Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies.

BACKGROUND: Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. RESULTS: Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. CONCLUSION: By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

GOexpress: an R/Bioconductor package for the identification and visualisation of robust gene ontology signatures through supervised learning of gene expression data.

BACKGROUND: Identification of gene expression profiles that differentiate experimental groups is critical for discovery and analysis of key molecular pathways and also for selection of robust diagnostic or prognostic biomarkers. While integration of differential expression statistics has been used to refine gene set enrichment analyses, such approaches are typically limited to single gene lists resulting from simple two-group comparisons or time-series analyses. In contrast, functional class scoring and machine learning approaches provide powerful alternative methods to leverage molecular measurements for pathway analyses, and to compare continuous and multi-level categorical factors. RESULTS: We introduce GOexpress, a software package for scoring and summarising the capacity of gene ontology features to simultaneously classify samples from multiple experimental groups. GOexpress integrates normalised gene expression data (e.g., from microarray and RNA-seq experiments) and phenotypic information of individual samples with gene ontology annotations to derive a ranking of genes and gene ontology terms using a supervised learning approach. The default random forest algorithm allows interactions between all experimental factors, and competitive scoring of expressed genes to evaluate their relative importance in classifying predefined groups of samples. CONCLUSIONS: GOexpress enables rapid identification and visualisation of ontology-related gene panels that robustly classify groups of samples and supports both categorical (e.g., infection status, treatment) and continuous (e.g., time-series, drug concentrations) experimental factors. The use of standard Bioconductor extension packages and publicly available gene ontology annotations facilitates straightforward integration of GOexpress within existing computational biology pipelines.

Definition of the cattle killer cell Ig-like receptor gene family: comparison with aurochs and human counterparts.

Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig-like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle.

DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

BACKGROUND: In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation. RESULTS: To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization. CONCLUSION: The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro.

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples.

BACKGROUND: The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. RESULTS: In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). CONCLUSIONS: The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.

Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, an intestinal disease of ruminants with major economic consequences. Infectious bacilli are phagocytosed by host macrophages upon exposure where they persist, resulting in lengthy subclinical phases of infection that can lead to immunopathology and disease dissemination. Consequently, analysis of the macrophage transcriptome in response to M. avium subsp. paratuberculosis infection can provide valuable insights into the molecular mechanisms that underlie Johne's disease. Here, we investigate pan-genomic gene expression in bovine monocyte-derived macrophages (MDM) purified from seven age-matched females, in response to in vitro infection with M. avium subsp. paratuberculosis (multiplicity of infection 2:1) at intervals of 2 hours, 6 hours and 24 hours post-infection (hpi). Differentially expressed genes were identified by comparing the transcriptomes of the infected MDM to the non-infected control MDM at each time point (adjusted P-value threshold ≤ 0.10). 1050 differentially expressed unique genes were identified 2 hpi, with 974 and 78 differentially expressed unique genes detected 6 and 24 hpi, respectively. Furthermore, in the infected MDM the number of upregulated genes exceeded the number of downregulated genes at each time point, with the fold-change in expression for the upregulated genes markedly higher than that for the downregulated genes. Inspection and systems biology analysis of the differentially expressed genes revealed an enrichment of genes involved in the inflammatory response, cell signalling pathways and apoptosis. The transcriptional changes associated with cellular signalling and the inflammatory response may reflect different immuno-modulatory mechanisms that underlie host-pathogen interactions during infection.

High-resolution transcriptomics of bovine purified protein derivative-stimulated peripheral blood from cattle infected with Mycobacterium bovis across an experimental time course.

OBJECTIVES: Improved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood-an important component of current bTB diagnostics-will provide new information for development of better diagnostics. METHODS: RNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (-1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle. RESULTS: In vitro PPD-b stimulation of peripheral blood from M. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters. CONCLUSIONS: A transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection.

Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis reveals suppression of host immune genes.

BACKGROUND: Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with 24,072 gene probe sets representing more than 23,000 gene transcripts. RESULTS: Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of gene transcripts showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity® Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group. CONCLUSIONS: This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection.

Zebu cattle are an exclusive legacy of the South Asia neolithic.

Animal domestication was a major step forward in human prehistory, contributing to the emergence of more complex societies. At the time of the Neolithic transition, zebu cattle (Bos indicus) were probably the most abundant and important domestic livestock species in Southern Asia. Although archaeological evidence points toward the domestication of zebu cattle within the Indian subcontinent, the exact geographic origins and phylogenetic history of zebu cattle remains uncertain. Here, we report evidence from 844 zebu mitochondrial DNA (mtDNA) sequences surveyed from 19 Asiatic countries comprising 8 regional groups, which identify 2 distinct mitochondrial haplogroups, termed I1 and I2. The marked increase in nucleotide diversity (P < 0.001) for both the I1 and I2 haplogroups within the northern part of the Indian subcontinent is consistent with an origin for all domestic zebu in this area. For haplogroup I1, genetic diversity was highest within the Indus Valley among the three hypothesized domestication centers (Indus Valley, Ganges, and South India). These data support the Indus Valley as the most likely center of origin for the I1 haplogroup and a primary center of zebu domestication. However, for the I2 haplogroup, a complex pattern of diversity is detected, preventing the unambiguous pinpointing of the exact place of origin for this zebu maternal lineage. Our findings are discussed with respect to the archaeological record for zebu domestication within the Indian subcontinent.

DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits.

BACKGROUND: Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. RESULTS: SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following adjustment for multiple-testing, significant association (q ≤ 0.05) remained between the rs41694646 SNP and four traits (animal stature, body depth, direct calving difficulty and milk yield) only. Notably, the single SNP in the bovine NESP55 gene (rs41694656) was associated (P ≤ 0.01) with somatic cell count--an often-cited indicator of resistance to mastitis and overall health status of the mammary system--and previous studies have demonstrated that the chromosomal region to where the GNAS domain maps underlies an important quantitative trait locus for this trait. This association, however, was not significant after adjustment for multiple testing. The three remaining SNPs assayed were not associated with any of the performance traits analysed in this study. Analysis of all pairwise linkage disequilibrium (r2) values suggests that most allele substitution effects for the assayed SNPs observed are independent. Finally, the polymorphic coding SNP in the putative bovine NESP55 gene was used to test the imprinting status of this gene across a range of foetal bovine tissues. CONCLUSIONS: Previous studies in other mammalian species have shown that DNA sequence variation within the imprinted GNAS gene cluster contributes to several physiological and metabolic disorders, including obesity in humans and mice. Similarly, the results presented here indicate an important role for the imprinted GNAS cluster in underlying complex performance traits in cattle such as animal growth, calving, fertility and health. These findings suggest that GNAS domain-associated polymorphisms may serve as important genetic markers for future livestock breeding programs and support previous studies that candidate imprinted loci may act as molecular targets for the genetic improvement of agricultural populations. In addition, we present new evidence that the bovine NESP55 gene is epigenetically regulated as a maternally expressed imprinted gene in placental and intestinal tissues from 8-10 week old bovine foetuses.

Single nucleotide polymorphisms within the bovine DLK1-DIO3 imprinted domain are associated with economically important production traits in cattle.

Previous studies show that DNA sequence variation within the mammalian DLK1-DIO3 imprinted domain influences production traits in domestic livestock, most notably the ovine callipyge phenotype. We assessed genotype-phenotype associations between 7 single nucleotide polymorphisms (SNPs) within the orthologous bovine DLK1-DIO3 domain and performance traits in 848 progeny-tested Holstein-Friesian dairy sires. One SNP (MEG3_01) located proximal to the maternally expressed 3 (MEG3/Gtl2) gene was associated with milk yield, subcutaneous fat levels, and progeny carcass conformation (P ≤ 0.01) and also tended to be associated with milk fat and protein yield (P ≤ 0.10). A single SNP (CLPG_01) within the putative CLPG1 locus was associated with progeny carcass fat (P ≤ 0.05), whereas a single SNP (PEG11_01) located proximal to the paternally expressed 11 (PEG11/Rtl) gene was associated with progeny carcass weight (P ≤ 0.05). The MEG3_01 SNP together with an additional 2 SNPs (MEG8_01 and MEG8_02) located proximal to the putative maternally expressed 8 (MEG8/Rian) ortholog were associated (P ≤ 0.05) with perinatal mortality. Finally, one SNP (MEG3_03) was associated (P ≤ 0.05) with gestation length, whereas both the CLPG_01 and MEG8_01 SNPs also tended to be associated with calving interval (P ≤ 0.10). Linkage disequilibrium analysis suggests that some phenotypic associations observed at these loci are independent. To our knowledge, this is one of the first studies demonstrating associations between the bovine DLK1-DIO3 domain and milk, carcass, fertility and, health traits in cattle. This imprinted domain may serve as a potential target for future genetic selection strategies.

Single nucleotide polymorphisms at the imprinted bovine insulin-like growth factor 2 (IGF2) locus are associated with dairy performance in Irish Holstein-Friesian cattle.

The imprinted insulin-like growth factor 2 gene (IGF2) encodes a fetal mitogenic hormone protein (IGF-II) and has previously been shown to be associated with performance in dairy cattle. In this study we assessed genotype-phenotype associations between four single nucleotide polymorphisms (SNPs) located within the bovine IGF2 locus on chromosome 29 and a range of performance traits related to milk production, animal growth and body size, fertility and progeny survival in 848 progeny-tested Irish Holstein-Friesian sires. Two of the four SNPs (rs42196909 and IGF2.g-3815A>G), which were in strong linkage disequilibrium (r2 = 0·995), were associated with milk yield (P ≤ 0·01) and milk protein yield (P ≤ 0·05); the rs42196901 SNP was also associated (P ≤ 0·05) with milk fat yield. Associations (P ≤ 0·05) with milk fat percentage and milk protein percentage were observed at the rs42196901 and IGF2.g-3815A>G SNPs, respectively. The rs42196909 and IGF2.g-3815A>G SNPs were also associated with progeny carcass conformation (P ≤ 0·05), while an association (P ≤ 0·01) with progeny carcass weight was observed at the rs42194733 SNP locus. None of the four SNPs were associated with body size, fertility and progeny survival. These findings support previous work which suggests that the IGF2 locus is an important biological regulator of milk production in dairy cattle and add to an accumulating body of research showing that imprinted genes influence many complex performance traits in cattle.

RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course.

Bovine tuberculosis, caused by infection with members of the Mycobacterium tuberculosis complex, particularly Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including RNA sequencing, has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analysed the transcriptome of bovine whole peripheral blood samples collected at -1 week pre-infection and +1, +2, +6, +10, and +12 weeks post-infection time points. Differentially expressed genes were catalogued and evaluated at each post-infection time point relative to the -1 week pre-infection time point and used for the identification of putative candidate host transcriptional biomarkers for M. bovis infection. Differentially expressed gene sets were also used for examination of cellular pathways associated with the host response to M. bovis infection, construction of de novo gene interaction networks enriched for host differentially expressed genes, and time-series analyses to identify functionally important groups of genes displaying similar patterns of expression across the infection time course. A notable outcome of these analyses was identification of a 19-gene transcriptional biosignature of infection consisting of genes increased in expression across the time course from +1 week to +12 weeks post-infection.

Polymorphisms in bovine immune genes and their associations with somatic cell count and milk production in dairy cattle.

BACKGROUND: Mastitis, an inflammation of the mammary gland, is a major source of economic loss on dairy farms. The aim of this study was to quantify the associations between two previously identified polymorphisms in the bovine toll-like receptor 2 (TLR2) and chemokine receptor 1 (CXCR1) genes and mammary health indictor traits in (a) 246 lactating dairy cow contemporaries representing five breeds from one research farm and (b) 848 Holstein-Friesian bulls that represent a large proportion of the Irish dairy germplasm. To expand the study, a further 14 polymorphisms in immune genes were included for association studies in the bull population. RESULTS: TLR4-2021 associated (P < 0.05) with both milk protein and fat percentage in late lactation (P < 0.01) within the cow cohort. No association was observed between this polymorphism and either yield or composition of milk within the bull population. CXCR1-777 significantly associated (P < 0.05) with fat yield in the bull population and tended to associate (P < 0.1) with somatic cell score (SCS) in the cows genotyped. CD14-1908 A allele was found to associate with increased (P < 0.05) milk fat and protein yield and also tended to associate with increased (P < 0.1) milk yield. A SERPINA1 haplotype with superior genetic merit for milk protein yield and milk fat percentage (P < 0.05) was also identified. CONCLUSION: Of the sixteen polymorphisms in seven immune genes genotyped, just CXCR1-777 tended to associate with SCS, albeit only in the on-farm study. The lack of an association between the polymorphisms with SCS in the Holstein-Friesian data set would question the potential importance of these variants in selection for improved mastitis resistance in the Holstein-Friesian cow.

DNA sequence polymorphisms in a panel of eight candidate bovine imprinted genes and their association with performance traits in Irish Holstein-Friesian cattle.

BACKGROUND: Studies in mice and humans have shown that imprinted genes, whereby expression from one of the two parentally inherited alleles is attenuated or completely silenced, have a major effect on mammalian growth, metabolism and physiology. More recently, investigations in livestock species indicate that genes subject to this type of epigenetic regulation contribute to, or are associated with, several performance traits, most notably muscle mass and fat deposition. In the present study, a candidate gene approach was adopted to assess 17 validated single nucleotide polymorphisms (SNPs) and their association with a range of performance traits in 848 progeny-tested Irish Holstein-Friesian artificial insemination sires. These SNPs are located proximal to, or within, the bovine orthologs of eight genes (CALCR, GRB10, PEG3, PHLDA2, RASGRF1, TSPAN32, ZIM2 and ZNF215) that have been shown to be imprinted in cattle or in at least one other mammalian species (i.e. human/mouse/pig/sheep). RESULTS: Heterozygosities for all SNPs analysed ranged from 0.09 to 0.46 and significant deviations from Hardy-Weinberg proportions (P ≤ 0.01) were observed at four loci. Phenotypic associations (P ≤ 0.05) were observed between nine SNPs proximal to, or within, six of the eight analysed genes and a number of performance traits evaluated, including milk protein percentage, somatic cell count, culled cow and progeny carcass weight, angularity, body conditioning score, progeny carcass conformation, body depth, rump angle, rump width, animal stature, calving difficulty, gestation length and calf perinatal mortality. Notably, SNPs within the imprinted paternally expressed gene 3 (PEG3) gene cluster were associated (P ≤ 0.05) with calving, calf performance and fertility traits, while a single SNP in the zinc finger protein 215 gene (ZNF215) was associated with milk protein percentage (P ≤ 0.05), progeny carcass weight (P ≤ 0.05), culled cow carcass weight (P ≤ 0.01), angularity (P ≤ 0.01), body depth (P ≤ 0.01), rump width (P ≤ 0.01) and animal stature (P ≤ 0.01). CONCLUSIONS: Of the eight candidate bovine imprinted genes assessed, DNA sequence polymorphisms in six of these genes (CALCR, GRB10, PEG3, RASGRF1, ZIM2 and ZNF215) displayed associations with several of the phenotypes included for analyses. The genotype-phenotype associations detected here are further supported by the biological function of these six genes, each of which plays important roles in mammalian growth, development and physiology. The associations between SNPs within the imprinted PEG3 gene cluster and traits related to calving, calf performance and gestation length suggest that this domain on chromosome 18 may play a role regulating pre-natal growth and development and fertility. SNPs within the bovine ZNF215 gene were associated with bovine growth and body conformation traits and studies in humans have revealed that the human ZNF215 ortholog belongs to the imprinted gene cluster associated with Beckwith-Wiedemann syndrome--a genetic disorder characterised by growth abnormalities. Similarly, the data presented here suggest that the ZNF215 gene may have an important role in regulating bovine growth. Collectively, our results support previous work showing that (candidate) imprinted genes/loci contribute to heritable variation in bovine performance traits and suggest that DNA sequence polymorphisms within these genes/loci represents an important reservoir of genomic markers for future genetic improvement of dairy and beef cattle populations.

A Population Genomics Analysis of the Native Irish Galway Sheep Breed.

The Galway sheep population is the only native Irish sheep breed and this livestock genetic resource is currently categorised as 'at-risk'. In the present study, comparative population genomics analyses of Galway sheep and other sheep populations of European origin were used to investigate the microevolution and recent genetic history of the breed. These analyses support the hypothesis that British Leicester sheep were used in the formation of the Galway. When compared to conventional and endangered breeds, the Galway breed was intermediate in effective population size, genomic inbreeding and runs of homozygosity. This indicates that, although the Galway breed is declining, it is still relatively genetically diverse and that conservation and management plans informed by genomic information may aid its recovery. The Galway breed also exhibited distinct genomic signatures of artificial or natural selection when compared to other breeds, which highlighted candidate genes that may be involved in production and health traits.

Genomic Characterisation of the Indigenous Irish Kerry Cattle Breed.

Kerry cattle are an endangered landrace heritage breed of cultural importance to Ireland. In the present study we have used genome-wide SNP array data to evaluate genomic diversity within the Kerry population and between Kerry cattle and other European breeds. Patterns of genetic differentiation and gene flow among breeds using phylogenetic trees with ancestry graphs highlighted historical gene flow from the British Shorthorn breed into the ancestral population of modern Kerry cattle. Principal component analysis (PCA) and genetic clustering emphasised the genetic distinctiveness of Kerry cattle relative to comparator British and European cattle breeds. Modelling of genetic effective population size (Ne) revealed a demographic trend of diminishing Ne over time and that recent estimated Ne values for the Kerry breed may be less than the threshold for sustainable genetic conservation. In addition, analysis of genome-wide autozygosity (FROH) showed that genomic inbreeding has increased significantly during the 20 years between 1992 and 2012. Finally, signatures of selection revealed genomic regions subject to natural and artificial selection as Kerry cattle adapted to the climate, physical geography and agro-ecology of southwest Ireland.

RNA Sequencing (RNA-Seq) Reveals Extremely Low Levels of Reticulocyte-Derived Globin Gene Transcripts in Peripheral Blood From Horses (Equus caballus) and Cattle (Bos taurus).

RNA-seq has emerged as an important technology for measuring gene expression in peripheral blood samples collected from humans and other vertebrate species. In particular, transcriptomics analyses of whole blood can be used to study immunobiology and develop novel biomarkers of infectious disease. However, an obstacle to these methods in many mammalian species is the presence of reticulocyte-derived globin mRNAs in large quantities, which can complicate RNA-seq library sequencing and impede detection of other mRNA transcripts. A range of supplementary procedures for targeted depletion of globin transcripts have, therefore, been developed to alleviate this problem. Here, we use comparative analyses of RNA-seq data sets generated from human, porcine, equine, and bovine peripheral blood to systematically assess the impact of globin mRNA on routine transcriptome profiling of whole blood in cattle and horses. The results of these analyses demonstrate that total RNA isolated from equine and bovine peripheral blood contains very low levels of globin mRNA transcripts, thereby negating the need for globin depletion and greatly simplifying blood-based transcriptomic studies in these two domestic species.

Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli.

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection.