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.

An integrative atlas of chicken long non-coding genes and their annotations across 25 tissues.

Long non-coding RNAs (LNC) regulate numerous biological processes. In contrast to human, the identification of LNC in farm species, like chicken, is still lacunar. We propose a catalogue of 52,075 chicken genes enriched in LNC ( http://www.fragencode.org/ ), built from the Ensembl reference extended using novel LNC modelled here from 364 RNA-seq and LNC from four public databases. The Ensembl reference grew from 4,643 to 30,084 LNC, of which 59% and 41% with expression ≥ 0.5 and ≥ 1 TPM respectively. Characterization of these LNC relatively to the closest protein coding genes (PCG) revealed that 79% of LNC are in intergenic regions, as in other species. Expression analysis across 25 tissues revealed an enrichment of co-expressed LNC:PCG pairs, suggesting co-regulation and/or co-function. As expected LNC were more tissue-specific than PCG (25% vs. 10%). Similarly to human, 16% of chicken LNC hosted one or more miRNA. We highlighted a new chicken LNC, hosting miR155, conserved in human, highly expressed in immune tissues like miR155, and correlated with immunity-related PCG in both species. Among LNC:PCG pairs tissue-specific in the same tissue, we revealed an enrichment of divergent pairs with the PCG coding transcription factors, as for example LHX5, HXD3 and TBX4, in both human and chicken.

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.

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.

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

An integrated analysis of gut microbiota, blood biochemical and metabolome in 52 endurance horses was performed. Clustering by gut microbiota revealed the existence of two communities mainly driven by diet as host properties showed little effect. Community 1 presented lower richness and diversity, but higher dominance and rarity of species, including some pathobionts. Moreover, its microbiota composition was tightly linked to host blood metabolites related to lipid metabolism and glycolysis at basal time. Despite the lower fiber intake, community type 1 appeared more specialized to produce acetate as a mean of maintaining the energy supply as glucose concentrations fell during the race. On the other hand, community type 2 showed an enrichment of fibrolytic and cellulolytic bacteria as well as anaerobic fungi, coupled to a higher production of propionate and butyrate. The higher butyrate proportion in community 2 was not associated with protective effects on telomere lengths but could have ameliorated mucosal inflammation and oxidative status. The gut microbiota was neither associated with the blood biochemical markers nor metabolome during the endurance race, and did not provide a biomarker for race ranking or risk of failure to finish the race.

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.

Survey of allele specific expression in bovine muscle.

Allelic imbalance is a common phenomenon in mammals that plays an important role in gene regulation. An Allele Specific Expression (ASE) approach can be used to detect variants with a cis-regulatory effect on gene expression. In cattle, this type of study has only been done once in Holstein. In our study we performed a genome-wide analysis of ASE in 19 Limousine muscle samples. We identified 5,658 ASE SNPs (Single Nucleotide Polymorphisms showing allele specific expression) in 13% of genes with detectable expression in the Longissimus thoraci muscle. Interestingly we found allelic imbalance in AOX1, PALLD and CAST genes. We also found 2,107 ASE SNPs located within genomic regions associated with meat or carcass traits. In order to identify causative cis-regulatory variants explaining ASE we searched for SNPs altering binding sites of transcription factors or microRNAs. We identified one SNP in the 3'UTR region of PRNP that could be a causal regulatory variant modifying binding sites of several miRNAs. We showed that ASE is frequent within our muscle samples. Our data could be used to elucidate the molecular mechanisms underlying gene expression imbalance.

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.

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.

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.

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.

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.

Identification of a t(3;4)(p1.3;q1.5) translocation breakpoint in pigs using somatic cell hybrid mapping and high-resolution mate-pair sequencing.

Reciprocal translocations are the most frequently occurring constitutional structural rearrangements in mammalian genomes. In phenotypically normal pigs, an incidence of 1/200 is estimated for such rearrangements. Even if constitutional translocations do not necessarily induce defects and diseases, they are responsible for significant economic losses in domestic animals due to reproduction failures. Over the last 30 years, advances in molecular and cytogenetic technologies have led to major improvements in the resolution of the characterization of translocation events. Characterization of translocation breakpoints helps to decipher the mechanisms that lead to such rearrangements and the functions of the genes that are involved in the translocation. Here, we describe the fine characterization of a reciprocal translocation t(3;4) (p1.3;q1.5) detected in a pig line. The breakpoint was identified at the base-pair level using a positional cloning and chromosome walking strategy in somatic cell hybrids that were generated from an animal that carries this translocation. We show that this translocation occurs within the ADAMTSL4 gene and results in a loss of expression in homozygous carriers. In addition, by taking this translocation as a model, we used a whole-genome next-generation mate-pair sequencing approach on pooled individuals to evaluate this strategy for high-throughput screening of structural rearrangements.

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.

The Effects of Weaning Methods on Gut Microbiota Composition and Horse Physiology.

Weaning has been described as one of the most stressful events in the life of horses. Given the importance of the interaction between the gut-brain axis and gut microbiota under stress, we evaluated (i) the effect of two different weaning methods on the composition of gut microbiota across time and (ii) how the shifts of gut microbiota composition after weaning affect the host. A total of 34 foals were randomly subjected to a progressive (P) or an abrupt (A) weaning method. In the P method, mares were separated from foals at progressively increasing intervals every day, starting from five min during the fourth week prior to weaning and ending with 6 h during the last week before weaning. In the A method, mares and foals were never separated prior to weaning (0 d). Different host phenotypes and gut microbiota composition were studied across 6 age strata (days -30, 0, 3, 5, 7, and 30 after weaning) by 16S rRNA gene sequencing. Results revealed that the beneficial species belonging to Prevotella, Paraprevotella, and Ruminococcus were more abundant in the A group prior to weaning compared to the P group, suggesting that the gut microbiota in the A cohort was better adapted to weaning. Streptococcus, on the other hand, showed the opposite pattern after weaning. Fungal loads, which are thought to increase the capacity for fermenting the complex polysaccharides from diet, were higher in P relative to A. Beyond the effects of weaning methods, maternal separation at weaning markedly shifted the composition of the gut microbiota in all foals, which fell into three distinct community types at 3 days post-weaning. Most genera in community type 2 (i.e., Eubacterium, Coprococcus, Clostridium XI, and Blautia spp.) were negatively correlated with salivary cortisol levels, but positively correlated with telomere length and N-butyrate production. Average daily gain was also greater in the foals harboring a community type 2 microbiota. Therefore, community type 2 is likely to confer better stress response adaptation following weaning. This study identified potential microbial biomarkers that could predict the likelihood for physiological adaptations to weaning in horses, although causality remains to be addressed.

Transcriptome of melanoma cells from two mouse models, Tyr:NRasQ61K and Tyr:Rack1-HA, Tyr:NRasQ61K.

The transcriptome sequencing of melanoma cells from two mouse models differing in the expression level of the scaffold protein Receptor for activated C kinase (RACK1) are presented. Primary melanoma cells were harvested from Tyr:NRasQ61K; Pax3GFP/+ mice, with or without the Tyr:Rack1-HA transgene. Cells were cultured and infected with scramble shRNA or Rack1-targeting shRNA, on technical triplicates of viral infection. Libraries were prepared by selecting polyadenylated mRNAs and RNA Sequencing (RNASeq) was performed. Samples are described in the SRA portal (SRP096162) and FASTQ files have been deposited in Sequence Read Archive (accession numbers: SRR5150106 to SRR5150117). The interpretation of these data is presented in the following research article: "RACK1 cooperates with NRASQ61K to promote melanoma in vivo" (Campagne et al., 2017, doi: 10.1016/j.cellsig.2017.03.015) [1].

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.

Genetic variability of the activity of bidirectional promoters: a pilot study in bovine muscle.

Bidirectional promoters are regulatory regions co-regulating the expression of two neighbouring genes organized in a head-to-head orientation. In recent years, these regulatory regions have been studied in many organisms; however, no investigation to date has been done to analyse the genetic variation of the activity of this type of promoter regions. In our study, we conducted an investigation to first identify bidirectional promoters sharing genes expressed in bovine Longissimus thoracis and then to find genetic variants affecting the activity of some of these bidirectional promoters. Combining bovine gene information and expression data obtained using RNA-Seq, we identified 120 putative bidirectional promoters active in bovine muscle. We experimentally validated in vitro 16 of these bidirectional promoters. Finally, using gene expression and whole-genome genotyping data, we explored the variability of the activity in muscle of the identified bidirectional promoters and discovered genetic variants affecting their activity. We found that the expression level of 77 genes is correlated with the activity of 12 bidirectional promoters. We also identified 57 single nucleotide polymorphisms associated with the activity of 5 bidirectional promoters. To our knowledge, our study is the first analysis in any species of the genetic variability of the activity of bidirectional promoters.