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Additional file 1: Table S1. Sequencing and alignment performance for all individual samples and species pools. Cheetah_pool, tiger_pool and snowleopard_pool refer to species pools consisting of 4 multiplexed individuals each. Figure S1. Mapped and total reads across WGS samples and pools. Total number of mapped reads (black) includes singletons and pairs. Number of paired reads mapped indicated by the grey line. Tiger_zoo, snowleopard_zoo and cheetah_zoo refer to multiplexed pool samples. Figure S2. Plots of genome coverage for each sample .bam file aligned to the felCat9 reference assembly for a. cheetah, b. Sumatran tiger and c. snow leopard cohorts. On each panel, the key indicates coloured line of each sample and their sequencing depth in brackets. Table S2. Functional annotation of all fixed and within-species SNPs for each species. Table S3a. Top 20 gene ontology terms (GOterms) enriched for species-specific SNV within cheetahs. P-values were adjusted for multiple testing using Benjamini-Hochberg false discovery rate. Table S3b. Top 20 gene ontology terms (GOterms) enriched for species specific SNVs within Sumatran tigers. P-values were adjusted for multiple testing using Benjamini-Hochberg false discovery rate. Table S4. Genes under positive selection identified as those displaying elevated πNπSratios across all three species. Table S5a. Top 20 gene ontology terms (GOterms) enriched across fixed SNVs in snow leopards. P-values were adjusted for multiple testing using Benjamini-Hochberg FDR (false discovery rate). Table S5b. Top 20 gene ontology terms (GOterms) enriched across fixed SNVs in Sumatran tigers. P-values were adjusted for multiple testing using Benjamini-Hochberg FDR (false discovery rate). Table S6. Multi-species alignment of LCORL (ENSFCAG00000029474) revealed Panthera-specific conservation of six missense variants. Cheetah, snow leopard and Sumatran tiger refer to samples aligned to the domestic cat (felCat9) reference assembly. Protein positions are reported relative to the Ensembl transcript ENSFCAT00000081895.1. Table S7. Protocadherin genes containing fixed non-synonymous SNVs common to all big cat species relative to the domestic cat (felCat9) reference assembly. Table S8. Genes containing deleterious SNVs implicated in heritable conditions affecting big cats grouped by species. Figure S3a. Gene ontology annotation of clinically significant GOterms in cheetahs. Deleterious variants were observed in 201 genes included in custom list of GO terms relevant to known heritable conditions, immune and reproductive function. These genes were annotated for terms relevant to reproductive and immune function. Network interaction graph produced by GOnet ( https://tools.dice-database.org/GOnet/ ). Figure S3b. Gene ontology annotation of clinically significant GOterms in Sumatran tigers. Deleterious variants were observed in 44 genes included in custom list of GO terms relevant to known heritable conditions, immune and reproductive function. These genes were annotated for terms relevant to cilium structure. Network interaction graph produced by GOnet ( https://tools.dice-database.org/GOnet/ ). Table S9. Samples downloaded from Sequence Read Archive (SRA) comprised six cheetahs, one snow leopard and seven Sumatran tigers. Table S10.Genes associated with size in domestic species identified from a literature search. Table S11. Selected GO enrichment terms used to classify deleterious variants potentially implicated in reproductive success and overall health of captive bred big cats. Table S12. Known heritable conditions affecting captive bred big cat species and relevant GO terms.

Table S2. PCR primers and conditions used for DNA sequencing of the feline GNPTAB exons. (DOCX 25 kb)

BREED RISK, IMMUNOPHENOTYPES AND GENOMIC STUDIES OF CANINE ATOPIC DERMATITIS Canine atopic dermatitis (AD) is a complex genetically-linked immunological hypersensitivity which has similar clinical signs and pathological features to human AD, and involves immune dysregulation and skin barrier impairment. This thesis examines genetic factors underpinning canine AD and focuses on breed prevalence and evaluation of changes in the blood. Sixteen breeds with increased relative risk (≥1.5) were identified and gender predisposition in two dog breeds was revealed. One clade of dog breeds is highly represented amongst AD patients worldwide, and with increased RR in Australia. A 19 cytokine/chemokine multiplex bio-assay measured significantly elevated CXCL8, IL-7 and IL-15 concentrations, and reduced Stem-cell factor (SCF) in plasma of canine AD patients (n=27) compared to controls (n=11). Microarray gene expression data from leukocytes of atopic dogs (n=6) and controls (n=6) revealed 603 differentially expressed (DE) genes. Amongst these, candidate immune-related genes were highlighted, including the most under-represented gene, IL-7 receptor alpha subunit (IL7R). Quantitative real time PCR confirmed reduced gene expression for genes of the IL-7/IL7R pathway. A significant enrichment cluster consisting of immune-related pathways was identified, including T-cell receptor and B-cell receptor pathways. Expression analysis of miRNA from leukocytes of AD dogs identified15 with differential patterns of expression. The miRNA cfa-miR-31 had low levels in atopic dogs, suggesting a potential immune impairment dysregulation. Integrating the data gathered in the array studies reported in this thesis together with the genetic structure of dog breeds provides a powerful model to enable better characterisation of Australian dog breed susceptibility to canine AD and breed-related phenotypes. The IL-7/IL7R pathway may play a key role in the immune response in canine AD, may provide biomarkers to assist with diagnoses, and is a potential target for therapeutic agents.

The efficacy of the pedometer to predict lameness earlier than the appearance of the clinical signs in a herd of dairy cows was investigated by correlating pedometric activity (PA) with clinical cases of lameness. The computer program was set to identify cows with a reduction of 5% or more in PA compared with their own previous 10 days average; these animals were then examined for clinical lameness. At the same time, every lame cow was checked to see if and when its PA was reduced. Forty-six cows showed a reduced PA; 38 cases of lameness were identified by either a reduction in PA or clinical observation; of these, 21 lame cows (45.7%) showed a reduction in PA of 5% or more, 7 to 10 days prior to the appearance of clinical signs. This cohort comprised 55.3% of the lame cows. In 92% of the lame cows identified by PA, the decrease was above 15%.

Table S1. Lysosomal enzymes assays: substrates, buffers, conditions and references. (DOCX 15 kb)

Table S1. Lysosomal enzymes assays: substrates, buffers, conditions and references. (DOCX 15 kb)