Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Unraveling the history of the genus Gallus through whole genome sequencing.

The genus Gallus is distributed across a large part of Southeast Asia and has received special interest because the domestic chicken, Gallus gallus domesticus, has spread all over the world and is a major protein source for humans. There are four species: the red junglefowl (G. gallus), the green junglefowl (G. varius), the Lafayette's junglefowl (G. lafayettii) and the grey junglefowl (G. sonneratii). The aim of this study is to reconstruct the history of these species by a whole genome sequencing approach and resolve inconsistencies between well supported topologies inferred using different data and methods. Using deep sequencing, we identified over 35 million SNPs and reconstructed the phylogeny of the Gallus genus using both distance (BioNJ) and maximum likelihood (ML) methods. We observed discrepancies according to reconstruction methods and genomic components. The two most supported topologies were previously reported and were discriminated by using phylogenetic and gene flow analyses, based on ABBA statistics. Terminology fix requested by the deputy editor led to support a scenario with G. gallus as the earliest branching lineage of the Gallus genus, instead of G. varius. We discuss the probable causes for the discrepancy. A likely one is that G. sonneratii samples from parks or private collections are all recent hybrids, with roughly 10% of their autosomal genome originating from G. gallus. The removal of those regions is needed to provide reliable data, which was not done in previous studies. We took care of this and additionally included two wild G. sonneratii samples from India, showing no trace of introgression. This reinforces the importance of carefully selecting and validating samples and genomic components in phylogenomics.

Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Bovine and murine models highlight novel roles for SLC25A46 in mitochondrial dynamics and metabolism, with implications for human and animal health.

Neuropathies are neurodegenerative diseases affecting humans and other mammals. Many genetic causes have been identified so far, including mutations of genes encoding proteins involved in mitochondrial dynamics. Recently, the "Turning calves syndrome", a novel sensorimotor polyneuropathy was described in the French Rouge-des-Prés cattle breed. In the present study, we determined that this hereditary disease resulted from a single nucleotide substitution in SLC25A46, a gene encoding a protein of the mitochondrial carrier family. This mutation caused an apparent damaging amino-acid substitution. To better understand the function of this protein, we knocked out the Slc25a46 gene in a mouse model. This alteration affected not only the nervous system but also altered general metabolism, resulting in premature mortality. Based on optic microscopy examination, electron microscopy and on biochemical, metabolic and proteomic analyses, we showed that the Slc25a46 disruption caused a fusion/fission imbalance and an abnormal mitochondrial architecture that disturbed mitochondrial metabolism. These data extended the range of phenotypes associated with Slc25a46 dysfunction. Moreover, this Slc25a46 knock-out mouse model should be useful to further elucidate the role of SLC25A46 in mitochondrial dynamics.

Multi-species annotation of transcriptome and chromatin structure in domesticated animals.

BACKGROUND: Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). RESULTS: RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. CONCLUSIONS: We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

RNA-seq analysis of hepatic gene expression of common Pekin, Muscovy, mule and hinny ducks fed ad libitum or overfed.

BACKGROUND: Duck species are known to have different susceptibility to fatty liver production in response to overfeeding. In order to better describe mechanisms involved in the development of hepatic steatosis and differences between species, transcriptome analyses were conducted on RNAs extracted from the livers of Pekin and Muscovy duck species and of their reciprocal hybrids, Mule and Hinny ducks fed ad libitum or overfed to identify differentially expressed genes and associated functions. RESULTS: After extraction from the liver of ducks from the four genetic types, RNAs were sequenced and sequencing data were analyzed. Hierarchic clustering and principal component analyses of genes expression levels indicated that differences between individuals lie primarily in feeding effect, differences between genetic types being less important. However, Muscovy ducks fed ad libitum and overfed were clustered together. Interestingly, Hinny and Mule hybrid ducks could not be differentiated from each other, according to feeding. Many genes with expression differences between overfed and ad libitum fed ducks were identified in each genetic type. Functional annotation analyses of these differentially expressed genes highlighted some expected functions (carbohydrate and lipid metabolisms) but also some unexpected ones (cell proliferation and immunity). CONCLUSIONS: These analyses evidence differences in response to overfeeding between different genetic types and help to better characterize functions involved in hepatic steatosis in ducks.

Correction to: Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

After publication of this work [1], we noted that there was an error in Table 3 Line 4.

Genetic and transcriptomic analyses provide new insights on the early antiviral response to VHSV in resistant and susceptible rainbow trout.

BACKGROUND: The viral hemorrhagic septicemia virus (VHSV) is a major threat for salmonid farming and for wild fish populations worldwide. Previous studies have highlighted the importance of innate factors regulated by a major quantitative trait locus (QTL) for the natural resistance to waterborne VHSV infection in rainbow trout. The aim of this study was to analyze the early transcriptomic response to VHSV inoculation in cell lines derived from previously described resistant and susceptible homozygous isogenic lines of rainbow trout to obtain insights into the molecular mechanisms responsible for the resistance to the viral infection. RESULTS: We first confirmed the presence of the major QTL in a backcross involving a highly resistant fish isogenic line (B57) and a highly susceptible one (A22), and were able to define the confidence interval of the QTL and to identify its precise position. We extended the definition of the QTL since it controls not only resistance to waterborne infection but also the kinetics of mortality after intra-peritoneal injection. Deep sequencing of the transcriptome of B57 and A22 derived cell lines exposed to inactivated VHSV showed a stronger response to virus inoculation in the resistant background. In line with our previous observations, an early and strong induction of interferon and interferon-stimulated genes was correlated with the resistance to VHSV, highlighting the major role of innate immune factors in natural trout resistance to the virus. Interestingly, major factors of the antiviral innate immunity were much more expressed in naive B57 cells compared to naive A22 cells, which likely contributes to the ability of B57 to mount a fast antiviral response after viral infection. These observations were further extended by the identification of several innate immune-related genes localized close to the QTL area on the rainbow trout genome. CONCLUSIONS: Taken together, our results improve our knowledge in virus-host interactions in vertebrates and provide novel insights in the molecular mechanisms explaining the resistance to VHSV in rainbow trout. Our data also provide a collection of potential markers for resistance and susceptibility of rainbow trout to VHSV infection.

Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

BACKGROUND: Bacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms. RESULTS: Two isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A "compensation-like" effect was detected between several pairs of QTL for the two modes of infection. An "enhancing-like" interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes). CONCLUSIONS: These results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes.

Identification of copy number variation in French dairy and beef breeds using next-generation sequencing.

BACKGROUND: Copy number variations (CNV) are known to play a major role in genetic variability and disease pathogenesis in several species including cattle. In this study, we report the identification and characterization of CNV in eight French beef and dairy breeds using whole-genome sequence data from 200 animals. Bioinformatics analyses to search for CNV were carried out using four different but complementary tools and we validated a subset of the CNV by both in silico and experimental approaches. RESULTS: We report the identification and localization of 4178 putative deletion-only, duplication-only and CNV regions, which cover 6% of the bovine autosomal genome; they were validated by two in silico approaches and/or experimentally validated using array-based comparative genomic hybridization and single nucleotide polymorphism genotyping arrays. The size of these variants ranged from 334 bp to 7.7 Mb, with an average size of ~ 54 kb. Of these 4178 variants, 3940 were deletions, 67 were duplications and 171 corresponded to both deletions and duplications, which were defined as potential CNV regions. Gene content analysis revealed that, among these variants, 1100 deletions and duplications encompassed 1803 known genes, which affect a wide spectrum of molecular functions, and 1095 overlapped with known QTL regions. CONCLUSIONS: Our study is a large-scale survey of CNV in eight French dairy and beef breeds. These CNV will be useful to study the link between genetic variability and economically important traits, and to improve our knowledge on the genomic architecture of cattle.

A missense variant in the coil1A domain of the keratin 25 gene is associated with the dominant curly hair coat trait (Crd) in horse.

BACKGROUND: Curly horses present a variety of curl phenotypes that are associated with various degrees of curliness of coat, mane, tail and ear hairs. Their origin is still a matter of debate and several genetic hypotheses have been formulated to explain the diversity in phenotype, including the combination of autosomal dominant and recessive alleles. Our purpose was to map the autosomal dominant curly hair locus and identify the causal variant using genome-wide association study (GWAS) and whole-genome sequencing approaches. RESULTS: A GWAS was performed using a Bayesian sparse linear mixed model, based on 51 curly and 19 straight-haired French and North American horses from 13 paternal families genotyped on the Illumina EquineSNP50 BeadChip. A single strong signal was observed on equine chromosome 11, in a region that encompasses the type I keratin gene cluster. This region was refined by haplotype analysis to a segment including 36 genes, among which are 10 keratin genes (KRT-10, -12, -20, -23, -24, -25, -26, -27, -28, -222). To comprehensively identify candidate causal variants within all these genes, whole-genome sequences were obtained for one heterozygous curly stallion and its straight-haired son. Among the four non-synonymous candidate variants identified and validated in the curly region, only variant g.21891160G>A in the KRT25 gene (KRT25:p.R89H) was in perfect agreement with haplotype status in the whole pedigree. Genetic association was then confirmed by genotyping a larger population consisting of 353 horses. However, five discordant curly horses were observed, which carried neither the variant nor the main haplotype associated with curliness. Sequencing of KRT25 for two discordant horses did not identify any other deleterious variant, which suggests locus rather than allelic heterogeneity for the curly phenotype. CONCLUSIONS: We identified the KRT25:p.R89H variant as responsible for the dominant curly trait, but a second dominant locus may also be involved in the shape of hairs within North American Curly horses.

Long noncoding RNA repertoire in chicken liver and adipose tissue.

BACKGROUND: Improving functional annotation of the chicken genome is a key challenge in bridging the gap between genotype and phenotype. Among all transcribed regions, long noncoding RNAs (lncRNAs) are a major component of the transcriptome and its regulation, and whole-transcriptome sequencing (RNA-Seq) has greatly improved their identification and characterization. We performed an extensive profiling of the lncRNA transcriptome in the chicken liver and adipose tissue by RNA-Seq. We focused on these two tissues because of their importance in various economical traits for which energy storage and mobilization play key roles and also because of their high cell homogeneity. To predict lncRNAs, we used a recently developed tool called FEELnc, which also classifies them with respect to their distance and strand orientation to the closest protein-coding genes. Moreover, to confidently identify the genes/transcripts expressed in each tissue (a complex task for weakly expressed molecules such as lncRNAs), we probed a particularly large number of biological replicates (16 per tissue) compared to common multi-tissue studies with a larger set of tissues but less sampling. RESULTS: We predicted 2193 lncRNA genes, among which 1670 were robustly expressed across replicates in the liver and/or adipose tissue and which were classified into 1493 intergenic and 177 intragenic lncRNAs located between and within protein-coding genes, respectively. We observed similar structural features between chickens and mammals, with strong synteny conservation but without sequence conservation. As previously reported, we confirm that lncRNAs have a lower and more tissue-specific expression than mRNAs. Finally, we showed that adjacent lncRNA-mRNA genes in divergent orientation have a higher co-expression level when separated by less than 1 kb compared to more distant divergent pairs. Among these, we highlighted for the first time a novel lncRNA candidate involved in lipid metabolism, lnc_DHCR24, which is highly correlated with the DHCR24 gene that encodes a key enzyme of cholesterol biosynthesis. CONCLUSIONS: We provide a comprehensive lncRNA repertoire in the chicken liver and adipose tissue, which shows interesting patterns of co-expression between mRNAs and lncRNAs. It contributes to improving the structural and functional annotation of the chicken genome and provides a basis for further studies on energy storage and mobilization traits in the chicken.

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences.

BACKGROUND: In recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability. RESULTS: In this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data. CONCLUSIONS: To our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection.

Identification of large intergenic non-coding RNAs in bovine muscle using next-generation transcriptomic sequencing.

BACKGROUND: The advent of large-scale gene expression technologies has helped to reveal in eukaryotic cells, the existence of thousands of non-coding transcripts, whose function and significance remain mostly poorly understood. Among these non-coding transcripts, long non-coding RNAs (lncRNAs) are the least well-studied but are emerging as key regulators of diverse cellular processes. In the present study, we performed a survey in bovine Longissimus thoraci of lincRNAs (long intergenic non-coding RNAs not overlapping protein-coding transcripts). To our knowledge, this represents the first such study in bovine muscle. RESULTS: To identify lincRNAs, we used paired-end RNA sequencing (RNA-Seq) to explore the transcriptomes of Longissimus thoraci from nine Limousin bull calves. Approximately 14-45 million paired-end reads were obtained per library. A total of 30,548 different transcripts were identified. Using a computational pipeline, we defined a stringent set of 584 different lincRNAs with 418 lincRNAs found in all nine muscle samples. Bovine lincRNAs share characteristics seen in their mammalian counterparts: relatively short transcript and gene lengths, low exon number and significantly lower expression, compared to protein-encoding genes. As for the first time, our study identified lincRNAs from nine different samples from the same tissue, it is possible to analyse the inter-individual variability of the gene expression level of the identified lincRNAs. Interestingly, there was a significant difference when we compared the expression variation of the 418 lincRNAs with the 10,775 known selected protein-encoding genes found in all muscle samples. In addition, we found 2,083 pairs of lincRNA/protein-encoding genes showing a highly significant correlated expression. Fourteen lincRNAs were selected and 13 were validated by RT-PCR. Some of the lincRNAs expressed in muscle are located within quantitative trait loci for meat quality traits. CONCLUSIONS: Our study provides a glimpse into the lincRNA content of bovine muscle and will facilitate future experimental studies to unravel the function of these molecules. It may prove useful to elucidate their effect on mechanisms underlying the genetic variability of meat quality traits. This catalog will complement the list of lincRNAs already discovered in cattle and therefore will help to better annotate the bovine genome.

Involvement of mitochondrial dysfunction and ER-stress in the physiopathology of equine osteochondritis dissecans (OCD).

Osteochondrosis (OC) is a developmental bone disorder affecting several mammalian species including the horse. Equine OC is described as a focal disruption of endochondral ossification, leading to osteochondral lesions (osteochondritis dissecans, OCD) that may release free bodies within the joint. OCD lesions trigger joint swelling, stiffness and lameness and affects about 30% of the equine population. OCD is considered as multifactorial but its physiopathology is still poorly understood and genes involved in genetic predisposition are still unknown. Our study compared two healthy and two OC-affected 18-month-old French Trotters diagnosed with OCD lesions at the intermediate ridge of the distal tibia. A comparative shot-gun proteomic analysis of non-wounded cartilage and sub-chondral bone from healthy (healthy samples) and OC-affected foals (predisposed samples) identified 83 and 53 modulated proteins, respectively. These proteins are involved in various biological pathways including matrix structure and maintenance, protein biosynthesis, folding and transport, mitochondrial activity, energy and calcium metabolism. Transmission electron microscopy revealed typical features of mitochondrial swelling and ER-stress, such as large, empty mitochondria, and hyper-dilated rough endoplasmic reticulum, in the deep zone of both OC lesions and predisposed cartilage. Abnormal fibril organization surrounding chondrocytes and abnormal features at the ossification front were also observed. Combining these findings with quantitative trait loci and whole genome sequencing results identified about 140 functional candidate genes carrying putative damaging mutations in 30 QTL regions. In summary, our study suggests that OCD lesions may result from defective hypertrophic terminal differentiation associated with mitochondrial dysfunction and ER-stress, leading to impaired cartilage and bone biomechanical properties, making them prone to fractures. In addition, 11 modulated proteins and several candidate mutations located in QTL regions were identified, bringing new insight into the molecular physiopathology and genetic basis of OCD.

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling.

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

Gene-based single nucleotide polymorphism discovery in bovine muscle using next-generation transcriptomic sequencing.

BACKGROUND: Genetic information based on molecular markers has increasingly being used in cattle breeding improvement programmes, as a mean to improve conventionally phenotypic selection. Advances in molecular genetics have led to the identification of several genetic markers associated with genes affecting economic traits. Until recently, the identification of the causative genetic variants involved in the phenotypes of interest has remained a difficult task. The advent of novel sequencing technologies now offers a new opportunity for the identification of such variants. Despite sequencing costs plummeting, sequencing whole-genomes or large targeted regions is still too expensive for most laboratories. A transcriptomic-based sequencing approach offers a cheaper alternative to identify a large number of polymorphisms and possibly to discover causative variants. In the present study, we performed a gene-based single nucleotide polymorphism (SNP) discovery analysis in bovine Longissimus thoraci, using RNA-Seq. To our knowledge, this represents the first study done in bovine muscle. RESULTS: Messenger RNAs from Longissimus thoraci from three Limousin bull calves were subjected to high-throughput sequencing. Approximately 36-46 million paired-end reads were obtained per library. A total of 19,752 transcripts were identified and 34,376 different SNPs were detected. Fifty-five percent of the SNPs were found in coding regions and ~22% resulted in an amino acid change. Applying a very stringent SNP quality threshold, we detected 8,407 different high-confidence SNPs, 18% of which are non synonymous coding SNPs. To analyse the accuracy of RNA-Seq technology for SNP detection, 48 SNPs were selected for validation by genotyping. No discrepancies were observed when using the highest SNP probability threshold. To test the usefulness of the identified SNPs, the 48 selected SNPs were assessed by genotyping 93 bovine samples, representing mostly the nine major breeds used in France. Principal component analysis indicates a clear separation between the nine populations. CONCLUSIONS: The RNA-Seq data and the collection of newly discovered coding SNPs improve the genomic resources available for cattle, especially for beef breeds. The large amount of variation present in genes expressed in Limousin Longissimus thoracis, especially the large number of non synonymous coding SNPs, may prove useful to study the mechanisms underlying the genetic variability of meat quality traits.

Development and characterisation of an expressed sequence tags (EST)-derived single nucleotide polymorphisms (SNPs) resource in rainbow trout.

BACKGROUND: There is considerable interest in developing high-throughput genotyping with single nucleotide polymorphisms (SNPs) for the identification of genes affecting important ecological or economical traits. SNPs are evenly distributed throughout the genome and are likely to be functionally relevant. In rainbow trout, in silico screening of EST databases represents an attractive approach for de novo SNP identification. Nevertheless, EST sequencing errors and assembly of EST paralogous sequences can lead to the identification of false positive SNPs which renders the reliability of EST-derived SNPs relatively low. Further validation of EST-derived SNPs is therefore required. The objective of this work was to assess the quality of and to validate a large number of rainbow trout EST-derived SNPs. RESULTS: A panel of 1,152 EST-derived SNPs was selected from the INRA Sigenae SNP database and was genotyped in standard and double haploid individuals from several populations using the Illumina GoldenGate BeadXpress assay. High-quality genotyping data were obtained for 958 SNPs representing a genotyping success rate of 83.2 %, out of which, 350 SNPs (36.5 %) were polymorphic in at least one population and were designated as true SNPs. They also proved to be a potential tool to investigate genetic diversity of the species, as the set of SNP successfully sorted individuals into three main groups using STRUCTURE software. Functional annotations revealed 28 non-synonymous SNPs, out of which four substitutions were predicted to affect protein functions. A subset of 223 true SNPs were polymorphic in the two INRA mapping reference families and were integrated into the INRA microsatellite-based linkage map. CONCLUSIONS: Our results represent the first study of EST-derived SNPs validation in rainbow trout, a species whose genome sequences is not yet available. We designed several specific filters in order to improve the genotyping yield. Nevertheless, our selection criteria should be further improved in order to reduce the observed high rate of false positive SNPs which results from the occurrence of whole genome duplications.

Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response.

BACKGROUND: Designing sustainable animal production systems that better balance productivity and resistance to disease is a major concern. In order to address questions related to immunity and resistance to disease in pig, it is necessary to increase knowledge on its immune system and to produce efficient tools dedicated to this species. RESULTS: A long-oligonucleotide-based chip referred to as SLA-RI/NRSP8-13K was produced by combining a generic set with a newly designed SLA-RI set that targets all annotated loci of the pig major histocompatibility complex (MHC) region (SLA complex) in both orientations as well as immunity genes outside the SLA complex. The chip was used to study the immune response of pigs following stimulation of porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) or a mixture of phorbol myristate acetate (PMA) and ionomycin for 24 hours. Transcriptome analysis revealed that ten times more genes were differentially expressed after PMA/ionomycin stimulation than after LPS stimulation. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than of B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 (tumor necrosis factor receptor superfamily, member 9) genes. In addition, a very intense repression of THBS1 (thrombospondin 1) was observed. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing. Repression of MHC class II genes was observed after both stimulations. Our results provide preliminary data suggesting that antisense transcripts mapping to the SLA complex may have a role during immune response. CONCLUSION: The SLA-RI/NRSP8-13K chip was found to accurately decipher two distinct immune response activations of PBMCs indicating that it constitutes a valuable tool to further study immunity and resistance to disease in pig. The transcriptome analysis revealed specific and common features of the immune responses depending on the stimulation agent that increase knowledge on pig immunity.

Publisher Correction: Unraveling the effects of the gut microbiota composition and function on horse endurance physiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.