Innovative construction of the first reliable catalogue of bovine circular RNAs.

The aim of this study was to compare the circular transcriptome of divergent tissues in order to understand: i) the presence of circular RNAs (circRNAs) that are not exonic circRNAs, i.e. originated from backsplicing involving known exons and, ii) the origin of artificial circRNA (artif_circRNA), i.e. circRNA not generated in-vivo. CircRNA identification is mostly an in-silico process, and the analysis of data from the BovReg project (https://www.bovreg.eu/) provided an opportunity to explore new ways to identify reliable circRNAs. By considering 117 tissue samples, we characterized 23,926 exonic circRNAs, 337 circRNAs from 273 introns (191 ciRNAs, 146 intron circles), 108 circRNAs from small non-coding genes and nearly 36.6K circRNAs classified as other_circRNAs. Furthermore, for 63 of those samples we analysed in parallel data from total-RNAseq (ribosomal RNAs depleted prior to library preparation) with paired mRNAseq (library prepared with poly(A)-selected RNAs). The high number of circRNAs detected in mRNAseq, and the significant number of novel circRNAs, mainly other_circRNAs, led us to consider all circRNAs detected in mRNAseq as artificial. This study provided evidence of 189 false entries in the list of exonic circRNAs: 103 artif_circRNAs identified by total RNAseq/mRNAseq comparison using two circRNA tools, 26 probable artif_circRNAs, and 65 identified by deep annotation analysis. Extensive benchmarking was performed (including analyses with CIRI2 and CIRCexplorer-2) and confirmed 94% of the 23,737 reliable exonic circRNAs. Moreover, this study demonstrates the effectiveness of a panel of highly expressed exonic circRNAs (5-8%) in analysing the tissue specificity of the bovine circular transcriptome.

Transcriptomic Response of Differentiating Porcine Myotubes to Thermal Stress and Donor Piglet Age.

Climate change is a current concern that directly and indirectly affects agriculture, especially the livestock sector. Neonatal piglets have a limited thermoregulatory capacity and are particularly stressed by ambient temperatures outside their optimal physiological range, which has a major impact on their survival rate. In this study, we focused on the effects of thermal stress (35 °C, 39 °C, and 41 °C compared to 37 °C) on differentiating myotubes derived from the satellite cells of Musculus rhomboideus, isolated from two different developmental stages of thermolabile 5-day-old (p5) and thermostable 20-day-old piglets (p20). Analysis revealed statistically significant differential expression genes (DEGs) between the different cultivation temperatures, with a higher number of genes responding to cold treatment. These DEGs were involved in the macromolecule degradation and actin kinase cytoskeleton categories and were observed at lower temperatures (35 °C), whereas at higher temperatures (39 °C and 41 °C), the protein transport system, endoplasmic reticulum system, and ATP activity were more pronounced. Gene expression profiling of HSP and RBM gene families, which are commonly associated with cold and heat responses, exhibited a pattern dependent on temperature variability. Moreover, thermal stress exhibited an inhibitory effect on cell cycle, with a more pronounced downregulation during cold stress driven by ADGR genes. Additionally, our analysis revealed DEGs from donors with an undeveloped thermoregulation capacity (p5) and those with a fully developed thermoregulation capacity (p20) under various cultivation temperature. The highest number of DEGs and significant GO terms was observed under temperatures of 35 °C and 37 °C. In particular, under 35 °C, the DEGs were enriched in insulin, thyroid hormone, and calcium signaling pathways. This result suggests that the different thermoregulatory capacities of the donor piglets determined the ability of the primary muscle cell culture to differentiate into myotubes at different temperatures. This work sheds new light on the underlying molecular mechanisms that govern piglet differentiating myotube response to thermal stress and can be leveraged to develop effective thermal management strategies to enhance skeletal muscle growth.

Brain Transcriptome Responses to Dexamethasone Depending on Dose and Sex Reveal Factors Contributing to Sex-Specific Vulnerability to Stress-Induced Disorders.

BACKGROUND: Glucocorticoid (GC) receptor (GR) signaling in the hypothalamus (Hyp) and in the superordinate limbic structures, such as the hippocampus (Hip), conveys feedback regulation of the neuroendocrine stress response and acts upon other neurobiological functions that ultimately influence mental health. These responses are strongly influenced by sex, but the molecular causes are still largely unexplored. METHODS: To investigate GR targets and their GC sensitivity in the Hyp and Hip, we treated juvenile male and female piglets with 10 (D10) or 60 (D60) µg/kg dexamethasone (DEX), a selective GR agonist, and analyzed transcriptome responses compared to a saline control group using RNA sequencing. RESULTS: Both doses influenced similar biological functions, including cellular response to lipid and immune cell-related functions, but the transcriptional response to D10 was considerably weaker, particularly in the Hip. Weighted Gene Co-expression Network Analysis revealed a network of genes coordinately regulated by DEX in both structures, among which the alpha-arrestin ARRDC2 takes a central position. Distinct functional groups of genes were differentially regulated by DEX between sexes depending on the dose; at D10, these included particularly mitochondrial genes, whereas at D60 interferon signaling and lipid homeostasis genes were enriched. The general and sex-specific transcriptional responses to DEX highlight microglia as the prominent target. Several key marker genes of disease-associated microglia were regulated by DEX depending on sex, such as TREM2 and LPL. CONCLUSION: The discovered expression signatures suggest that DEX induced a dysfunctional state of microglia in males, while in females microglia were primed, which could entail predisposition for different mental disorders.

Genetic background and production periods shape the microRNA profiles of the gut in laying hens.

There is growing evidence of the importance of miRNAs for intestinal functional properties and nutritional uptake. Comparative miRNAs profiles of the jejunal mucosa were established against two genetic backgrounds (Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL), which are similar in egg production but differ in physiological traits including mineral utilization, along the production periods of laying hens. The target genes of miRNAs higher expressed in LB vs. LSL (miR-126-3p, miR-214, miR-24-3p, miR-726-5p, miR-29b-3p) were enriched for energy pathways at all stages. The target genes of the miRNAs higher in LSL (miR-1788-5p, miR-103-3p, miR-22-5p, miR-221-3p, miR-375) were more enriched for immune and the bone signalling pathways. The most prominent expression differences were between 16 and 24 weeks of age before and after onset of laying. Our results evidence central roles of intestinal miRNAs as regulators of gene expression, influencing intestinal homeostasis and adaptation to environment in different strains and production phases.

MicroRNA-mRNA Networks in Pregnancy Complications: A Comprehensive Downstream Analysis of Potential Biomarkers.

Pregnancy complications are a major cause of fetal and maternal morbidity and mortality in humans. The majority of pregnancy complications initiate due to abnormal placental development and function. During the last decade, the role of microRNAs (miRNAs) in regulating placental and fetal development has become evident. Dysregulation of miRNAs in the placenta not only affects placental development and function, but these miRNAs can also be exported to both maternal and fetal compartments and affect maternal physiology and fetal growth and development. Due to their differential expression in the placenta and maternal circulation during pregnancy complications, miRNAs can be used as diagnostic biomarkers. However, the differential expression of a miRNA in the placenta may not always be reflected in maternal circulation, which makes it difficult to find a reliable biomarker for placental dysfunction. In this review, we provide an overview of differentially expressed miRNAs in the placenta and/or maternal circulation during preeclampsia (PE) and intrauterine growth restriction (IUGR), which can potentially serve as biomarkers for prediction or diagnosis of pregnancy complications. Using different bioinformatics tools, we also identified potential target genes of miRNAs associated with PE and IUGR, and the role of miRNA-mRNA networks in the regulation of important signaling pathways and biological processes.

Identification of the Key Molecular Drivers of Phosphorus Utilization Based on Host miRNA-mRNA and Gut Microbiome Interactions.

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1, SMAD3, IGSF10, Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

Epigenome-wide skeletal muscle DNA methylation profiles at the background of distinct metabolic types and ryanodine receptor variation in pigs.

BACKGROUND: Epigenetic variation may result from selection for complex traits related to metabolic processes or appear in the course of adaptation to mediate responses to exogenous stressors. Moreover epigenetic marks, in particular the DNA methylation state, of specific loci are driven by genetic variation. In this sense, polymorphism with major gene effects on metabolic and cell signaling processes, like the variation of the ryanodine receptors in skeletal muscle, may affect DNA methylation. METHODS: DNA-Methylation profiles were generated applying Reduced Representation Bisulfite Sequencing (RRBS) on 17 Musculus longissimus dorsi samples. We examined DNA methylation in skeletal muscle of pig breeds differing in metabolic type, Duroc and Pietrain. We also included F2 crosses of these breeds to get a first clue to DNA methylation sites that may contribute to breed differences. Moreover, we compared DNA methylation in muscle tissue of Pietrain pigs differing in genotypes at the gene encoding the Ca2+ release channel (RYR1) that largely affects muscle physiology. RESULTS: More than 2000 differently methylated sites were found between breeds including changes in methylation profiles of METRNL, IDH3B, COMMD6, and SLC22A18, genes involved in lipid metabolism. Depending on RYR1 genotype there were 1060 differently methylated sites including some functionally related genes, such as CABP2 and EHD, which play a role in buffering free cytosolic Ca2+ or interact with the Na+/Ca2+ exchanger. CONCLUSIONS: The change in the level of methylation between the breeds is probably the result of the long-term selection process for quantitative traits involving an infinite number of genes, or it may be the result of a major gene mutation that plays an important role in muscle metabolism and triggers extensive compensatory processes.

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue.

BACKGROUND: In the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue. RESULTS: Selective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns. CONCLUSIONS: The implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.

Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage.

With the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.

Unmapped reads from cattle RNAseq data: A source for missing and misassembled sequences in the reference assemblies and for detection of pathogens in the host.

Usually, reads from transcriptome sequencing data unmapped to the target species' reference genome are disregarded. A recent RNAseq project on the new fatal disease Bovine Neonatal Pancytopenia had indicated an unexplained immune response signature to a double-stranded RNA virus. To unravel its background, contigs were de novo assembled from unmapped RNAseq reads and aligned against the bovine genome assemblies and multispecies NCBI databases. Lack of genuine virus sequence contigs rejected the hypothesis of a live virus being causal for the unexplained immune response. Alignment data also demonstrated incomplete bovine reference genome assemblies. In addition, we found that several parasite and virus genome reference assemblies in NCBI were contaminated with bovine DNA and confirmed recombination of bovine DNA into BVD virus strains. Exploring unmapped reads can extract useful biological information regarding the presence of microorganisms and can highlight issues with reference genome assemblies of host and pathogen species.

A novel RNAseq-assisted method for MHC class I genotyping in a non-model species applied to a lethal vaccination-induced alloimmune disease.

BACKGROUND: MHC class I genotyping is essential for a wide range of biomedical, immunological and biodiversity applications. Whereas in human a comprehensive MHC class I allele catalogue is available, respective data in non-model species is scarce in spite of decades of research. RESULTS: Taking advantage of the new high-throughput RNA sequencing technology (RNAseq), we developed a novel RNAseq-assisted method (RAMHCIT) for MHC class I typing at nucleotide level. RAMHCIT is performed on white blood cells, which highly express MHC class I molecules enabling reliable discovery of new alleles and discrimination of closely related alleles due to the high coverage of alleles with reads. RAMHCIT is more comprehensive than previous methods, because no targeted PCR pre-amplification of MHC loci is necessary, which avoids preselection of alleles as usually encountered, when amplification with MHC class I primers is performed prior to sequencing. In addition to allele identification, RAMHCIT also enables quantification of MHC class I expression at allele level, which was remarkably consistent across individuals. CONCLUSIONS: Successful application of RAMHCIT is demonstrated on a data set from cattle with different phenotype regarding a lethal, vaccination-induced alloimmune disease (bovine neonatal pancytopenia), for which MHC class I alleles had been postulated as causal agents.

MicroRNA-mRNA regulatory networking fine-tunes the porcine muscle fiber type, muscular mitochondrial respiratory and metabolic enzyme activities.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in diverse biological processes via regulation of gene expression including in skeletal muscles. In the current study, miRNA expression profile was investigated in longissimus muscle biopsies of malignant hyperthermia syndrome-negative Duroc and Pietrain pigs with distinct muscle metabolic properties in order to explore the regulatory role of miRNAs related to mitochondrial respiratory activity and metabolic enzyme activity in skeletal muscle. RESULTS: A comparative analysis of the miRNA expression profile between Duroc and Pietrain pigs was performed, followed by integration with mRNA profiles based on their pairwise correlation and computational target prediction. The identified target genes were enriched in protein ubiquitination pathway, stem cell pluripotency and geranylgeranyl diphosphate biosynthesis, as well as skeletal and muscular system development. Next, we analyzed the correlation between individual miRNAs and phenotypical traits including muscle fiber type, mitochondrial respiratory activity, metabolic enzyme activity and adenosine phosphate concentrations, and constructed the regulatory miRNA-mRNA networks associated with energy metabolism. It is noteworthy that miR-25 targeting BMPR2 and IRS1, miR-363 targeting USP24, miR-28 targeting HECW2 and miR-210 targeting ATP5I, ME3, MTCH1 and CPT2 were highly associated with slow-twitch oxidative fibers, fast-twitch oxidative fibers, ADP and ATP concentration suggesting an essential role of the miRNA-mRNA regulatory networking in modulating the mitochondrial energy expenditure in the porcine muscle. In the identified miRNA-mRNA network, a tight relationship between mitochondrial and ubiquitin proteasome system at the level of gene expression was observed. It revealed a link between these two systems contributing to energy metabolism of skeletal muscle under physiological conditions. CONCLUSIONS: We assembled miRNA-mRNA regulatory networks based on divergent muscle properties between different pig breeds and further with the correlation analysis of expressed genes and phenotypic measurements. These complex networks relate to muscle fiber type, metabolic enzyme activity and ATP production and may contribute to divergent muscle phenotypes by fine-tuning the expression of genes. Altogether, the results provide an insight into a regulatory role of miRNAs in muscular energy metabolisms and may have an implication on meat quality and production.

Epistatic interactions between at least three loci determine the "rat-tail" phenotype in cattle.

BACKGROUND: The "rat-tail" syndrome (RTS) is an inherited hypotrichosis in cattle, which is exclusively expressed in diluted coloured hair. The affected animals also suffer from disturbed thermoregulation, which impairs their health and growth performance. Phenotypic features that are similar to RTS are observed in dogs with black hair follicle dysplasia. RESULTS: We used a resource cross population between German Holstein and Charolais cattle breeds to prove that epistatic interactions between at least three independent genetic loci are required for the expression of the RTS phenotype. In this population, the RTS is exclusively expressed in animals with a eumelanic background that is due to the dominant E (D) allele at the melanocortin 1 receptor gene located on Bos taurus autosome (BTA) 18. In addition, only the individuals that are heterozygous at the dilution locus on BTA5 that corresponds to the premelanosome protein or silver gene variant c.64G>A were classified as displaying a RTS phenotype. Linkage and whole-genome association analyses using different models and different pedigrees allowed us to map a third locus (hereafter referred to as the RTS locus) that is essential for the expression of the RTS phenotype to the chromosomal region between 14 and 22 Mb on BTA5. Our findings clearly demonstrate that the RTS and dilution loci are distinct loci on BTA5. CONCLUSIONS: Our study provides evidence that the RTS locus has effects on hair conformation and coat colour dilution and that the effect on coat colour dilution is clearly independent from that of the dilution locus. Finally, our results excluded several other loci that were previously reported to be associated with or to underlie hair conformation or pigmentation traits as the causal mutations of RTS and also several major functional candidate genes that are associated with hypotrichosis in humans. Our finding on the identification of a three-locus interaction that underlies RTS provides a prime example of epistatic interaction between several independent loci that is required for the expression of a distinct phenotype.

Differential expression of miRNAs and their target mRNAs in endometria prior to maternal recognition of pregnancy associates with endometrial receptivity for in vivo- and in vitro-produced bovine embryos.

Endometrial receptivity is a prerequisite for successful embryo implantation and pregnancy. Receptivity involves complex processes promoted by many transcripts that are key components of molecular pathways that depend on ovarian hormones and that contribute to shaping structural, metabolic, and communication properties of endometrial cells toward reception of embryos. MicroRNAs (miRNAs) are important regulators of the expression of these transcripts encoding effector molecules. We acquired miRNA and mRNA signatures, miRNA-mRNA pairs, and regulatory networks linked with the emergence and maintenance of postimplantation pregnancy. Endometrial tissue samples were obtained at Days 3 and 7 of the estrous cycle of cows that did or did not become pregnant after transfer of either in vivo-produced (IVV) or in vitro-produced (IVT) embryos in the next cycle following the biopsy. We report a list of endometrial miRNAs that were differentially expressed between Day 3 and Day 7 of the bovine estrous cycle (including miR-1290, miR-3437, miR-1246, miR-486, miR-3107, and miR-382), that differed with high or low endometrial receptivity (miR-3902-3p, miR-1825, miR-H14-3p, miR-885-3p, miR-504-3p, and miR-186), or that differed among the IVT and IVV transfers (miR-449a/b/c, miR-138, miR-874, miR-4342, miR-2231, and miR-2751). Moreover, mRNA transcripts were also analyzed, and pairs of negatively correlated miRNAs and mRNAs were predicted in silico. The miRNA-mRNA target pairs had roles in response to hormonal stimuli and oxidative stress, chromatin organization, miRNA-mediated epigenetic histone changes, cell proliferation, p53 signaling, and apoptosis. Overall, we identified significant miRNAs, miRNA-mRNA pairs, and functional networks that are associated with the state of pregnancy at Day 28 as a parameter of endometrial receptivity and that are affected by estrous cycle and embryo culture systems.

Dynamics of DNA methylation during osteogenic differentiation of porcine synovial membrane mesenchymal stem cells from two metabolically distinct breeds.

Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell's metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including KLF1, NFATC3, ZNF148, ASCL1, FOXI1, and KLF5. These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation.

Identification of novel transcripts and noncoding RNAs in bovine skin by deep next generation sequencing.

BACKGROUND: Deep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue. RESULTS: A total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin. CONCLUSIONS: The results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge.

Correlated mRNAs and miRNAs from co-expression and regulatory networks affect porcine muscle and finally meat properties.

BACKGROUND: Physiological processes aiding the conversion of muscle to meat involve many genes associated with muscle structure and metabolic processes. MicroRNAs regulate networks of genes to orchestrate cellular functions, in turn regulating phenotypes. RESULTS: We applied weighted gene co-expression network analysis to identify co-expression modules that correlated to meat quality phenotypes and were highly enriched for genes involved in glucose metabolism, response to wounding, mitochondrial ribosome, mitochondrion, and extracellular matrix. Negative correlation of miRNA with mRNA and target prediction were used to select transcripts out of the modules of trait-associated mRNAs to further identify those genes that are correlated with post mortem traits. CONCLUSIONS: Porcine muscle co-expression transcript networks that correlated to post mortem traits were identified. The integration of miRNA and mRNA expression analyses, as well as network analysis, enabled us to interpret the differentially-regulated genes from a systems perspective. Linking co-expression networks of transcripts and hierarchically organized pairs of miRNAs and mRNAs to meat properties yields new insight into several biological pathways underlying phenotype differences. These pathways may also be diagnostic for many myopathies, which are accompanied by deficient nutrient and oxygen supply of muscle fibers.

Monitoring the immune response to vaccination with an inactivated vaccine associated to bovine neonatal pancytopenia by deep sequencing transcriptome analysis in cattle.

Bovine neonatal pancytopenia (BNP) is a new fatal, alloimmune/alloantibody mediated disease of new-born calves induced by ingestion of colostrum from cows, which had been vaccinated with a specific vaccine against the Bovine Virus Diarrhoea Virus (BVDV). The hypothesis of pathogenic MHC class I molecules in the vaccine had been put up, but no formal proof of specific causal MHC class I alleles has been provided yet. However, the unique features of the vaccine obviously result in extremely high specific antibody titres in the vaccinated animals, but apparently also in further molecules inducing BNP. Thus, a comprehensive picture of the immune response to the vaccine is essential. Applying the novel approach of next generation RNA sequencing (RNAseq), our study provides a new holistic, comprehensive analysis of the blood transcriptome regulation after vaccination with the specific BVDV vaccine. Our RNAseq approach identified a novel cytokine-like gene in the bovine genome that is highly upregulated after vaccination. This gene has never been described before in any other species and might be specific to ruminant immune response. Furthermore, our data revealed a very coordinated immune response to double-stranded (ds) RNA or a dsRNA analogue after vaccination with the inactivated single-stranded (ss) RNA vaccine. This would suggest either a substantial contamination of the vaccine with dsRNA from host cells after virus culture or a dsRNA analogue applied to the vaccine. The first option would highlight the potential risks associated with virus culture on homologous cells during vaccine production; the latter option would emphasise the potential risks associated with immune stimulating adjuvants used in vaccine production.

Exploration of genotype-by-environment interactions affecting gene expression responses in porcine immune cells.

As one of the keys to healthy performance, robustness of farm animals is gaining importance, and with this comes increasing interest in genetic dissection of genotype-by-environment interactions (G×E). Changes in gene expression are among the most sensitive responses conveying adaptation to environmental stimuli. Environmentally responsive regulatory variation thus likely plays a central role in G×E. In the present study, we set out to detect action of environmentally responsive cis-regulatory variation by the analysis of condition-dependent allele specific expression (cd-ASE) in porcine immune cells. For this, we harnessed mRNA-sequencing data of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with lipopolysaccharide, dexamethasone, or their combination. These treatments mimic common challenges such as bacterial infection or stress, and induce vast transcriptome changes. About two thirds of the examined loci showed significant ASE in at least one treatment, and out of those about ten percent exhibited cd-ASE. Most of the ASE variants were not yet reported in the PigGTEx Atlas. Genes showing cd-ASE were enriched in cytokine signaling in immune system and include several key candidates for animal health. In contrast, genes showing no ASE featured cell-cycle related functions. We confirmed LPS-dependent ASE for one of the top candidates, SOD2, which ranks among the major response genes in LPS-stimulated monocytes. The results of the present study demonstrate the potential of in vitro cell models coupled with cd-ASE analysis for the investigation of G×E in farm animals. The identified loci may benefit efforts to unravel the genetic basis of robustness and improvement of health and welfare in pigs.

RNA-Seq-based discovery of genetic variants and allele-specific expression of two layer lines and broiler chicken.

Recent advances in the selective breeding of broilers and layers have made poultry production one of the fastest-growing industries. In this study, a transcriptome variant calling approach from RNA-seq data was used to determine population diversity between broilers and layers. In total, 200 individuals were analyzed from three different chicken populations (Lohmann Brown (LB), n = 90), Lohmann Selected Leghorn (LSL, n = 89), and Broiler (BR, n = 21). The raw RNA-sequencing reads were pre-processed, quality control checked, mapped to the reference genome, and made compatible with Genome Analysis ToolKit for variant detection. Subsequently, pairwise fixation index (F ST) analysis was performed between broilers and layers. Numerous candidate genes were identified, that were associated with growth, development, metabolism, immunity, and other economically significant traits. Finally, allele-specific expression (ASE) analysis was performed in the gut mucosa of LB and LSL strains at 10, 16, 24, 30, and 60 weeks of age. At different ages, the two-layer strains showed significantly different allele-specific expressions in the gut mucosa, and changes in allelic imbalance were observed across the entire lifespan. Most ASE genes are involved in energy metabolism, including sirtuin signaling pathways, oxidative phosphorylation, and mitochondrial dysfunction. A high number of ASE genes were found during the peak of laying, which were particularly enriched in cholesterol biosynthesis. These findings indicate that genetic architecture as well as biological processes driving particular demands relate to metabolic and nutritional requirements during the laying period shape allelic heterogeneity. These processes are considerably affected by breeding and management, whereby elucidating allele-specific gene regulation is an essential step towards deciphering the genotype to phenotype map or functional diversity between the chicken populations. Additionally, we observed that several genes showing significant allelic imbalance also colocalized with the top 1% of genes identified by the FST approach, suggesting a fixation of genes in cis-regulatory elements.