Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds.

In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10-10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.

A strategy to discover new organizers identifies a putative heart organizer.

Organizers are regions of the embryo that can both induce new fates and impart pattern on other regions. So far, surprisingly few organizers have been discovered, considering the number of patterned tissue types generated during development. This may be because their discovery has relied on transplantation and ablation experiments. Here we describe a new approach, using chick embryos, to discover organizers based on a common gene expression signature, and use it to uncover the anterior intestinal portal (AIP) endoderm as a putative heart organizer. We show that the AIP can induce cardiac identity from non-cardiac mesoderm and that it can pattern this by specifying ventricular and suppressing atrial regional identity. We also uncover some of the signals responsible. The method holds promise as a tool to discover other novel organizers acting during development.

The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor receptor.

BACKGROUND: Macrophages have many functions in development and homeostasis as well as innate immunity. Recent studies in mammals suggest that cells arising in the yolk sac give rise to self-renewing macrophage populations that persist in adult tissues. Macrophage proliferation and differentiation is controlled by macrophage colony-stimulating factor (CSF1) and interleukin 34 (IL34), both agonists of the CSF1 receptor (CSF1R). In the current manuscript we describe the origin, function and regulation of macrophages, and the role of CSF1R signaling during embryonic development, using the chick as a model. RESULTS: Based upon RNA-sequencing comparison to bone marrow-derived macrophages grown in CSF1, we show that embryonic macrophages contribute around 2% of the total embryo RNA in day 7 chick embryos, and have similar gene expression profiles to bone marrow-derived macrophages. To explore the origins of embryonic and adult macrophages, we injected Hamburger-Hamilton stage 16 to 17 chick embryos with either yolk sac-derived blood cells, or bone marrow cells from EGFP+ donors. In both cases, the transferred cells gave rise to large numbers of EGFP+ tissue macrophages in the embryo. In the case of the yolk sac, these cells were not retained in hatched birds. Conversely, bone marrow EGFP+ cells gave rise to tissue macrophages in all organs of adult birds, and regenerated CSF1-responsive marrow macrophage progenitors. Surprisingly, they did not contribute to any other hematopoietic lineage. To explore the role of CSF1 further, we injected embryonic or hatchling CSF1R-reporter transgenic birds with a novel chicken CSF1-Fc conjugate. In both cases, the treatment produced a large increase in macrophage numbers in all tissues examined. There were no apparent adverse effects of chicken CSF1-Fc on embryonic or post-hatch development, but there was an unexpected increase in bone density in the treated hatchlings. CONCLUSIONS: The data indicate that the yolk sac is not the major source of macrophages in adult birds, and that there is a macrophage-restricted, self-renewing progenitor cell in bone marrow. CSF1R is demonstrated to be limiting for macrophage development during development in ovo and post-hatch. The chicken provides a novel and tractable model to study the development of the mononuclear phagocyte system and CSF1R signaling.

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project.

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

Phylogenomic analyses data of the avian phylogenomics project.

BACKGROUND: Determining the evolutionary relationships among the major lineages of extant birds has been one of the biggest challenges in systematic biology. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders. We used these genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomic analyses. FINDINGS: Here we present the datasets associated with the phylogenomic analyses, which include sequence alignment files consisting of nucleotides, amino acids, indels, and transposable elements, as well as tree files containing gene trees and species trees. Inferring an accurate phylogeny required generating: 1) A well annotated data set across species based on genome synteny; 2) Alignments with unaligned or incorrectly overaligned sequences filtered out; and 3) Diverse data sets, including genes and their inferred trees, indels, and transposable elements. Our total evidence nucleotide tree (TENT) data set (consisting of exons, introns, and UCEs) gave what we consider our most reliable species tree when using the concatenation-based ExaML algorithm or when using statistical binning with the coalescence-based MP-EST algorithm (which we refer to as MP-EST*). Other data sets, such as the coding sequence of some exons, revealed other properties of genome evolution, namely convergence. CONCLUSIONS: The Avian Phylogenomics Project is the largest vertebrate phylogenomics project to date that we are aware of. The sequence, alignment, and tree data are expected to accelerate analyses in phylogenomics and other related areas.

Major transcriptome re-organisation and abrupt changes in signalling, cell cycle and chromatin regulation at neural differentiation in vivo.

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome re-organisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGFß). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation.

Peeling back the evolutionary layers of molecular mechanisms responsive to exercise-stress in the skeletal muscle of the racing horse.

The modern horse (Equus caballus) is the product of over 50 million yrs of evolution. The athletic abilities of the horse have been enhanced during the past 6000 yrs under domestication. Therefore, the horse serves as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. The structure and function of skeletal muscle show remarkable plasticity to the physical and metabolic challenges following exercise. Here, we reveal an evolutionary layer of responsiveness to exercise-stress in the skeletal muscle of the racing horse. We analysed differentially expressed genes and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. By estimating genome-wide dN/dS ratios using six mammalian genomes, and FST and iHS using re-sequencing data derived from 20 horses, we were able to peel back the evolutionary layers of adaptations to exercise-stress in the horse. We found that the oldest and thickest layer (dN/dS) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (FST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.

Decreased expression of the satiety signal receptor CCKAR is responsible for increased growth and body weight during the domestication of chickens.

Animal domestication has resulted in changes in growth and size. It has been suggested that this may have involved selection for differences in appetite. Divergent growth between chickens selected for egg laying or meat production is one such example. The neurons expressing AGRP and POMC in the basal hypothalamus are important components of appetite regulation, as are the satiety feedback pathways that carry information from the intestine, including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between a fast and a relatively slow growing strain of chicken has identified a region on chromosome 4 downstream of the CCKAR gene, which is responsible for up to a 19% difference in body weight at 12 wk of age. Animals possessing the high-growth haplotype at the locus have lower expression of mRNA and immunoreactive CCKAR in the brain, intestine, and exocrine organs, which is correlated with increased levels of orexigenic AGRP in the hypothalamus. Animals with the high-growth haplotype are resistant to the anorectic effect of exogenously administered CCK, suggesting that their satiety set point has been altered. Comparison with traditional breeds shows that the high-growth haplotype has been present in the founders of modern meat-type strains and may have been selected early in domestication. This is the first dissection of the physiological consequences of a genetic locus for a quantitative trait that alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds and also mammals.

Systemic virus distribution and host responses in brain and intestine of chickens infected with low pathogenic or high pathogenic avian influenza virus.

BACKGROUND: Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens.Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses. METHODS: To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains. RESULTS: Both H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection. CONCLUSIONS: Although both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.

The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of Shh during limb development.

Point mutations in the intronic ZRS region of Lmbr1, a limb specific cis-regulatory element of Sonic hedgehog (Shh), are associated with polydactyly in humans, cats, and mice. We and others have recently mapped the dominant preaxial polydactyly (Po) locus in Silkie chickens to a single nucleotide polymorphism (SNP) in the ZRS region. Using polymorphisms in the chicken Shh sequence, we confirm that the ZRS region directly regulates Shh expression in the developing limb causing ectopic Shh expression in the anterior leg, prolonged Shh expression in the posterior limb, and allelic imbalance between wt and Slk Shh alleles in heterozygote limbs. Using Silkie legs, we have explored the consequences of increased Shh expression in the posterior leg on the patterning of the toes, and the induction of preaxial polydactyly.

Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering.

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body.

Identification of melatonin-regulated genes in the ovine pituitary pars tuberalis, a target site for seasonal hormone control.

The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef / Nampt (nicotinamide phosphoribosyltransferase), Hif1alpha (hypoxia-inducible factor-1alpha), and Kcnq5 (K+ channel) and down-regulation of Rorbeta, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorbeta in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1alpha, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT.

Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution.

BACKGROUND: Comparative genomics is a powerful means of establishing inter-specific relationships between gene function/location and allows insight into genomic rearrangements, conservation and evolutionary phylogeny. The availability of the complete sequence of the chicken genome has initiated the development of detailed genomic information in other birds including turkey, an agriculturally important species where mapping has hitherto focused on linkage with limited physical information. No molecular study has yet examined conservation of avian microchromosomes, nor differences in copy number variants (CNVs) between birds. RESULTS: We present a detailed comparative cytogenetic map between chicken and turkey based on reciprocal chromosome painting and mapping of 338 chicken BACs to turkey metaphases. Two inter-chromosomal changes (both involving centromeres) and three pericentric inversions have been identified between chicken and turkey; and array CGH identified 16 inter-specific CNVs. CONCLUSION: This is the first study to combine the modalities of zoo-FISH and array CGH between different avian species. The first insight into the conservation of microchromosomes, the first comparative cytogenetic map of any bird and the first appraisal of CNVs between birds is provided. Results suggest that avian genomes have remained relatively stable during evolution compared to mammalian equivalents.

Identification and functional characterization of a bovine orthologue to DC-SIGN.

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) C-type lectin is almost exclusively expressed at the cell surface of DC. In addition to its normal function facilitating contact of DC with T cells, DC-SIGN has been shown to bind a variety of pathogens, including Mycobacterium bovis, and HIV-1 envelope protein gp120. In this study, we identified the bovine ortholog of the human DC-SIGN gene within the bovine genome, which exists as a single copy. PCR amplified a product, showing a 100% match with the predicted sequences as well as a sequence predicted to be similar to that of SIGNR7. Furthermore, a protein with the same molecular weight as human DC-SIGN was detected by Western blot in cell lysate derived from bovine DC. To characterize this molecule functionally, the uptake of FITC-labeled OVA and FITC-labeled gp120 (FITC-gp120) by bovine and human DC was assessed. FITC-gp120 was shown to bind to bovine DC in a time- and temperature-dependent manner. Binding was blocked by a polyclonal anti-DC-SIGN antibody but not by a control antibody. Furthermore, blocking of this molecule also reduced the binding of M. bovis bacillus Calmette-Guerin expressing GFP. Confocal microscopy showed that DC-SIGN was expressed on the surface of bovine DC. Subsequent pulse-chase studies revealed that FITC-gp120 was internalized by bovine monocyte-derived DC as early as 10 min. Thus, there is evidence of a DC-SIGN-like molecule expressed specifically by bovine DC. This molecule may play an important role in the infection of bovine (DC) cells with M. bovis.

Molecular immunophenotyping of lungs and spleens in naive and vaccinated chickens early after pulmonary avian influenza A (H9N2) virus infection.

In a respiratory-infection-model with the avian influenza A H9N2 virus we studied lung and splenic immune reactions in chickens using a recently developed 5K chicken immuno-microarray. Groups of chickens were either mock-immunized (referred to as non-immune), vaccinated with inactivated viral antigen only (immune) or with viral antigen in a water-in-oil (W/O) immunopotentiator (immune potentiated). Three weeks after vaccination all animals were given a respiratory infection. Immune potentiated birds developed inhibitory antiviral antibodies, showed minimal lung histopathology and no detectable viral sequences, while non-immune animals showed microscopic immunopathology and detectable virus. Immune birds, receiving antigen in saline only, showed minimal microscopic histopathology, and intermediate levels of virus detection. These classical features in the different groups were mirrored by overlapping or specific mRNA gene expression profiles in lungs and spleen using microarray analysis. To our knowledge this is the first study demonstrating pneumonia-associated lung pathology of the low pathogenic avian influenza H9N2 virus. Our data provide insights into the molecular interaction of this virus with its natural host when naive or primed by vaccination.

The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid3 locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid3 mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid3 protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins.

Simultaneous mapping of epistatic QTL in chickens reveals clusters of QTL pairs with similar genetic effects on growth.

We used simultaneous mapping of interacting quantitative trait locus (QTL) pairs to study various growth traits in a chicken F2 intercross. The method was shown to increase the number of detected QTLs by 30 % compared with a traditional method detecting QTLs by their marginal genetic effects. Epistasis was shown to be an important contributor to the genetic variance of growth, with the largest impact on early growth (before 6 weeks of age). There is also evidence for a discrete set of interacting loci involved in early growth, supporting the previous findings of different genetic regulation of early and late growth in chicken. The genotype-phenotype relationship was evaluated for all interacting QTL pairs and 17 of the 21 evaluated QTL pairs could be assigned to one of four clusters in which the pairs in a cluster have very similar genetic effects on growth. The genetic effects of the pairs indicate commonly occurring dominance-by-dominance, heterosis and multiplicative interactions. The results from this study clearly illustrate the increase in power obtained by using this novel method for simultaneous detection of epistatic QTL, and also how visualization of genotype-phenotype relationships for epistatic QTL pairs provides new insights to biological mechanisms underlying complex traits.

Molecular cytogenetic definition of the chicken genome: the first complete avian karyotype.

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.