Microarray Expression Profiles of 20.000 Genes across 23 Healthy Porcine Tissues

BACKGROUND: Gene expression microarrays have been intensively applied to screen for genes involved in specific biological processes of interest such as diseases or responses to environmental stimuli. For mammalian species, cataloging of the global gene expression profiles in large tissue collections under normal conditions have been focusing on human and mouse genomes but is lacking for the pig genome. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the results from a large-scale porcine study establishing microarray cDNA expression profiles of approximately 20.000 genes across 23 healthy tissues. As expected, a large portion of the genes show tissue specific expression in agreement with mappings to gene descriptions, Gene Ontology terms and KEGG pathways. Two-way hierarchical clustering identified expected tissue clusters in accordance with tissue type and a number of cDNA clusters having similar gene expression patterns across tissues. For one of these cDNA clusters, we demonstrate that possible tissue associated gene function can be inferred for previously uncharacterized genes based on their shared expression patterns with functionally annotated genes. We show that gene expression in common porcine tissues is similar to the expression in homologous tissues of human. CONCLUSIONS/SIGNIFICANCE: The results from this study constitute a valuable and publicly available resource of basic gene expression profiles in normal porcine tissues and will contribute to the identification and functional annotation of porcine genes

Comparative analysis of protein coding sequences from human, mouse and the domesticated pig

BACKGROUND: The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish Fugu rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution. RESULTS: We provide evidence that the evolutionary splits among primates, rodents and artiodactyls happened shortly after each other, with most gene trees favouring a topology with rodents as outgroup to primates and artiodactyls. Using an unrooted topology of the three mammalian species we show that since their diversification, the pig and mouse lineages have on average experienced 1.44 and 2.86 times as many synonymous substitutions as humans, respectively, whereas the rates of non-synonymous substitutions are more similar. The analysis shows the highest average dN/dS ratio in the human lineage, followed by the pig and then the mouse lineages. Using codon based models we detect signals of positive Darwinian selection in approximately 5.3%, 4.9% and 6.0% of the genes on the human, pig and mouse lineages respectively. Approximately 16.8% of all the genes studied here are not currently annotated as functional genes in humans. Our analyses indicate that a large fraction of these genes may have lost their function quite recently or may still be functional genes in some or all of the three mammalian species. CONCLUSIONS: We present a comparative analysis of protein coding genes from three major mammalian lineages. Our study demonstrates the usefulness of codon-based likelihood models in detecting selection and it illustrates the value of sequencing organisms at different phylogenetic distances for comparative studies

Using microarrays to identify positional candidate genes for QTL: the case study of ACTH response in pigs

Background: Microarray studies can supplement QTL studies by suggesting potential candidate genes in the QTL regions, which by themselves are too large to provide a limited selection of candidate genes. Here we provide a case study where we explore ways to integrate QTL data and microarray data for the pig, which has only a partial genome sequence. We outline various procedures to localize differentially expressed genes on the pig genome and link this with information on published QTL. The starting point is a set of 237 differentially expressed cDNA clones in adrenal tissue from two pig breeds, before and after treatment with adrenocorticotropic hormone (ACTH). Results: Different approaches to localize the differentially expressed (DE) genes to the pig genome showed different levels of success and a clear lack of concordance for some genes between the various approaches. For a focused analysis on 12 genes, overlapping QTL from the public domain were presented. Also, differentially expressed genes underlying QTL for ACTH response were described. Using the latest version of the draft sequence, the differentially expressed genes were mapped to the pig genome. This enabled co-location of DE genes and previously studied QTL regions, but the draft genome sequence is still incomplete and will contain many errors. A further step to explore links between DE genes and QTL at the pathway level was largely unsuccessful due to the lack of annotation of the pig genome. This could be improved by further comparative mapping analyses but this would be time consuming. Conclusion: This paper provides a case study for the integration of QTL data and microarray data for a species with limited genome sequence information and annotation. The results illustrate the challenges that must be addressed but also provide a roadmap for future work that is applicable to other non-model species

Gene expression profiles in liver of pigs with extreme high and low levels of androstenone

<p>Abstract</p> <p>Background</p> <p>Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue.</p> <p>Results</p> <p>Liver tissue samples from 58 boars of the two breeds Duroc and Norwegian Landrace, 29 with extreme high and 29 with extreme low levels of androstenone, were selected from more than 2500 individuals. The samples were hybridised to porcine cDNA microarrays and the 1% most significant differentially expressed genes were considered significant. Among the differentially expressed genes were metabolic phase I related genes belonging to the cytochrome P450 family and the flavin-containing monooxygenase <it>FMO1</it>. Additionally, phase II conjugation genes including UDP-glucuronosyltransferases <it>UGT1A5</it>, <it>UGT2A1 </it>and <it>UGT2B15</it>, sulfotransferase <it>STE</it>, N-acetyltransferase <it>NAT12 </it>and glutathione S-transferase were identified. Phase I and phase II metabolic reactions increase the water solubility of steroids and play a key role in their elimination. Differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases (<it>HSD17B2</it>, <it>HSD17B4</it>, <it>HSD17B11 </it>and <it>HSD17B13</it>) and plasma proteins alpha-1-acid glycoprotein (<it>AGP</it>) and orosomucoid (<it>ORM1</it>). 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. Differences in the expression of <it>FMO1</it>, <it>NAT12</it>, <it>HSD17B2 </it>and <it>HSD17B13 </it>were verified by quantitative real competitive PCR.</p> <p>Conclusion</p> <p>A number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase I oxidation metabolism, phase II conjugation metabolism, and regulation of steroid availability. The study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint.</p

Molecular characterisation of the early response in pigs to experimental infection with Actinobacillus pleuropneumoniae using cDNA microarrays

<p>Abstract</p> <p>Background</p> <p>The bacterium <it>Actinobacillus pleuropneumoniae </it>is responsible for porcine pleuropneumonia, a widespread, highly contagious and often fatal respiratory disease of pigs. The general porcine innate immune response after <it>A. pleuropneumoniae </it>infection is still not clarified. The objective of this study was hence to characterise the transcriptional response, measured by using cDNA microarrays, in pigs 24 hours after experimental inoculation with <it>A. pleuropneumoniae</it>.</p> <p>Methods</p> <p>Microarray analyses were conducted to reveal genes being differentially expressed in inflamed versus non-inflamed lung tissue sampled from inoculated animals as well as in liver and tracheobronchial lymph node tissue sampled from three inoculated animals versus two non-inoculated animals. The lung samples were studied using a porcine cDNA microarray with 5375 unique PCR products while liver tissue and tracheobronchial lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products.</p> <p>Results</p> <p>A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus non-infected animals and 130 genes differed in expression in tracheobronchial lymph node tissue from infected versus non-infected animals. Among these genes, several have previously been described to be part of a general host response to infections encoding immune response related proteins. In inflamed lung tissue, genes encoding immune activating proteins and other pro-inflammatory mediators of the innate immune response were found to be up-regulated. Genes encoding different acute phase reactants were found to be differentially expressed in the liver.</p> <p>Conclusion</p> <p>The obtained results are largely in accordance with previous studies of the mammalian immune response. Furthermore, a number of differentially expressed genes have not previously been associated with infection or are presently unidentified. Determination of their specific roles during infection may lead to a better understanding of innate immunity in pigs. Although additional work including more animals is clearly needed to elucidate host response to porcine pleuropneumonia, the results presented in this study demonstrate three subsets of genes consistently expressed at different levels depending upon infection status.</p

A robust linkage map of the porcine autosomes based on gene-associated SNPs

<p>Abstract</p> <p>Background</p> <p>Genetic linkage maps are necessary for mapping of mendelian traits and quantitative trait loci (QTLs). To identify the actual genes, which control these traits, a map based on gene-associated single nucleotide polymorphism (SNP) markers is highly valuable. In this study, the SNPs were genotyped in a large family material comprising more than 5,000 piglets derived from 12 Duroc boars crossed with 236 Danish Landrace/Danish Large White sows. The SNPs were identified in sequence alignments of 4,600 different amplicons obtained from the 12 boars and containing coding regions of genes derived from expressed sequence tags (ESTs) and genomic shotgun sequences.</p> <p>Results</p> <p>Linkage maps of all 18 porcine autosomes were constructed based on 456 gene-associated and six porcine EST-based SNPs. The total length of the averaged-sex whole porcine autosome was estimated to 1,711.8 cM resulting in an average SNP spacing of 3.94 cM. The female and male maps were estimated to 2,336.1 and 1,441.5 cM, respectively. The gene order was validated through comparisons to the cytogenetic and/or physical location of 203 genes, linkage to evenly spaced microsatellite markers as well as previously reported conserved synteny. A total of 330 previously unmapped genes and ESTs were mapped to the porcine autosome while ten genes were mapped to unexpected locations.</p> <p>Conclusion</p> <p>The linkage map presented here shows high accuracy in gene order. The pedigree family network as well as the large amount of meiotic events provide good reliability and make this map suitable for QTL and association studies. In addition, the linkage to the RH-map of microsatellites makes it suitable for comparison to other QTL studies.</p

Pigs in sequence space: A 0.66X coverage pig genome survey based on shotgun sequencing

BACKGROUND: Comparative whole genome analysis of Mammalia can benefit from the addition of more species. The pig is an obvious choice due to its economic and medical importance as well as its evolutionary position in the artiodactyls. RESULTS: We have generated ~3.84 million shotgun sequences (0.66X coverage) from the pig genome. The data are hereby released (NCBI Trace repository with center name "SDJVP", and project name "Sino-Danish Pig Genome Project") together with an initial evolutionary analysis. The non-repetitive fraction of the sequences was aligned to the UCSC human-mouse alignment and the resulting three-species alignments were annotated using the human genome annotation. Ultra-conserved elements and miRNAs were identified. The results show that for each of these types of orthologous data, pig is much closer to human than mouse is. Purifying selection has been more efficient in pig compared to human, but not as efficient as in mouse, and pig seems to have an isochore structure most similar to the structure in human. CONCLUSION: The addition of the pig to the set of species sequenced at low coverage adds to the understanding of selective pressures that have acted on the human genome by bisecting the evolutionary branch between human and mouse with the mouse branch being approximately 3 times as long as the human branch. Additionally, the joint alignment of the shot-gun sequences to the human-mouse alignment offers the investigator a rapid way to defining specific regions for analysis and resequencing