 ], suggesting that gene flow was not solely responsible for the observed patterns of genetic structure.…”

“…Herein, we build on the work of Goldsmith et al [ 37 ] and Donaldson et al [ 53 ], using the same immunogenetic GBS assay, by increasing the number of red fox sampled per location to better assess frequency differences of genetic variants among the sampled locations and to gain further insight into the role of red fox in AR maintenance in Alaska. We also aimed to put the interrelationships of AR and red foxes in Alaska into context by including red fox from Ontario (Canada) as a potential outgroup.…”