Gene expression atlas of the domestic goat and comparative analysis of immune signatures with sheep
Item statusRestricted Access
Embargo end date30/11/2021
Muriuki, Charity Muthoni
Goats are an economically important livestock species providing a resource of meat and milk across the world. They are of particular importance in developing countries contributing to sustainable agriculture, alleviation of poverty and utilisation of marginal grazing. Recently, a highly contiguous refence genome (ARS1) of the domestic goat was released. However, gene expression information on the domestic goat is particularly limited when compared to other ruminants. Despite great genetic similarity, ruminants vary in their susceptibility to similar pathogens, but the underlying molecular mechanisms remain largely unknown. To elucidate the molecular basis of variation in disease response in small ruminants, a gene expression atlas of the domestic goat was generated from a subset of 22 tissue and cell types and compared to the previously developed sheep gene expression atlas. Fifty-four mRNA-Seq (poly-A selected) 75bp paired-end libraries spanning all major organ systems in the domestic goat were produced, generating a total of 8.7×108 paired end sequence reads. The tissues and cell-types sampled were all transcriptionally complex, with each expressing at least 50% of the total protein coding genes at detectable levels. 18,528 protein coding genes (out of a possible 21,343) had detectable expression in at least one tissue sampled, enabling the capture of 90% of the reference transcriptome. Additionally, of the 21,343 protein coding genes in the ARS1 reference transcriptome 7,036 (33%) had no informative gene name. Using the HISAT2 annotation pipeline, informative gene names were assigned to 1,114 (15%) of the previously un-annotated protein coding genes in ARS1, greatly expanding the previously available genetic and genomic resources available for goat. Using network cluster analysis, genes were assigned to specific biological pathways or cell populations based on expression profiles. Clusters of genes in the liver, gastro-intestinal tract and those involved in innate immunity are analysed and discussed in detail. Additionally, a protocol to isolate goat bone marrow derived macrophages (BMDM) and culture them in the presence of macrophage colony stimulating factor (CSF1) was developed and optimized. The goat BMDM were characterised using light microscopy to confirm morphology as well as flow cytometry to investigate the cell surface markers. Flow cytometry results revealed that goat BMDM express CD14, CD16 and CD172a on the surface similar to sheep macrophages. When exposed to bacterial lipopolysaccharide (LPS), goat BMDM responded by inducing inflammatory cytokines such as TNF, interferon-associated genes including IFI6, IFIT3 and IFNG and interleukins such as IL10RA, IL12B, IL16 and IL1RAP similar to sheep BMDM. However, unlike sheep, goat BMDM produced detectable levels of nitric oxide (NO) post-LPS stimulation. The goat BMDM post-LPS stimulation were also analysed with RNA-Seq to reveal hundreds of upregulated genes further expanding the transcriptional data available for goat. Finally, the data generated from the network cluster analysis of the goat was used to run a comparative analysis with the larger gene expression atlas of the domestic sheep, revealing transcriptional differences between the two species which may underlie the mechanisms controlling disease variation.