DOI: 10.5068/d1w39k
title: Epigenetic models developed for plains zebras predict age in domestic horses and endangered equids

description: Effective conservation and management of threatened wildlife populations
 require an accurate assessment of age structure to estimate demographic
 trends and population viability. Epigenetic aging models are promising
 developments because they estimate individual age with high accuracy,
 accurately predict age in related species, and do not require invasive
 sampling or intensive long-term studies. Using blood and biopsy samples
 from known age plains zebras (Equus quagga), we modeled epigenetic aging
 using two approaches: the epigenetic clock (EC) and the epigenetic
 pacemaker (EPM). The plains zebra EC has the potential for broad
 application within the genus Equus given that five of the seven extant
 wild species of the genus are threatened. We test the ECs ability to
 predict age in sister taxa, including two endangered species and the more
 distantly related domestic horse, demonstrating high accuracy in each
 case. Comparing estimated and chronological age (age acceleration) can
 indicate health status in known age populations. Our epigenetic models
 leverage samples from a population with a complex pedigree, allowing us to
 measure the association between inbreeding and age acceleration. The EPM
 model highlights an interaction between age and inbreeding associated with
 accelerated aging, suggesting that the effects of inbreeding on epigenetic
 aging increase with age.
description: We obtained both whole blood (96) and remote biopsy (24) samples
 from a captive population of zebras maintained in a semi-wild state by the
 Quagga Project<sup>38</sup> in the Western Cape of South
 Africa. The collection of 188 whole-blood samples from domestic horses is
 described in detail in<sup>39</sup>. The Grevy’s zebra (n=5)
 and Somali wild ass (n=7) are samples from zoo-based animals that were
 opportunistically collected and banked during routine health exams. The
 DNA methylation profiles from these samples have been reported
 previously<sup>40</sup> We generated All
 DNA methylation data (plains zebra, horse, Somali wild ass, Grevy's
 zebra) using a custom Illumina methylation array
 (HorvathMammalMethylChip40). The array contains 36 thousand probes, 31,836
 of which mapped uniquely to the horse genome. We normalized methylation
 values from each species (plains zebra, horse, Somali wild ass, and
 Grevy’s zebra) and tissue (blood and biopsy) using SeSAMe.
 We studied epigenetic aging in plains zebras using both
 epigenetic clock (EC) and epigenetic pacemaker (EPM) models. The ECs were
 developed by fitting a generalized linear model with elastic-net
 penalization (alpha=0.5) using leave-one-out (LOO)
 cross-validation in glmnet v.4.0-2 in R v.4.1.0.
 We derived the genotypes used to estimate inbreeding (F and
 F<sub>ROH</sub>) from two sources: RAD sequencing (42 samples)
 and imputed genotypes at the same set of loci from low-coverage
 whole-genome sequencing data (28 additional samples). F and ROH were
 estimated using PLINK. We used multiple linear regressions to assess
 whether inbreeding is associated with age acceleration in the plains zebra
 population. Age acceleration was the dependent variable and was calculated
 as the residuals of chronological age regressed on predicted age. The
 independent variables were sex, chronological age, inbreeding, and the
 interaction between chronological age and inbreeding.
 More details are available in Larison et. al "Epigenetic
 models developed for plains zebras predict age in domestic horses and
 endangered equids" on
 bioRxiv https://www.biorxiv.org/content/10.1101/2021.03.29.437607v1
 [currently in review: Communications Biology]
description: Data are provided in three zipped folders EC, EPM, and
 Inbreeding. Each folder contains a README file. In brief:
 Scripts are provided to process each data set. They can be
 found on Zenodo at https://doi.org/10.5281/zenodo.5815909 The data in the EC folder include everything necessary to create plains zebra epigenetic clocks. The methylation data provided in the EC folder has already been normalized. The non-normalized data can be found on Gene Expression Omnibus (GEO, GSE184223). Also in the EC script are tests of the plains zebra blood clock in three other equids. These commands should be run using normalized data - using SeSAMe. The non-normalized data is available on GEO (GSE184223).  Data in the EPM folder contain everything needed to run the epigenetic pacemaker models, and an excel sheet with all the pearson coefficients for plains zebra methylation (CpGs mapping to horse) and age.  Data in the inbreeding folder include files needed to estimate ROH and F and assess the relationship between inbreeding and age acceleration. The other files are found in the EC and EPM folders as noted.