- Journal Article (209)
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- Conference Contribution (51)
- Book or Chapter (10)
- Other (2)
- Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human
BACKGROUND: Despite the significance of chicken as a model organism, our understanding of the chicken transcriptome is limited compared to human. This issue is common to all non-human vertebrate annotations due to the difficulty in transcript identification from short read RNAseq data. While previous studies have used single molecule long read sequencing for transcript discovery, they did not perform RNA normalization and 5'-cap selection which may have resulted in lower transcriptome coverage and truncated transcript sequences.
RESULTS: We sequenced normalised chicken brain and embryo RNA libraries with Pacific Bioscience Iso-Seq. 5' cap selection was performed on the embryo library to provide methodological comparison. From these Iso-Seq sequencing projects, we have identified 60 k transcripts and 29 k genes within the chicken transcriptome. Of these, more than 20 k are novel lncRNA transcripts with ~3 k classified as sense exonic overlapping lncRNA, which is a class that is underrepresented in many vertebrate annotations. The relative proportion of alternative transcription events revealed striking similarities between the chicken and human transcriptomes while also providing explanations for previously observed genomic differences.
CONCLUSIONS: Our results indicate that the chicken transcriptome is similar in complexity compared to human, and provide insights into other vertebrate biology. Our methodology demonstrates the potential of Iso-Seq sequencing to rapidly expand our knowledge of transcriptomics.DOI
- A New Chicken Genome Assembly Provides Insight into Avian Genome Structure
The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3) built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31 and 33 and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. Gallus_gallus-5.0 when annotated for gene content shows an increase of 4,679 annotated genes, 2,768 non-coding and 1,911 protein-coding, over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B encompassing two segments; one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts.DOI
- A new look at the LTR retrotransposon content of the chicken genome
BACKGROUND: LTR retrotransposons contribute approximately 10 % of the mammalian genome, but it has been previously reported that there is a deficit of these elements in the chicken relative to both mammals and other birds. A novel LTR retrotransposon classification pipeline, LocaTR, was developed and subsequently utilised to re-examine the chicken LTR retrotransposon annotation, and determine if the proposed chicken deficit is biologically accurate or simply a technical artefact.
RESULTS: Using LocaTR 3.01 % of the chicken galGal4 genome assembly was annotated as LTR retrotransposon-derived elements (nearly double the previous annotation), including 1,073 that were structurally intact. Element distribution is significantly correlated with chromosome size and is non-random within each chromosome. Elements are significantly depleted within coding regions and enriched in gene sparse areas of the genome. Over 40 % of intact elements are found in clusters, unrelated by age or genera, generally in poorly recombining regions. The transcription of most LTR retrotransposons were suppressed or incomplete, but individual domain and full length retroviral transcripts were produced in some cases, although mostly with regularly interspersed stop codons in all reading frames. Furthermore, RNAseq data from 23 diverse tissues enabled greater characterisation of the co-opted endogenous retrovirus Ovex1. This gene was shown to be expressed ubiquitously but at variable levels across different tissues. LTR retrotransposon content was found to be very variable across the avian lineage and did not correlate with either genome size or phylogenetic position. However, the extent of previous, species-specific LTR retrotransposon annotation appears to be a confounding factor.
CONCLUSIONS: Use of the novel LocaTR pipeline has nearly doubled the annotated LTR retrotransposon content of the chicken genome compared to previous estimates. Further analysis has described element distribution, clustering patterns and degree of expression in a variety of adult tissues, as well as in three embryonic stages. This study also enabled better characterisation of the co-opted gamma retroviral envelope gene Ovex1. Additionally, this work suggests that there is no deficit of LTR retrotransposons within the Galliformes relative to other birds, or to mammalian genomes when scaled for the three-fold difference in genome size.DOI
- 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.DOI
- Novel insights into chromosome evolution in birds, archosaurs, and reptiles
Homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs) in mammalian chromosomes are enriched for distinct DNA features, contributing to distinct phenotypes. To reveal HSB and EBR roles in avian evolution, we performed a sequence-based comparison of 21 avian and five outgroup species using recently sequenced genomes across the avian family tree and a newly-developed algorithm. We identified EBRs and HSBs in ancestral bird, archosaurian (bird, crocodile, dinosaur), and reptile chromosomes. Genes involved in the regulation of gene expression and biosynthetic processes were preferably located in HSBs, for example the avian-specific HSBs were enriched for genes involved in limb development. Within birds, some lineage-specific EBRs rearranged genes related to distinct phenotypes, such as forebrain development in parrots. Our findings provide novel evolutionary insights into genome evolution in birds, particularly how chromosome rearrangements likely contributed to the formation of novel phenotypes. DOI
- Commercial chicken breeds exhibit highly divergent patterns of linkage disequilibrium
The analysis of linkage disequilibrium (LD) underpins the development of effective genotyping technologies, trait mapping and understanding of biological mechanisms such as those driving recombination and the impact of selection. We apply the Malécot-Morton model of LD to create additive LD maps which describe the high-resolution LD landscape of commercial chickens. We investigated LD in chickens (Gallus gallus) at the highest resolution to date for broiler, white egg and brown egg layer commercial lines. There is minimal concordance between breeds of fine scale LD patterns (correlation coefficient < 0.21), and even between discrete broiler lines. Regions of LD breakdown, which may align with recombination hotspots, are enriched near CpG islands and transcription start sites (p < 2.2x10-16), consistent with recent evidence described in finches, but concordance in hotspot locations between commercial breeds is only marginally greater than random. As in other birds functional elements in the chicken genome are associated with recombination, but, unlike evidence from other bird species, the LD landscape is not stable in the populations studied. The development of optimal genotyping panels for genome-led selection programmes will depend on careful analysis of the LD structure of each line of interest. Further study is required to fully elucidate the mechanisms underlying highly divergent LD patterns found in commercial chickens. DOI
- Quantitative trait loci with sex-specific effects for internal organs weights and hematocrit value in a broiler-layer cross
- Animal genomics and infectious disease resistance in poultry
Avian pathogens are responsible for major costs to society, both in terms of huge economic losses to the poultry industry and their implications for human health. The health and welfare of millions of birds is under continued threat from many infectious diseases, some of which are increasing in virulence and thus becoming harder to control, such as Marek's disease virus and avian influenza viruses. The current era in animal genomics has seen huge developments in both technologies and resources, which means that researchers have never been in a better position to investigate the genetics of disease resistance and determine the underlying genes/mutations which make birds susceptible or resistant to infection. Avian genomics has reached a point where the biological mechanisms of infectious diseases can be investigated and understood in poultry and other avian species. Knowledge of genes conferring disease resistance can be used in selective breeding programmes or to develop vaccines which help to control the effects of these pathogens, which have such a major impact on birds and humans alike.DOI
- Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project
- Binary Switching of Calendar Cells in the Pituitary Defines the Phase of the Circannual Cycle in Mammals
Persistent free-running circannual (approximately year-long) rhythms have evolved in animals to regulate hormone cycles, drive metabolic rhythms (including hibernation), and time annual reproduction. Recent studies have defined the photoperiodic input to this rhythm, wherein melatonin acts on thyrotroph cells of the pituitary pars tuberalis (PT), leading to seasonal changes in the control of thyroid hormone metabolism in the hypothalamus. However, seasonal rhythms persist in constant conditions in many species in the absence of a changing photoperiod signal, leading to the generation of circannual cycles. It is not known which cells, tissues, and pathways generate these remarkable long-term rhythmic processes. We show that individual PT thyrotrophs can be in one of two binary states reflecting either a long (EYA3+) or short (CHGA+) photoperiod, with the relative proportion in each state defining the phase of the circannual cycle. We also show that a morphogenic cycle driven by the PT leads to extensive re-modeling of the PT and hypothalamus over the circannual cycle. We propose that the PT may employ a recapitulated developmental pathway to drive changes in morphology of tissues and cells. Our data are consistent with the hypothesis that the circannual timer may reside within the PT thyrotroph and is encoded by a binary switch timing mechanism, which may regulate the generation of circannual neuroendocrine rhythms, leading to dynamic re-modeling of the hypothalamic interface. In summary, the PT-ventral hypothalamus now appears to be a prime structure involved in long-term rhythm generation. DOI
- Genome-wide analysis reveals the extent of EAV-HP integration in domestic chicken
BACKGROUND: EAV-HP is an ancient retrovirus pre-dating Gallus speciation, which continues to circulate in modern chicken populations, and led to the emergence of avian leukosis virus subgroup J causing significant economic losses to the poultry industry. We mapped EAV-HP integration sites in Ethiopian village chickens, a Silkie, Taiwan Country chicken, red junglefowl Gallus gallus and several inbred experimental lines using whole-genome sequence data.
RESULTS: An average of 75.22 ± 9.52 integration sites per bird were identified, which collectively group into 279 intervals of which 5 % are common to 90 % of the genomes analysed and are suggestive of pre-domestication integration events. More than a third of intervals are specific to individual genomes, supporting active circulation of EAV-HP in modern chickens. Interval density is correlated with chromosome length (P < 2.31(-6)), and 27 % of intervals are located within 5 kb of a transcript. Functional annotation clustering of genes reveals enrichment for immune-related functions (P < 0.05).
CONCLUSIONS: Our results illustrate a non-random distribution of EAV-HP in the genome, emphasising the importance it may have played in the adaptation of the species, and provide a platform from which to extend investigations on the co-evolutionary significance of endogenous retroviral genera with their hosts.DOI
- The early immune response to infection of chickens with Infectious Bronchitis Virus (IBV) in susceptible and resistant birds
BACKGROUND: Infectious Bronchitis is a highly contagious respiratory disease which causes tracheal lesions and also affects the reproductive tract and is responsible for large economic losses to the poultry industry every year. This is due to both mortality (either directly provoked by IBV itself or due to subsequent bacterial infection) and lost egg production. The virus is difficult to control by vaccination, so new methods to curb the impact of the disease need to be sought. Here, we seek to identify genes conferring resistance to this coronavirus, which could help in selective breeding programs to rear chickens which do not succumb to the effects of this disease.
METHODS: Whole genome gene expression microarrays were used to analyse the gene expression differences, which occur upon infection of birds with Infectious Bronchitis Virus (IBV). Tracheal tissue was examined from control and infected birds at 2, 3 and 4 days post-infection in birds known to be either susceptible or resistant to the virus. The host innate immune response was evaluated over these 3 days and differences between the susceptible and resistant lines examined.
RESULTS: Genes and biological pathways involved in the early host response to IBV infection were determined andgene expression differences between susceptible and resistant birds were identified. Potential candidate genes for resistance to IBV are highlighted.
CONCLUSIONS: The early host response to IBV is analysed and potential candidate genes for disease resistance are identified. These putative resistance genes can be used as targets for future genetic and functional studies to prove a causative link with resistance to IBV.DOI
- Evolution of the avian β-defensin and cathelicidin genes
BACKGROUND: β-defensins and cathelicidins are two families of cationic antimicrobial peptides (AMPs) with a broad range of antimicrobial activities that are key components of the innate immune system. Due to their important roles in host defense against rapidly evolving pathogens, the two gene families provide an ideal system for studying adaptive gene evolution. In this study we performed phylogenetic and selection analyses on β-defensins and cathelicidins from 53 avian species representing 32 orders to examine the evolutionary dynamics of these peptides in birds.
RESULTS AND CONCLUSIONS: Avian β-defensins are found in a gene cluster consisting of 13 subfamiles. Nine of these are conserved as one to one orthologs in all birds, while the others (AvBD1, AvBD3, AvBD7 and AvBD14) are more subject to gene duplication or pseudogenisation events in specific avian lineages. Avian cathelicidins are found in a gene cluster consisting of three subfamilies with species-specific duplications and gene loss. Evidence suggested that the propiece and mature peptide domains of avian cathelicidins are possibly co-evolving in such a way that the cationicity of the mature peptide is partially neutralised by the negative charge of the propiece prior to peptide secretion (further evidence obtained by repeating the analyses on primate cathelicidins). Negative selection (overall mean dN < dS) was detected in most of the gene domains examined, conserving certain amino acid residues that may be functionally crucial for the avian β-defensins and cathelicidins, while episodic positive selection was also involved in driving the diversification of specific codon sites of certain AMPs in avian evolutionary history. These findings have greatly improved our understanding of the molecular evolution of avian AMPs and will be useful to understand their role in the avian innate immune response. Additionally, the large dataset of β-defensin and cathelicidin peptides may also provide a valuable resource for translational research and development of novel antimicrobial agents in the future.DOI
- Transcriptomic Profiling of Virus-Host Cell Interactions following Chicken Anaemia Virus (CAV) Infection in an In Vivo Model
Chicken Anaemia Virus (CAV) is an economically important virus that targets lymphoid and erythroblastoid progenitor cells leading to immunosuppression. This study aimed to investigate the interplay between viral infection and the host’s immune response to better understand the pathways that lead to CAV-induced immunosuppression. To mimic vertical transmission of CAV in the absence of maternally-derived antibody, day-old chicks were infected and their responses measured at various time-points post-infection by qRT-PCR and gene expression microarrays. The kinetics of mRNA expression levels of signature cytokines of innate and adaptive immune responses were determined by qRT-PCR. The global gene expression profiles of mock-infected (control) and CAV-infected chickens at 14 dpi were also compared using a chicken immune-related 5K microarray. Although in the thymus there was evidence of induction of an innate immune response following CAV infection, this was limited in magnitude. There was little evidence of a Th1 adaptive immune response in any lymphoid tissue, as would normally be expected in response to viral infection. Most cytokines associated with Th1, Th2 or Treg subsets were down-regulated, except IL-2, IL-13, IL-10 and IFNγ, which were all up-regulated in thymus and bone marrow. From the microarray studies, genes that exhibited significant (greater than 1.5-fold, false discovery rate <0.05) changes in expression in thymus and bone marrow on CAV infection were mainly associated with T-cell receptor signalling, immune response, transcriptional regulation, intracellular signalling and regulation of apoptosis. Expression levels of a number of adaptor proteins, such as src-like adaptor protein (SLA), a negative regulator of T-cell receptor signalling and the transcription factor Special AT-rich Binding Protein 1 (SATB1), were significantly down-regulated by CAV infection, suggesting potential roles for these genes as regulators of viral infection or cell defence. These results extend our understanding of CAV-induced immunosuppression and suggest a global immune dysregulation following CAV infection. DOI
- Third Report on Chicken Genes and Chromosomes 2015
- Functional classification of 15 million SNPs detected from diverse chicken populations
Next-generation sequencing has prompted a surge of discovery of millions of genetic variants from vertebrate genomes. Besides applications in genetic association and linkage studies, a fraction of these variants will have functional consequences. This study describes detection and characterization of 15 million SNPs from chicken genome with the goal to predict variants with potential functional implications (pfVars) from both coding and non-coding regions. The study reports: 183K amino acid-altering SNPs of which 48% predicted as evolutionary intolerant, 13K splicing variants, 51K likely to alter RNA secondary structures, 500K within most conserved elements and 3K from non-coding RNAs. Regions of local fixation within commercial broiler and layer lines were investigated as potential selective sweeps using genome-wide SNP data. Relationships with phenotypes, if any, of the pfVars were explored by overlaying the sweep regions with known QTLs. Based on this, the candidate genes and/or causal mutations for a number of important traits are discussed. Although the fixed variants within sweep regions were enriched with non-coding SNPs, some non-synonymous-intolerant mutations reached fixation, suggesting their possible adaptive advantage. The results presented in this study are expected to have important implications for future genomic research to identify candidate causal mutations and in poultry breeding.DOI
- Characterization of the avian trojan gene family reveals contrasting evolutionary constraints
"Trojan" is a leukocyte-specific, cell surface protein originally identified in the chicken. Its molecular function has been hypothesized to be related to anti-apoptosis and the proliferation of immune cells. The Trojan gene has been localized onto the Z sex chromosome. The adjacent two genes also show significant homology to Trojan, suggesting the existence of a novel gene/protein family. Here, we characterize this Trojan family, identify homologues in other species and predict evolutionary constraints on these genes. The two Trojan-related proteins in chicken were predicted as a receptor-type tyrosine phosphatase and a transmembrane protein, bearing a cytoplasmic immuno-receptor tyrosine-based activation motif. We identified the Trojan gene family in ten other bird species and found related genes in three reptiles and a fish species. The phylogenetic analysis of the homologues revealed a gradual diversification among the family members. Evolutionary analyzes of the avian genes predicted that the extracellular regions of the proteins have been subjected to positive selection. Such selection was possibly a response to evolving interacting partners or to pathogen challenges. We also observed an almost complete lack of intracellular positively selected sites, suggesting a conserved signaling mechanism of the molecules. Therefore, the contrasting patterns of selection likely correlate with the interaction and signaling potential of the molecules.DOI
- Analysis of the crow lung transcriptome in response to infection with highly pathogenic H5N1 avian influenza virus
The highly pathogenic avian influenza (HPAI) H5N1 virus, currently circulating in Asia, causes severe disease in domestic poultry as well as wild birds like crow. However, the molecular pathogenesis of HPAIV infection in crows and other wild birds is not well known. Thus, as a step to explore it, a comprehensive global gene expression analysis was performed on crow lungs, infected with HPAI H5N1 crow isolate (A/Crow/India/11TI11/2011) using high throughput next generation sequencing (NGS) (GS FLX Titanium XLR70). The reference genome of crow is not available, so RNA seq analysis was performed on the basis of a de novo assembled transcriptome. The RNA seq result shows, 4052 genes were expressed uniquely in noninfected, 6277 genes were expressed uniquely in HPAIV infected sample and of the 6814 genes expressed in both samples, 2279 genes were significantly differentially expressed. Our transcriptome profile data allows for the ability to understand the molecular mechanism behind the recent lethal HPAIV outbreak in crows which was, until recently, thought to cause lethal infections only in gallinaceous birds such as chickens, but not in wild birds. The pattern of differentially expressed genes suggest that this isolate of H5N1 virus evades the host innate immune response by attenuating interferon (IFN)-inducible signalling possibly by down regulating the signalling from type I IFN (IFNAR1 and IFNAR2) and type II IFN receptors, upregulation of the signalling inhibitors suppressor of cytokine signalling 1 (SOCS1) and SOCS3 and altering the expression of toll-like receptors (TLRs). This may be the reason for disease and mortality in crows.DOI
- Analysis of the early immune response to infection by Infectious Bursal Disease Virus (IBDV) in chickens differing in their resistance to the disease
Chicken whole genome gene expression arrays were used to analyse the host response to infection by Infectious Bursal Disease Virus (IBDV). Spleen and bursal tissue were examined from control and infected birds at 2, 3 and 4 days post-infection from two lines that differ in their resistance to IBDV infection. The host response was evaluated over this period and differences between susceptible and resistant chicken lines were examined. Anti-viral genes, including IFNA, IFNG, MX1, IFITM1, IFITM3 and IFITM5 were up-regulated in response to infection. Evaluation of this gene expression data has allowed us to predicted several genes as candidates for involvement in resistance to IBDV.
IMPORTANCE: Infectious bursal disease (IBD) is of economic importance to the poultry industry and thus is also important for food security. Vaccines are available but field strains of the virus are of increasing virulence. There is thus an urgent need to explore new control solutions, one of which would be to breed birds with greater resistance to IBD. A goal which is perhaps uniquely achievable with poultry, of all farm animal species, as the genetics of 85% of the 60 billion chickens produced worldwide each year is under the control of essentially two breeding companies. This is the most comprehensive study to try to identify global transcriptomic differences in the target organ of the virus between chicken lines that differ in resistance, and to predict candidate resistance genes.DOI
- The development and maintenance of the mononuclear phagocyte system of the chick is controlled by signals from the macrophage colony-stimulating factor (CSF1) 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 signalling during embryonic development, using the chick as a model.
RESULTS: Based upon RNAseq comparison to bone marrow-derived macrophages (BMDM) 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 BMDM. To explore the origins of embryonic and adult macrophages, we injected HH16 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 chCSF1-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 signalling.DOI
- SNP and INDEL detection in a QTL region on chicken chromosome 2 associated with muscle deposition
Genetic improvement is important for the poultry industry, contributing to increased efficiency of meat production and quality. Because breast muscle is the most valuable part of the chicken carcass, knowledge of polymorphisms influencing this trait can help breeding programs. Therefore, the complete genome of 18 chickens from two different experimental lines (broiler and layer) from EMBRAPA was sequenced, and SNPs and INDELs were detected in a QTL region for breast muscle deposition on chicken chromosome 2 between microsatellite markers MCW0185 and MCW0264 (105 849-112 649 kb). Initially, 94 674 unique SNPs and 10 448 unique INDELs were identified in the target region. After quality filtration, 77% of the SNPs (85 765) and 60% of the INDELs (7828) were retained. The studied region contains 66 genes, and functional annotation of the filtered variants identified 517 SNPs and three INDELs in exonic regions. Of these, 357 SNPs were classified as synonymous, 153 as non-synonymous, three as stopgain, four INDELs as frameshift and three INDELs as non-frameshift. These exonic mutations were identified in 37 of the 66 genes from the target region, three of which are related to muscle development (DTNA, RB1CC1 and MOS). Fifteen non-tolerated SNPs were detected in several genes (MEP1B, PRKDC, NSMAF, TRAPPC8, SDR16C5, CHD7, ST18 and RB1CC1). These loss-of-function and exonic variants present in genes related to muscle development can be considered candidate variants for further studies in chickens. Further association studies should be performed with these candidate mutations as should validation in commercial populations to allow a better explanation of QTL effects.DOI
- 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.DOI
- Avianbase: a community resource for bird genomics
Giving access to sequence and annotation data for genome assemblies is important because, while facilitating research, it places both assembly and annotation quality under scrutiny, resulting in improvements to both. Therefore we announce Avianbase, a resource for bird genomics, which provides access to data released by the Avian Phylogenomics Consortium. DOI
- Cetaceans evolution: insights from the genome sequences of common minke whales
Background: Whales have captivated the human imagination for millennia. These incredible cetaceans are the only mammals that have adapted to life in the open oceans and have been a source of human food, fuel and tools around the globe. The transition from land to water has led to various aquatic specializations related to hairless skin and ability to regulate their body temperature in cold water.
Results: We present four common minke whale (Balaenoptera acutorostrata) genomes with depth of x13 similar to x17 coverage and perform resequencing technology without a reference sequence. Our results indicated the time to the most recent common ancestors of common minke whales to be about 2.3574 (95% HPD, 1.1521 - 3.9212) million years ago. Further, we found that genes associated with epilation and tooth-development showed signatures of positive selection, supporting the morphological uniqueness of whales.
Conclusions: This whole-genome sequencing offers a chance to better understand the evolutionary journey of one of the largest mammals on earth.DOI
- Variant discovery in a QTL region on chromosome 3 associated with fatness in chickens
Abdominal fat content is an economically important trait in commercially bred chickens. Although many quantitative trait loci (QTL) related to fat deposition have been detected, the resolution for these regions is low and functional variants are still unknown. The current study was conducted aiming at increasing resolution for a region previously shown to have a QTL associated with fat deposition, to detect novel variants from this region and to annotate those variants to delineate potentially functional ones as candidates for future studies. To achieve this, 18 chickens from a parental generation used in a reciprocal cross between broiler and layer lines were sequenced using the Illumina next-generation platform with an initial coverage of 18X/chicken. The discovery of genetic variants was performed in a QTL region located on chromosome 3 between microsatellite markers LEI0161 and ADL0371 (33 595 706-42 632 651 bp). A total of 136 054 unique SNPs and 15 496 unique INDELs were detected in this region, and after quality filtering, 123 985 SNPs and 11 298 INDELs were retained. Of these variants, 386 SNPs and 15 INDELs were located in coding regions of genes related to important metabolic pathways. Loss-of-function variants were identified in several genes, and six of those, namely LOC771163, EGLN1, GNPAT, FAM120B, THBS2 and GGPS1, were related to fat deposition. Therefore, these loss-of-function variants are candidate mutations for conducting further studies on this important trait in chickens.DOI
- Detection and characterization of small insertion and deletion genetic variants in modern layer chicken genomes
BACKGROUND: Small insertions and deletions (InDels) constitute the second most abundant class of genetic variants and have been found to be associated with many traits and diseases. The present study reports on the detection and characterisation of about 883 K high quality InDels from the whole-genome analysis of several modern layer chicken lines from diverse breeds.
RESULTS: To reduce the error rates seen in InDel detection, this study used the consensus set from two InDel-calling packages: SAMtools and Dindel, as well as stringent post-filtering criteria. By analysing sequence data from 163 chickens from 11 commercial and 5 experimental layer lines, this study detected about 883 K high quality consensus InDels with 93 % validation rate and an average density of 0.78 InDels/kb over the genome. Certain chromosomes, viz, GGAZ, 16, 22 and 25 showed very low densities of InDels whereas the highest rate was observed on GGA6. In spite of the higher recombination rates on microchromosomes, the InDel density on these chromosomes was generally lower relative to macrochromosomes possibly due to their higher gene density. About 43-87 % of the InDels were found to be fixed within each line. The majority of detected InDels (86 %) were 1-5 bases and about 63 % were non-repetitive in nature while the rest were tandem repeats of various motif types. Functional annotation identified 613 frameshift, 465 non-frameshift and 10 stop-gain/loss InDels. Apart from the frameshift and stopgain/loss InDels that are expected to affect the translation of protein sequences and their biological activity, 33 % of the non-frameshift were predicted as evolutionary intolerant with potential impact on protein functions. Moreover, about 2.5 % of the InDels coincided with the most-conserved elements previously mapped on the chicken genome and are likely to define functional elements. InDels potentially affecting protein function were found to be enriched for certain gene-classes e.g. those associated with cell proliferation, chromosome and Golgi organization, spermatogenesis, and muscle contraction.
CONCLUSIONS: The large catalogue of InDels presented in this study along with their associated information such as functional annotation, estimated allele frequency, etc. are expected to serve as a rich resource for application in future research and breeding in the chicken.DOI
- A comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance
BACKGROUND: Chickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains.
RESULTS: Transcriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection.
CONCLUSIONS: Taken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken.DOI
- Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment
BACKGROUND: Penguins are flightless aquatic birds widely distributed in the Southern Hemisphere. The distinctive morphological and physiological features of penguins allow them to live an aquatic life, and some of them have successfully adapted to the hostile environments in Antarctica. To study the phylogenetic and population history of penguins and the molecular basis of their adaptations to Antarctica, we sequenced the genomes of the two Antarctic dwelling penguin species, the Adélie penguin [Pygoscelis adeliae] and emperor penguin [Aptenodytes forsteri].
RESULTS: Phylogenetic dating suggests that early penguins arose ~60 million years ago, coinciding with a period of global warming. Analysis of effective population sizes reveals that the two penguin species experienced population expansions from ~1 million years ago to ~100 thousand years ago, but responded differently to the climatic cooling of the last glacial period. Comparative genomic analyses with other available avian genomes identified molecular changes in genes related to epidermal structure, phototransduction, lipid metabolism, and forelimb morphology.
CONCLUSIONS: Our sequencing and initial analyses of the first two penguin genomes provide insights into the timing of penguin origin, fluctuations in effective population sizes of the two penguin species over the past 10 million years, and the potential associations between these biological patterns and global climate change. The molecular changes compared with other avian genomes reflect both shared and diverse adaptations of the two penguin species to the Antarctic environment.DOI
- Whole-genome analyses resolve early branches in the tree of life of modern birds
To better determine the history of modern birds, we performed a genome-scale phylogenetic analysis of 48 species representing all orders of Neoaves using phylogenomic methods created to handle genome-scale data. We recovered a highly resolved tree that confirms previously controversial sister or close relationships. We identified the first divergence in Neoaves, two groups we named Passerea and Columbea, representing independent lineages of diverse and convergently evolved land and water bird species. Among Passerea, we infer the common ancestor of core landbirds to have been an apex predator and confirm independent gains of vocal learning. Among Columbea, we identify pigeons and flamingoes as belonging to sister clades. Even with whole genomes, some of the earliest branches in Neoaves proved challenging to resolve, which was best explained by massive protein-coding sequence convergence and high levels of incomplete lineage sorting that occurred during a rapid radiation after the Cretaceous-Paleogene mass extinction event about 66 million years ago.DOI
- Comparative genomics reveals insights into avian genome evolution and adaptation
Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.DOI
- Reconstruction of gross avian genome structure, organization and evolution suggests that the chicken lineage most closely resembles the dinosaur avian ancestor
Background: The availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed.
Results: Focusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species' genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra-and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n = 80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes.
Conclusions: Results suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g., in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor.DOI
- 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 TGFβ). 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.DOI
- All creatures great and small: 10 years on
- Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage
We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. DOI
- 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 d(N)/d(S) ratios using six mammalian genomes, and F-ST 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 (d(N)/d(S)) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (F-ST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.DOI
- Chick genomics
- 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 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 which carry information from the intestine including CCK and its receptor CCKAR (CCK1 receptor). Using 16 generations of a cross between fast and a relatively slow growing strain of chicken has identified a region on chromosome 4, downstream of the CCKAR gene, responsible for up to a 19% difference in body weight at 12 weeks 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 their satiety set point has been altered. Comparison with traditional breeds shows 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 loci for a quantitative trait which alters appetite and gives us an insight into the domestication of animals. This will allow elucidation of how differences in appetite occur in birds but also mammals. DOI
- Npas4 is activated by melatonin, and drives the clock gene Cry1 in the ovine pars tuberalis
Seasonal mammals integrate changes in the duration of nocturnal melatonin secretion to drive annual physiological cycles. Melatonin receptors within the proximal pituitary region, the pars tuberalis (PT), are essential in regulating seasonal neuroendocrine responses. In the ovine PT, melatonin is known to influence acute changes in transcriptional dynamics coupled to the onset (dusk) and offset (dawn) of melatonin secretion, leading to a potential interval-timing mechanism capable of decoding changes in day-length (photoperiod). Melatonin offset at dawn is linked to cAMP accumulation, which directly induces transcription of the clock gene Per1. The rise of melatonin at dusk induces a separate and distinct cohort, including the clock-regulated genes Cry1 and Nampt, but little is known of the up-stream mechanisms involved. Here, we used next generation sequencing of the ovine PT transcriptome at melatonin onset, and identified Npas4 as a rapidly induced bHLH-PAS domain transcription factor. In vivo we show nuclear localisation of NPAS4 protein in presumptive melatonin target cells of the PT (αGSU-expressing cells), while in situ hybridisation studies identified acute and transient expression in the PT of Npas4 in response to melatonin. In vitro, NPAS4 forms functional dimers with bHLH-PAS domain co-factors ARNT, ARNT2 and ARNTL, transactivating both Cry1 and Nampt ovine promoter reporters. Using a combination of 5` deletions and site-directed mutagenesis we show NPAS4:ARNT transactivation to be co-dependent upon two conserved central midline elements (CMEs) within the Cry1 promoter. Our data thus reveal NPAS4 as a candidate immediate early-response gene in the ovine PT, driving molecular responses to melatonin. DOI
- Comparative analysis of quantitative trait loci for body weight, growth rate and growth curve parameters from 3 to 72 weeks of age in female chickens of a broiler-layer cross
Background: Comparisons of quantitative trait loci (QTL) for growth and parameters of growth curves assist in understanding the genetics and ultimately the physiology of growth. Records of body weight at 3, 6, 12, 24, 48 and 72 weeks of age and growth rate between successive age intervals of about 500 F-2 female chickens of the Roslin broiler-layer cross were available for analysis. These data were analysed to detect and compare QTL for body weight, growth rate and parameters of the Gompertz growth function.
Results: Over 50 QTL were identified for body weight at specific ages and most were also detected in the nearest preceding and/or subsequent growth stage. The sum of the significant and suggestive additive effects for bodyweight at specific ages accounted for 23-43% of the phenotypic variation. A single QTL for body weight on chromosome 4 at 48 weeks of age had the largest additive effect (550.4 +/- 68.0 g, 11.5% of the phenotypic variation) and a QTL at a similar position accounted 14.5% of the phenotypic variation at 12 weeks of age. Age specific QTL for growth rate were detected suggesting that there are specific genes that affect developmental processes during the different stages of growth. Relatively few QTL influencing Gompertz growth curve parameters were detected and overlapped with loci affecting growth rate. Dominance effects were generally not significant but from 12 weeks of age they exceeded the additive effect in a few cases. No evidence for epistatic QTL pairs was found.
Conclusions: The results confirm the location for body weight and body weight gain during growth that were identified in previous studies and were consistent with QTL for the parameters of the Gompertz growth function. Chromosome 4 explained a relatively large proportion of the observed growth variation across the different ages, and also harboured most of the detected QTL for Gompertz parameters, confirming its importance in controlling growth. Very few QTL were detected for body weight or gain at 48 and 72 weeks of age, probably reflecting the effect of differences in reproduction and random environmental effects.DOI
- Microarray resources for genetic and genomic studies in chicken: a review
Advent of microarray technologies revolutionized the nature and scope of genetic and genomic research in human and other species by allowing massively parallel analysis of thousands of genomic sites. They have been used for diverse purposes such as for transcriptome analysis, CNV detection, SNP and CNV genotyping, studying DNA-protein interaction, and detection of genome methylation. Microarrays have also made invaluable contributions to research in chicken which is an important model organism for studying embryology, immunology, oncology, virology, evolution, genetics, and genomics and also for other avian species. Despite their huge contributions in life science research, the future of microarrays is now being questioned with the advent of massively parallel next generation sequencing (NGS) technologies, which promise to overcome some of the limitations of microarray platforms. In this article we review the various microarray resources developed for chicken and their past and potential future applications. We also discuss about the future of microarrays in the NGS era particularly in the context of livestock genetics. We argue that even though NGS promises some major advantages-in particular, offers the opportunity to discover novel elements in the genome-microarrays will continue to be major tools for research and practice in the field of livestock genetics/genomics due to their affordability, high throughput nature, mature established technologies and ease of application. Moreover, with advent of new microarray technologies like capture arrays, the NGS and microarrays are expected to complement each other in future research in life science. genesis 00:1-20. © 2013 Wiley Periodicals, Inc. DOI
- Association of IGF1 and KDM5A polymorphisms with performance, fatness and carcass traits in chickens
Two functional and positional candidate genes were selected in a region of chicken chromosome 1 (GGA1), based on their biological roles, and also where several quantitative trait loci (QTL) have been mapped and associated with performance, fatness and carcass traits in chickens. The insulin-like growth factor 1 (IGF1) gene has been associated with several physiological functions related to growth. The lysine (K)-specific demethylase 5A (KDM5A) gene participates in the epigenetic regulation of genes involved with the cell cycle. Our objective was to find associations of selected single-nucleotide polymorphisms (SNPs) in these genes with performance, fatness and carcass traits in 165 F(2) chickens from a resource population. In the IGF1 gene, 17 SNPs were detected, and in the KDM5A gene, nine SNPs were detected. IGF1 SNP c.47673G > A was associated with body weight and haematocrit percentage, and also with feed intake and percentages of abdominal fat and gizzard genotype × sex interactions. KDM5A SNP c.34208C > T genotype × sex interaction affected body weight, feed intake, percentages of abdominal fat (p = 0.0001), carcass, gizzard and haematocrit. A strong association of the diplotype × sex interaction (p DOI
- Development of a high density 600K SNP genotyping array for chicken
BACKGROUND: High density (HD) SNP genotyping arrays are an important tool for genetic analyses of animals and plants. Although the chicken is one of the most important farm animals, no HD array is yet available for high resolution genetic analysis of this species.
RESULTS: We report here the development of a 600 K Affymetrix(R) Axiom(R) HD genotyping array designed using SNPs segregating in a wide variety of chicken populations. In order to generate a large catalogue of segregating SNPs, we re-sequenced 243 chickens from 24 chicken lines derived from diverse sources (experimental, commercial broiler and layer lines) by pooling 10--15 samples within each line. About 139 million (M) putative SNPs were detected by mapping sequence reads to the new reference genome (Gallus_gallus_4.0) of which ~78 M appeared to be segregating in different lines. Using criteria such as high SNP-quality score, acceptable design scores predicting high conversion performance in the final array and uniformity of distribution across the genome, we selected ~1.8 M SNPs for validation through genotyping on an independent set of samples (n = 282). About 64% of the SNPs were polymorphic with high call rates (>98%), good cluster separation and stable Mendelian inheritance. Polymorphic SNPs were further analysed for their population characteristics and genomic effects. SNPs with extreme breach of Hardy-Weinberg equilibrium (P <0.00001) were excluded from the panel. The final array, designed on the basis of these analyses, consists of 580,954 SNPs and includes 21,534 coding variants. SNPs were selected to achieve an essentially uniform distribution based on genetic map distance for both broiler and layer lines. Due to a lower extent of LD in broilers compared to layers, as reported in previous studies, the ratio of broiler and layer SNPs in the array was kept as 3:2. The final panel was shown to genotype a wide range of samples including broilers and layers with over 100 K to 450 K informative SNPs per line. A principal component analysis was used to demonstrate the ability of the array to detect the expected population structure which is an important pre-investigation step for many genome-wide analyses.
CONCLUSIONS: This Affymetrix(R) Axiom(R) array is the first SNP genotyping array for chicken that has been made commercially available to the public as a product. This array is expected to find widespread usage both in research and commercial application such as in genomic selection, genome-wide association studies, selection signature analyses, fine mapping of QTLs and detection of copy number variants. DOI
- Identification of Differentially Evolved Genes: An Alternative Approach to Detection of Accelerated Molecular Evolution from Genome-Wide Comparative Data
One of the most important measures for detecting molecular adaptations between species/lineages at the gene level is the comparison of relative fixation rates of synonymous (dS) and non-synonymous (dN) mutations. This study shows that the branch model is sensitive to tree topology and proposes an alternative approach, devogs, which does not require phylogenetic topology for analysis. We compared devogs with a branch model method using virtual data and a varying omega ratio, in which parameters were obtained from real data. The positive predictive value, sensitivity, and specificity of the branch model were affected by the phylogenic tree topology. Devogs showed greater positive predictive value, whereas the branch model method had greater sensitivity. In a working example using devogs, a group of human RNA polymerase II-related genes, which are important in mediating alternative splicing, were significantly accelerated compared to four other mammals.DOI
- The duck genome and transcriptome provide insight into an avian influenza virus reservoir species
The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A viruses. We present the duck genome sequence and perform deep transcriptome analyses to investigate immune-related genes. Our data indicate that the duck possesses a contractive immune gene repertoire, as in chicken and zebra finch, and this repertoire has been shaped through lineage-specific duplications. We identify genes that are responsive to influenza A viruses using the lung transcriptomes of control ducks and ones that were infected with either a highly pathogenic (A/duck/Hubei/49/05) or a weakly pathogenic (A/goose/Hubei/65/05) H5N1 1 virus. Further, we show how the duck’s defense mechanisms against influenza infection have been optimized through the diversification of its b-defensin and butyrophilin-like repertoires. These analyses, in combination with the genomic and transcriptomic data, provide a resource for characterizing the interaction between host and influenza viruses.
- eChickAtlas: An introduction to the database
The precise control of gene expression is critical in embryonic development. Quantitative assays, such as microarrays and RNA sequencing, provide gene expression levels for a large number of genes, but do not contain spatial information. In contrast, in situ methods, such as in situ hybridisation and immunohistochemistry, provide spatial resolution, but poor quantification and can only reveal the expression of one, or very few genes at a time. Furthermore, the usual methods of documenting the results, by photographing whole mounts or sections, makes it very difficult to assess the three-dimensional (3D) relationships between expressing and non-expressing cells. Optical projection tomography (OPT) can capture the full 3D expression pattern in a whole embryo at a reasonable level of resolution and at moderately high throughput. A large database containing spatio-temporal patterns of expression for the mouse (e-Mouse Atlas Project, EMAP, www.emouseatlas.org) has been created, incorporating 3D information. Like the mouse, the chick is an important model in developmental biology and translational studies. To facilitate comparisons between these important model organisms, we have created a 3D anatomical atlas, accompanied by an anatomical ontology of the chick embryo and a database of gene expression patterns during chick development. This database is publicly available (www.echickatlas.org). DOI
- Many quantitative trait loci for feather growth in an F broiler × layer cross collocate with body weight loci
1. A genome-wide scan of 467 F(2) progeny of a broiler x layer cross was conducted to identify quantitative trait loci (QTL) affecting the rate of growth of the tail, wing and back feathers, and the width of the breast feather tract, at three weeks of age. 2. Correlations between the traits ranged from 0·36 to 0·61. Males had longer tail and wing feathers and shorter back feathers than females. Breast feather tract width was greater in females than males. 3. QTL effects were generally additive and accounted for 11 to 45% of sex average feather lengths of the breeds, and 100% of the breast feather tract width. Positive and negative alleles were inherited from both lines, whereas the layer allele was larger than the broiler allele after adjusting for body weight. 4. A total of 4 genome-significant and 4 suggestive QTL were detected. At three or 6 weeks of age, 5 of the QTL were located in similar regions as QTL for body weight. 5. Analysis of a model with body weight at three weeks as a covariate identified 5 genome significant and 6 suggestive QTL, of which only two were coincident with body weight QTL. One QTL for feather length at 148 cM on GGA1 was identified at a similar location in the unadjusted analysis. 6. The results suggest that the rate of feather growth is largely controlled by body weight QTL, and that QTL specific for feather growth also exist. DOI
- Bone mineral density QTL at sexual maturity and end of lay
1. An F(2) cross of a broiler male line and a White Leghorn layer line was used to identify quantitative trait loci (QTL) for bone density at the onset of lay and at the end of the laying period. A total of 686 measures of humeral bone density were available for analysis. 2. There was no evidence for epistasis. 3. Genome-wide significant QTL for bone density at the onset of lay were identified on chromosomes 1 (311 cM) and 8 (2 cM) and on chromosomes 1 (311 cM), 3 (57 cM) and 8 (2 cM) with a covariate for the number of yellow follicles (a proxy for the concentration of circulating oestrogen). 4. Evidence for only 4 chromosome-wide suggestive QTL were detected at the end of lay (72 weeks). 5. Analysis of the combined data confirmed two genome-wide suggestive QTL on chromosome 1 (137 and 266 cM) and on chromosomes 8 (2 cM) and 9 (10 cM) in analyses with or without the covariate. 6. Positive QTL alleles came from the broiler line with the exception of 2 suggestive QTL at the onset of lay on chromosomes 3 and 5 in an analysis with the covariate. 7. In general, QTL acted additively, except that dominant effects were identified for three suggestive QTL at the onset of lay on chromosomes 3 (57 and 187 cM) and 5 (9 cM). 8. The significant QTL in this study were at similar locations to QTL identified in a range of crosses in other publications, suggesting that they are prime candidates for the search for genes and mutations that could be used as selection criteria to improve bone strength and decrease fractures in commercial laying hens. DOI
- A high resolution genome-wide scan for significant selective sweeps: an application to pooled sequence data in laying chickens
In most studies aimed at localizing footprints of past selection, outliers at tails of the empirical distribution of a given test statistic are assumed to reflect locus-specific selective forces. Significance cutoffs are subjectively determined, rather than being related to a clear set of hypotheses. Here, we define an empirical p-value for the summary statistic by means of a permutation method that uses the observed SNP structure in the real data. To illustrate the methodology, we applied our approach to a panel of 2.9 million autosomal SNPs identified from re-sequencing a pool of 15 individuals from a brown egg layer line. We scanned the genome for local reductions in heterozygosity, suggestive of selective sweeps. We also employed a modified sliding window approach that accounts for gaps in the sequence and increases scanning resolution by moving the overlapping windows by steps of one SNP only, and suggest to call this a "creeping window" strategy. The approach confirmed selective sweeps in the region of previously described candidate genes, i.e. TSHR, PRL, PRLHR, INSR, LEPR, IGF1, and NRAMP1 when used as positive controls. The genome scan revealed 82 distinct regions with strong evidence of selection (genome-wide p-value DOI
- Quantitative trait loci associated with chemical composition of the chicken carcass
Major objectives of the poultry industry are to increase meat production and to reduce carcass fatness, mainly abdominal fat. Information on growth performance and carcass composition are important for the selection of leaner meat chickens. To enhance our understanding of the genetic architecture underlying the chemical composition of chicken carcasses, an F(2) population developed from a broiler × layer cross was used to map quantitative trait loci (QTL) affecting protein, fat, water and ash contents in chicken carcasses. Two genetic models were applied in the QTL analysis: the line-cross and the half-sib models, both using the regression interval mapping method. Six significant and five suggestive QTL were mapped in the line-cross analysis, and four significant and six suggestive QTL were mapped in the half-sib analysis. A total of eleven QTL were mapped for fat (ether extract), five for protein, four for ash and one for water contents in the carcass using both analyses. No study to date has reported QTL for carcass chemical composition in chickens. Some QTL mapped here for carcass fat content match, as expected, QTL regions previously associated with abdominal fat in the same or in different populations, and novel QTL for protein, ash and water contents in the carcass are presented here. The results described here also reinforce the need for fine mapping and to perform multi-trait analyses to better understand the genetic architecture of these traits. DOI
- Complex traits analysis of chicken growth using targeted genetical genomics
Dissecting the genetic control of complex trait variation remains very challenging, despite many advances in technology. The aim of this study was to use a major growth quantitative trait locus (QTL) in chickens mapped to chromosome 4 as a model for a targeted approach to dissect the QTL. We applied a variant of the genetical genomics approach to investigate genome-wide gene expression differences between two contrasting genotypes of a marked QTL. This targeted approach allows the direct quantification of the link between the genotypes and the genetic responses, thus narrowing the QTL-phenotype gap using fewer samples (i.e. microarrays) compared with the genome-wide genetical genomics studies. Four differentially expressed genes were localized under the region of the QTL. One of these genes is a potential positional candidate gene (AADAT) that affects lysine and tryptophan metabolism and has alternative splicing variants between the two genotypes. In addition, the lysine and glycolysis metabolism pathways were significantly enriched for differentially expressed genes across the genome. The targeted approach provided a complementary route to fine mapping of QTL by characterizing the local and the global downstream effects of the QTL and thus generating further hypotheses about the action of that QTL. DOI
- Systemic virus distribution and host responses in brain and intestine of chickens infected with low pathogenic or high pathogenic avian influenza virus
ABSTRACT: 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. DOI
- Systems analysis of immune responses in Marek's disease virus-infected chickens identifies a gene involved in susceptibility and highlights a possible novel pathogenicity mechanism
Marek's disease virus (MDV) is a highly contagious oncogenic alphaherpesvirus that causes disease that is both a cancer model and a continuing threat to the world's poultry industry. This comprehensive gene expression study analyzes the host response to infection in both resistant and susceptible lines of chickens and inherent expression differences between the two lines following the infection of the host. A novel pathogenicity mechanism, involving the downregulation of genes containing HIC1 transcription factor binding sites as early as 4 days postinfection, was suggested from this analysis. HIC1 drives antitumor mechanisms, suggesting that MDV infection switches off genes involved in antitumor regulation several days before the expression of the MDV oncogene meq. The comparison of the gene expression data to previous QTL data identified several genes as candidates for involvement in resistance to MD. One of these genes, IRG1, was confirmed by single nucleotide polymorphism analysis to be involved in susceptibility. Its precise mechanism remains to be elucidated, although the analysis of gene expression data suggests it has a role in apoptosis. Understanding which genes are involved in susceptibility/resistance to MD and defining the pathological mechanisms of the disease gives us a much greater ability to try to reduce the incidence of this virus, which is costly to the poultry industry in terms of both animal welfare and economics. DOI
- QTL for percentage of carcass and carcass parts in a broiler x layer cross
P>An F-2 experimental population, developed from a broiler layer cross, was used in a genome scan of QTL for percentage of carcass, carcass parts, shank and head. Up to 649 F-2 chickens from four paternal half-sib families were genotyped with 128 genetic markers covering 22 linkage groups. Total map length was 2630 cM, covering approximately 63% of the genome. QTL interval mapping using regression methods was applied to line-cross and half-sib models. Under the line-cross model, 12 genome-wide significant QTL and 17 suggestive linkages for percentages of carcass parts, shank and head were mapped to 13 linkage groups (GGA1, 2, 3, 4, 5, 7, 8, 9, 11, 12, 14, 18 and 27). Under the paternal half-sib model, six genome-wide significant QTL and 18 suggestive linkages for percentages of carcass parts, shank and head were detected on nine chicken linkage groups (GGA1, 2, 3, 4, 5, 12, 14, 15 and 27), seven of which seemed to corroborate positions revealed by the previous model. Overall, three novel QTL of importance to the broiler industry were mapped (one significant for shank% on GGA3 and two suggestive for carcass and breast percentages on GGA14 and drums and thighs percentage on GGA15). One novel QTL for wings% was mapped to GGA3, six novel QTL (GGA1, 3, 7, 8, 9 and 27) and suggestive linkages (GGA2, 4, and 5) were mapped for head%, and suggestive linkages were identified for back% on GGA2, 11 and 12. In addition, many of the QTL mapped in this study confirmed QTL previously reported in other populations.DOI
- 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. DOI
- Mpdz null allele in an avian model of retinal degeneration and mutations in human leber congenital amaurosis and retinitis pigmentosa
Purpose. To identify the defective gene in the sex-linked, recessively inherited retinal dysplasia and degeneration (rdd) chicken and to search for the human equivalent disease. Methods. Microsatellites from chicken chromosome Z were genotyped in 77 progeny of a carrier male (rdd/+) and an affected female (rdd/W), and candidate genes were sequenced. Retinal cross-sections from rdd and wild-type birds were analyzed by immunohistology. The human orthologous gene was screened in a panel of archival DNAs from 276 patients with retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA) using melting curve analysis and DNA sequencing. Results. The rdd locus was refined to an approximately 3-Mb region on chromosome Z. Sequence analysis identified a C-->T change in the mpdz gene that created a premature stop codon (c.1372C-->T, p.R458X), which segregated with the disease phenotype. As expected, the full-length mpdz protein was absent in rdd retinas, but in wild-type birds, it localized to the retinal outer limiting membrane, where it may have a role in the interactions between photoreceptors and Muller glia cells. The screen to identify the human equivalent disease found 10 heterozygous variants in the orthologous gene in patients with RP (three missense and two null alleles) and LCA (four missense and one null allele). Conclusions. These findings reveal that MPDZ is essential for normal development of the retina and may have a role in maintaining photoreceptor integrity. The identification of human mutations suggests that MPDZ plays a role in human retinal disease, but the precise nature of this role remains to be determined. DOI
- Molecular evolution of the vertebrate TLR1 gene family - a complex history of gene duplication, gene conversion, positive selection and co-evolution
Background: The Toll-like receptors represent a large superfamily of type I transmembrane glycoproteins, some common to a wide range of species and others are more restricted in their distribution. Most members of the Toll-like receptor superfamily have few paralogues; the exception is the TLR1 gene family with four closely related genes in mammals TLR1, TLR2, TLR6 and TLR10, and four in birds TLR1A, TLR1B, TLR2A and TLR2B. These genes were previously thought to have arisen by a series of independent gene duplications. To understand the evolutionary pattern of the TLR1 gene family in vertebrates further, we cloned the sequences of TLR1A, TLR1B, TLR2A and TLR2B in duck and turkey, constructed phylogenetic trees, predicted codons under positive selection and identified co-evolutionary amino acid pairs within the TLR1 gene family using sequences from 4 birds, 28 mammals, an amphibian and a fish. Results: This detailed phylogenetic analysis not only clarifies the gene gains and losses within the TLR1 gene family of birds and mammals, but also defines orthologues between these vertebrates. In mammals, we predict amino acid sites under positive selection in TLR1, TLR2 and TLR6 but not TLR10. We detect co-evolution between amino acid residues in TLR2 and the other members of this gene family predicted to maintain their ability to form functional heterodimers. In birds, we predict positive selection in the TLR2A and TLR2B genes at functionally significant amino acid residues. We demonstrate that the TLR1 gene family has mostly been subject to purifying selection but has also responded to directional selection at a few sites, possibly in response to pathogen challenge. Conclusions: Our phylogenetic and structural analyses of the vertebrate TLR1 family have clarified their evolutionary origins and predict amino acid residues likely to be important in the host's defense against invading pathogens. DOI
- 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. Developmental Dynamics, 2011. (c) 2011 Wiley-Liss, Inc. DOI
- Overlap of quantitative trait loci for early growth rate, and for body weight and age at onset of sexual maturity in chickens
Critical age, weight and body composition have been suggested as necessary correlates of sexual maturity. A genome scan to identify quantitative trait loci (QTL) for age and body weight at first egg (AFE and WFE) was conducted on 912 birds from an F2 broiler–layer cross using 106 microsatellite markers. Without a covariate, QTL for body WFE were detected on chromosomes 2, 4, 8, 27 and Z and a single QTL for AFE was detected on chromosome 2. With AFE as a covariate, additional QTL for body WFE were found on chromosomes 1 and 13, with abdominal fat pad as covariate a QTL for body WFE was found on chromosome 1. With body WFE as covariate, additional QTL for AFE were found on chromosomes 1, 3, 4, 13 and 27. The QTL generally acted additively and there was no evidence for epistasis. Consistent with the original line differences, broiler alleles had positive effects on body WFE and negative effects on AFE, whereas the phenotypic correlation between the two traits was positive. The mapped QTL for body WFE cumulatively accounted for almost half the body weight difference between the chicken lines at puberty. Overlapping QTL for body WFE and body weight to 9 weeks of age indicate that most QTL affecting growth rate also affect body WFE. The co-localisation of QTL for body weight, growth and sexual maturity suggests that body weight and growth rate are closely related to the attainment of sexual maturity and that the genetic determination of growth rate has correlated effects on puberty. DOI
- Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis
A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (~1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest. DOI
- Pivotal Advance: Avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products
Macrophages are involved in many aspects of development, host defense, pathology, and homeostasis. Their normal differentiation, proliferation, and survival are controlled by CSF-1 via the activation of the CSF1R. A recently discovered cytokine, IL-34, was shown to bind the same receptor in humans. Chicken is a widely used model organism in developmental biology, but the factors that control avian myelopoiesis have not been identified previously. The CSF-1, IL-34, and CSF1R genes in chicken and zebra finch were identified from respective genomic/cDNA sequence resources. Comparative analysis of the avian CSF1R loci revealed likely orthologs of mammalian macrophage-specific promoters and enhancers, and the CSF1R gene is expressed in the developing chick embryo in a pattern consistent with macrophage-specific expression. Chicken CSF-1 and IL-34 were expressed in HEK293 cells and shown to elicit macrophage growth from chicken BM cells in culture. Comparative sequence and co-evolution analysis across all vertebrates suggests that the two ligands interact with distinct regions of the CSF1R. These studies demonstrate that there are two separate ligands for a functional CSF1R across all vertebrates. DOI
- The genome of a songbird
The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken-the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour. DOI
- Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis
Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation. DOI
- Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary
Seasonally breeding mammals such as sheep use photoperiod, encoded by the nocturnal secretion of the pineal hormone melatonin, as a critical cue to drive hormone rhythms and synchronize reproduction to the most optimal time of year [1, 2]. Melatonin acts directly on the pars tuberalis (PT) of the pituitary, regulating expression of thyrotropin, which then relays messages back to the hypothalamus to control reproductive circuits [3, 4]. In addition, a second local intrapituitary circuit controls seasonal prolactin (PRL) release via one or more currently uncharacterized low-molecular-weight peptides, termed "tuberalins," of PT origin [5-7]. Studies in birds have identified the transcription factor Eya3 as the first molecular response activated by long photoperiod (LP) . Using arrays and in situ hybridization studies, we demonstrate here that Eya3 is the strongest LP-activated gene in sheep, revealing a common photoperiodic molecular response in birds and mammals. We also demonstrate TAC1 (encoding the tachykinins substance P and neurokinin A) to be strongly activated by LP within the sheep PT. We show that these PRL secretagogues act on primary pituitary cells and thus are candidates for the elusive PT-expressed tuberalin seasonal hormone regulator. DOI
- Gene duplication and fragmentation in the zebra finch major histocompatibility complex
Background: Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines. Results: The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes. Conclusion: The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages. DOI
- Identification of chromosomal regions associated with growth and carcass traits in an F(3) full sib intercross line originating from a cross of chicken lines divergently selected on body weight
An F(3) resource population originating from a cross between two divergently selected lines for high (D+ line) or low (D- line) body weight at 8-weeks of age (BW55) was generated and used for Quantitative Trait Locus (QTL) mapping. From an initial cross of two founder F(0) animals from D(+) and D(-) lines, progeny were randomly intercrossed over two generations following a full sib intercross line (FSIL) design. One hundred and seventy-five genome-wide polymorphic markers were employed in the DNA pooling and selective genotyping of F(3) to identify markers with significant effects on BW55. Fifty-three markers on GGA2, 5 and 11 were then genotyped in the whole F(3) population of 503 birds, where interval mapping with GridQTL software was employed. Eighteen QTL for body weight, carcass traits and some internal organ weights were identified. On GGA2, a comparison between 2-QTL vs. 1-QTL analysis revealed two separate QTL regions for body, feet, breast muscle and carcass weight. Given co-localization of QTL for some highly correlated traits, we concluded that there were 11 distinct QTL mapped. Four QTL localized to already mapped QTL from other studies, but seven QTL have not been previously reported and are hence novel and unique to our selection line. This study provides a low resolution QTL map for various traits and establishes a genetic resource for future fine-mapping and positional cloning in the advanced FSIL generations. DOI
- Quantitative trait loci associated with fatness in a broiler-layer cross
An F(2) population established by crossing a broiler male line and a layer line was used to map quantitative trait loci (QTL) affecting abdominal fat weight, abdominal fat percentage and serum cholesterol and triglyceride concentrations. Two genetic models, the line-cross and the half-sib, were applied in the QTL analysis, both using the regression interval method. Three significant QTL and four suggestive QTL were mapped in the line-cross analysis and four significant and four suggestive QTL were mapped in the half-sib analysis. A total of five QTL were mapped for abdominal fat weight, six for abdominal fat percentage and four for triglyceride concentration in both analyses. New QTL associated with serum triglyceride concentration were mapped on GGA5, GGA23 and GG27. QTL mapped between markers LEI0029 and ADL0371 on GGA3 for abdominal fat percentage and abdominal fat weight and a suggestive QTL on GGA12 for abdominal fat percentage showed significant parent-of-origin effects. Some QTL mapped here match QTL regions mapped in previous studies using different populations, suggesting good candidate regions for fine-mapping and candidate gene searches. DOI
- The cattle genome reveals its secrets
The domesticated cow is the latest farm animal to have its genome sequenced and deciphered. The members of the Bovine Genome Consortium have published a series of papers on the assembly and what the sequence reveals so far about the biology of this ruminant and the consequences of its domestication. DOI
- Quantitative trait loci for performance traits in a broiler x layer cross
An F(2) resource population, derived from a broiler x layer cross, was used to map quantitative trait loci (QTL) for body weights at days 1, 35 and 41, weight gain, feed intake, feed efficiency from 35 to 41 days and intestinal length. Up to 577 F(2) chickens were genotyped with 103 genetic markers covering 21 linkage groups. A preliminary QTL mapping report using this same population focused exclusively on GGA1. Regression methods were applied to line-cross and half-sib models for QTL interval mapping. Under the line-cross model, eight QTL were detected for body weight at 35 days (GGA2, 3 and 4), body weight at 41 days (GGA2, 3, 4 and 10) and intestine length (GGA4). Under the half-sib model, using sire as common parent, five QTL were detected for body weight at day 1 (GGA3 and 18), body weight at 35 days (GGA2 and 3) and body weight at 41 days (GGA3). When dam was used as common parent, seven QTL were mapped for body weight at day 1 (GGA2), body weight at day 35 (GGA2, 3 and 4) and body weight at day 41 (GGA2, 3 and 4). Growth differences in chicken lines appear to be controlled by a chronological change in a limited number of chromosomal regions. DOI
- Towards the selection of chickens resistant to Salmonella and Campylobacter infections
Resistance to infection with enteric pathogens such as Salmonella and Campylobacter can be at many levels and include both non-immune and immune mechanisms. Immune resistance mechanisms can be specific, at the level of the adaptive immune response, or non-specific, at the level of the innate immune response. Whilst we can extrapolate to some degree in birds from what is known about immune responses to these pathogens in mammals, chickens are not "feathered mice", but have a different repertoire of genes, molecules, cells and organs involved in their immune response compared to mammals. Fundamental work on the chicken's immune response to enteric pathogens is therefore still required. Our studies focus particularly on the innate immune response, as responses of heterophils (the avian neutrophil equivalent) from commercial birds, and macrophages from inbred lines of chickens, correlate with resistance or susceptibility to Salmonella infection with a variety of Salmonella serovars and infection models. We work on two basic resistance mechanisms - resistance to colonization with Salmonella or Campylobacter, and resistance to systemic salmonellosis (or fowl typhoid). To map genes involved in resistance to colonization with Salmonella and Campylobacter, we are using a combination of expression quantitative trait loci (eQTLs) from microarray studies, allied with whole genome SNP arrays (WGA), a candidate gene approach and analysis of copy number variation across the genome. For resistance to systemic salmonellosis, we have refined the location ofa novel resistance locus on Chromosome 5, designated SAL1, using high density SNP panels, combined with advanced back-crossing of resistant and susceptible lines. Using a 6th generation backcross mapping population we have confirmed and refined the SAL1 locus to 8-00 kb of Chromosome 5. This region spans 14 genes, including two very striking functional candidates; CD27-binding protein (Siva) and the RAC-alpha serine/threonine protein kinase homologue, AKT1.
- The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation
The chicken talpid(3) mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid(3) mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid(3) mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid(3) mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid(3) mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid(3) mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling. DOI
- 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), Hif1 alpha (hypoxia-inducible factor-1 alpha), and Kcnq5 (K+ channel) and down-regulation of Ror beta, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef/Nampt, NeuroD1, and Ror beta in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef/Nampt, NeuroD1, and Hif1 alpha, 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. (Endocrinology 149: 5527-5539, 2008)DOI
- 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.DOI
- A genome-wide linkage study in families with major depression and co-morbid unexplained swelling
Major depressive disorder (MDD) is a common heritable condition. The diversity of the phenotype coupled with aetiological and genetic heterogeneity present formidable obstacles in the search for causative genetic loci. Studies of large families with many affected individuals, and the selection of well-defined clinical subgroups of depression, are two ways to reduce this complexity. Unexplained swelling symptoms (USS) are common in women and many patients give a strong personal and family history of depression. Co-morbid depression and swelling symptoms define a useful sub-phenotype for investigating genetic factors in depression. We have completed a genome-wide linkage analysis using 371 microsatellite markers in four families where MDD is co-morbid with USS. Of 47 affected individuals, 28 had both MDD and unexplained swelling, 11 had symptoms of swelling alone, and 8 had MDD alone. Parametric marker-specific analysis identified one suggestive locus, D8S260 (LOD = 2.02) and non-parametric multipoint variance component analysis identified a region on 7p (LOD = 2.10). A 47 cM suggestive linkage region on chromosome 14q (identified by both parametric and non-parametric methods) was identified and investigated further with fine-mapping markers but the evidence for linkage to this region decreased with increased marker information content. DOI
- Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution
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.
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.
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. DOI
- Evolution of the chicken Toll-like receptor gene family: a story of gene gain and gene loss
Toll-like receptors (TLRs) perform a vital role in disease resistance through their recognition of pathogen associated molecular patterns (PAMPs). Recent advances in genomics allow comparison of TLR genes within and between many species. This study takes advantage of the recently sequenced chicken genome to determine the complete chicken TLR repertoire and place it in context of vertebrate genomic evolution.
The chicken TLR repertoire consists of ten genes. Phylogenetic analyses show that six of these genes have orthologs in mammals and fish, while one is only shared by fish and three appear to be unique to birds. Furthermore the phylogeny shows that TLR1-like genes arose independently in fish, birds and mammals from an ancestral gene also shared by TLR6 and TLR10. All other TLRs were already present prior to the divergence of major vertebrate lineages 550 Mya (million years ago) and have since been lost in certain lineages. Phylogenetic analysis shows the absence of TLRs 8 and 9 in chicken to be the result of gene loss. The notable exception to the tendency of gene loss in TLR evolution is found in chicken TLRs 1 and 2, each of which underwent gene duplication about 147 and 65 Mya, respectively.
Comparative phylogenetic analysis of vertebrate TLR genes provides insight into their patterns and processes of gene evolution, with examples of both gene gain and gene loss. In addition, these comparisons clarify the nomenclature of TLR genes in vertebrates. DOI
- Evolution of avian innate immune system
- An epistatic QTL pair affects ovarian follicular numbers at the onset of lay in broiler breeders
- Genomic research and applications in the duck (Anas platyrhynchos)
As a major natural reservoir of influenza virus and an important food source, the duck is of great biological interest, e.g. in the area of host-pathogen interactions. Recently, preliminary genetic and cytogenetic maps of the duck have become available, providing for the first time a glimpse at a comparative map between the duck and chicken. These genetic tools have been used to detect QTLs related to duck growth, carcass and meat quality traits. However, molecular genetic research in the duck is only in its infancy. In the future we can expect the development of new duck resources, including a high-density genetic map, detailed comparative maps with the chicken and other vertebrates - and given the pace of genomics, possibly a genome sequence. These new resources will be used to evaluate the genetic diversity of global duck breeds, to define genetic markers to increase the quantity and quality of egg and meat products, and to aid in the battle against infectious diseases, such as avian influenza DOI
- A QTL for osteoporosis detected in an F2 population derived from White Leghorn chicken lines divergently selected for bone index
Osteoporosis, resulting from progressive loss of structural bone during the period of egg-laying in hens, is associated with an increased susceptibility to bone breakage. To study the genetic basis of bone strength, an F(2) cross was produced from lines of hens that had been divergently selected for bone index from a commercial pedigreed White Leghorn population. Quantitative trait loci (QTL) affecting the bone index and component traits of the index (tibiotarsal and humeral strength and keel radiographic density) were mapped using phenotypic data from 372 F(2) individuals in 32 F(1) families. Genotypes for 136 microsatellite markers in 27 linkage groups covering approximately 80% of the genome were analysed for association with phenotypes using within-family regression analyses. There was one significant QTL on chromosome 1 for bone index and the component traits of tibiotarsal and humeral breaking strength. Additive effects for tibiotarsal breaking strength represented 34% of the trait standard deviation and 7.6% of the phenotypic variance of the trait. These QTL for bone quality in poultry are directly relevant to commercial populations. DOI
- Towards a molecular genetic tool for health and performance monitoring of Atlantic salmon (Salmo salar): The TRAITS 17K cDNA microarray
- Emergence of the chicken as a model organism: implications for agriculture and biology
Many of the features of the chicken make it an ideal model organism for phylogenetics and embryology, along with applications in agriculture and medicine. The availability of new tools such as whole genome gene expression arrays and single nucleotide polymorphism panels, coupled with the genome sequence, will enhance this position. These advances are reviewed and their implications are discussed.
- Avian genomics in the 21st century
The chicken has long been an important model organism for developmental biology, as well as a major source of protein with billions of birds used in meat and egg production each year. Chicken genomics has been transformed in recent years, with the characterisation of large EST collections and most recently with the assembly of the chicken genome sequence. As the first livestock genome to be fully sequenced it leads the way for others to follow--with zebra finch later this year. The genome sequence and the availability of three million genetic polymorphisms are expected to aid the identification of genes that control traits of importance in poultry. As the first bird genome to be sequenced it is a model for the remaining 9,600 species thought to exist today. Many of the features of avian biology and organisation of the chicken genome make it an ideal model organism for phylogenetics and embryology, along with applications in agriculture and medicine. The availability of new tools such as whole-genome gene expression arrays and SNP panels, coupled with information resources on the genes and proteins are likely to enhance this position. DOI
- Analysis of talpid3 and wild-type chicken embryos reveals roles for Hedgehog signalling in development of the limb bud vasculature
Chicken talpid(3) mutant embryos have a wide range of Hedgehog-signalling related defects and it is now known that the talpid(3) gene product encodes a novel protein essential for Hedgehog signalling which is required for both activator and repressor functions of Gli transcription factors (Davey, M.G., Paton, I.R., Yin, Y., Schmidt, M., Bangs, F.K., Morrice, D.R., Gordon-Smith, T., Buxton, P., Stamataki, D., Tanaka, M., Münsterberg, A.E., Briscoe, J., Tickle, C., Burt, D.W. (2006). The chicken talpid(3) gene encodes a novel protein essential for Hedgehog signalling. Genes Dev 20 1365-77). Haemorrhaging, oedema and other severe vascular defects are a central aspect of the talpid(3) phenotype (Ede, D.A. and Kelly, W.A (1964a). Developmental abnormalities in the head region of the talpid(3) mutant fowl. J. Embryol. exp. Morp. 12:161-182) and, as Hedgehog (Hh) signalling has been implicated in every stage of development of the vascular system, the vascular defects seen in talpid(3) are also likely to be attributable to abnormal Hedgehog signalling. Gene expression of members of the VEGF and Angiopoietin families of angiogenic growth factors has been linked to haemorrhaging and oedema and we find widespread expression of VEGF-D, rigf and Ang2a in the talpid(3) limb. Furthermore, ectopic expression of these genes in talpid(3) limbs points to regulation via Gli repression rather than activation. We monitored specification of vessel identity in talpid(3) limb vasculature by examining expression of artery-specific genes, Np1 and EphrinB2, and the vein-specific genes, Np2a and Tie2. We show that there are supernumerary subclavian arteries in talpid(3) limb buds and abnormal expression of an artery-specific gene in the venous submarginal sinus, despite the direction of blood flow being normal. Furthermore, we show that Shh can induce Np1 expression but has no effect on Np2a. Finally, we demonstrate that induction of VEGF and Ang2a expression by Shh in normal limb buds is accompanied by vascular remodelling. Thus Hedgehog signalling has a pivotal role in the cascade of angiogenic events in a growing embryonic organ which is similar to that proposed in tumours. DOI
- Quantitative trait loci for bone traits segregating independently of those for growth in an F-2 broiler X layer cross
An F-2 broiler-layer cross was phenotyped for 18 skeletal traits at 6, 7 and 9 weeks of age and genotyped with 120 microsatellite markers. Interval mapping identified 61 suggestive and significant QTL on 16 of the 25 linkage groups for 16 traits. Thirty-six additional QTL were identified when the assumption that QTL were fixed in the grandparent lines was relaxed. QTL with large effects on the lengths of the tarsometatarsus, tibia and femur, and the weights of the tibia and femur were identified on GGA4 between 217 and 249 cM. Six QTL for skeletal traits were identified that did not co-locate with genome wide significant QTL for body weight and two body weight QTL did not coincide with skeletal trait QTL. Significant evidence of imprinting was found in ten of the QTL and QTL x sex interactions were identified for 22 traits. Six alleles from the broiler line for weight-and size-related skeletal QTL were positive. Negative alleles for bone quality traits such as tibial dyschondroplasia, leg bowing and tibia twisting generally originated from the layer line suggesting that the allele inherited from the broiler is more protective than the allele originating from the layer. Copyright (C) 2007 S. Karger AG, Basel.DOI
- Mutation in the guanine nucleotide-binding protein beta-3 causes retinal degeneration and embryonic mortality in chickens
PURPOSE. To identify the gene defect that causes blindness and the predisposition to embryonic death in the retinopathy globe enlarged (rge) chicken.
METHODS. Linkage analysis, with previously uncharacterized microsatellite markers from chicken chromosome 1, was performed on 138 progeny of an rge/+ and an rge/rge cross, and candidate genes were sequenced.
RESULTS. The rge locus was refined and the gene for guanine nucleotide-binding protein beta-3 (GNB3), which encodes a cone transducin beta subunit, was found to have a 3-bp deletion (D153del) that segregated with the rge phenotype. This mutation deleted a highly conserved aspartic acid residue in the third of seven WD domains in GNB3. In silico modeling suggested that this mutation destabilized the protein. Furthermore, a 70% reduction was found in immunoreactivity to anti-GNB3 in the rge-affected retina.
CONCLUSIONS. These findings implicate the beta-subunit of cone transducin as the defective protein underlying the rge phenotype. Furthermore, GNB3 is ubiquitously expressed, and the c. 825C -> T GNB3 splicing variant (MIM 139130) has been associated with hypertension, obesity, diabetes, low birth weight, coronary heart disease, and stroke in the human population. It therefore seems likely that the defect underlying these human diseases also causes reduced embryonic viability in the rge chicken, making it a powerful model for studying the pathology involved in these associations.DOI
- 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. DOI
- QTL analysis of body weight and conformation score in commercial broiler chickens using variance component and half-sib analyses
The aim of the study was to investigate quantitative trait loci (QTL) in previously identified regions of chicken chromosomes 1, 4 and 5 relating to 40-day body weights and conformation scores. Half-sib (HS) and variance component analyses were implemented and compared using QTL Express software. Data were from a two-generation design and consisted of 100 dam families nested in 46 sire families with trait values for 2,708 offspring. Chicken chromosome 4 showed nominal significance for QTL affecting body weight and conformation, and linkage was confirmed for both traits on chromosome 5. Results varied according to method of analysis and with common parent in the HS method. DOI
- Identification of a non-mammalian leptin-like gene: characterization and expression in the tiger salamander (Ambystoma tigrinum)
Leptin is well established as a multifunctional cytokine in mammals. However, little is known about the evolution of the leptin gene in other vertebrates. A recently published set of ESTs from the tiger salamander (Ambystoma tigrinum) contains a sequence sharing 56% nucleotide sequence identity with the human leptin cDNA. To confirm that the EST is naturally expressed in the salamander, a 409bp cDNA was amplified by RT-PCR of salamander testis and stomach mRNAs. The coding sequence of the cDNA is predicted to encode 169 amino acids, and the mature peptide to consist of 146 residues, as in mammals. Although the overall amino acid identity with mammalian leptins is only 29%, the salamander and mammalian peptides share common structural features. An intron was identified between coding exons providing evidence that the sequence is present in the salamander genome. Phylogenetic analysis showed a rate of molecular divergence consistent with the accepted view of vertebrate evolution. The pattern of tissue expression of the leptin-like cDNA differed between metamorphosed adult individuals of different sizes suggesting possible developmental regulation. Expression was most prominent in the skin and testis, but was also detected in tissues in which leptin mRNA is present in mammals, including the fat body, stomach, and muscle. The characterization of a salamander leptin-like gene provides a basis for understanding how the structure and functions of leptin have altered during the evolution of tetrapod vertebrates. DOI
- Development of a chicken 5 K microarray targeted towards immune function
Background: The development of microarray resources for the chicken is an important step in being able to profile gene expression changes occurring in birds in response to different challenges and stimuli. The creation of an immune-related array is highly valuable in determining the host immune response in relation to infection with a wide variety of bacterial and viral diseases.
Results: Here we report the development of chicken immune-related cDNA libraries and the subsequent construction of a microarray containing 5190 elements ( in duplicate). Clones on the array originate from tissues known to contain high levels of cells related to the immune system, namely Bursa, Peyers patch, thymus and spleen. Represented on the array are genes that are known to cluster with existing chicken ESTs as well as genes that are unique to our libraries. Some of these genes have no known homologies and represent novel genes in the chicken collection. A series of reference genes (ie. genes of known immune function) are also present on the array. Functional annotation data is also provided for as many of the genes on the array as is possible.
Conclusion: Six new chicken immune cDNA libraries have been created and nearly 10,000 sequences submitted to GenBank [ GenBank: AM063043-AM071350; AM071520-AM072286; AM075249-AM075607]. A 5 K immune-related array has been developed from these libraries. Individual clones and arrays are available from the ARK-Genomics resource centre.DOI
- The chicken genome
The chicken has long been an important model organism for developmental biology, as well as a major source of protein with billions of birds used in meat and egg production each year. Chicken genomics has been transformed in recent years, with the characterisation of large EST collections and most recently with the assembly of the chicken genome sequence. Since the chicken shared a common ancestor with mammals 310 million years ago it fills a gap in our knowledge in the evolution and conservation of vertebrate genomes. As the first livestock genome to be fully sequenced it leads the way for others to follow. The genome sequence and the availability of 3 million genetic polymorphisms are expected to aid the identification of genes that control traits of importance in poultry. As the first bird genome to be sequenced it is a model for the remaining 9,600 species thought to exist today. Many of the features of avian biology and organisation of the chicken genome make it an ideal model organism for phylogenetics and embryology, along with applications in agriculture and medicine. In this report these advances are reviewed and the implications of the chicken genome in current and future applications are discussed. DOI
- Integration of microsatellite-based genetic maps for the turkey (Meleagris gallopavo)
Integration of turkey genetic maps and their associated markers is essential to increase marker density in support of map-based genetic studies. The objectives of this study were to integrate 2 microsatellite-based turkey genetic maps--the Roslin map and the University of Minnesota (UMN) map--by genotyping markers from the Roslin study on the mapping families of the UMN study. A total of 279 markers was tested, and 240 were subsequently screened for polymorphisms in the UMN/Nicholas Turkey Breeding Farms (NTBF) mapping families. Of the 240 markers, 89 were genetically informative and were used for genotyping the F2 offspring. Significant genetic linkages (log of odds > 3.0) were found for 84 markers from the Roslin study. BLASTn comparison of marker sequences with the draft assembly of the chicken genome found 263 significant matches. The combination of genetic and in silico mapping allowed for the alignment of all linkage groups of the Roslin map with those of the UMN map. With the addition of the markers from the Roslin map, 438 markers are now genetically linked in the UMN/NTBF families, and more than 1700 turkey sequences have now been assigned to likely positions in the chicken-genome sequence. DOI
- Mapping QTLs on chicken chromosome 1 for performance and carcass traits in a broiler x layer cross
With the objective of mapping quantitative trait loci (QTLs) for performance and carcass traits, an F2 chicken population was developed by crossing broiler and layer lines. A total of 2063 F2 chicks in 21 full-sib families were reared as broilers and slaughtered at 42 days of age. Seventeen performance and carcass traits were measured. Parental F(0) and F1 individuals were genotyped with 80 microsatellites from chicken chromosome 1 to select informative markers. Thirty-three informative markers were used for selective genotyping of F2 individuals with extreme phenotypes for body weight at 42 days of age (BW42). Based on the regions identified by selective genotyping, seven full-sib families (649 F2 chicks) were genotyped with 26 markers. Quantitative trait loci affecting body weight, feed intake, carcass weight, drums and thighs weight and abdominal fat weight were mapped to regions already identified in other populations. Quantitative trait loci for weights of gizzard, liver, lungs, heart and feet, as well as length of intestine, not previously described in the literature were mapped on chromosome 1. This F2 population can be used to identify novel QTLs and constitutes a new resource for studies of genes related to growth and carcass traits in poultry. DOI
- Rskalpha-actin/hIGF-1 transgenic mice with increased IGF-I in skeletal muscle and blood: impact on regeneration, denervation and muscular dystrophy
Human IGF-I was over-expressed in skeletal muscles of C57/BL6xCBA mice under the control of the rat skeletal alpha-actin gene promoter. RT-PCR verified expression of the transgene in skeletal muscle but not in the liver of 1- and 21-day old heterozygote transgenic mice. The concentration of endogenous mouse IGF-I, measured by an immunoassay which does not detect human IGF-I, was not significantly different between transgenic mice and wild-type littermates (9.5 +/- 0.8 and 13.3 +/- 1.9 ng/g in muscle; 158.3 +/- 18.6 and 132.9 +/- 33.1 ng/ml in plasma, respectively). In contrast, quantitation with antibodies to human IGF-I showed an increase in IGF-I of about 100 ng/ml in plasma and 150 ng/g in muscle of transgenic mice at 6 months of age. Transgenic males, compared to their age matched wild-type littermates, had a significantly higher body weight (38.6 +/- 0.53 g vs. 35.8 +/- 0.64 g at 6 months of age; P <0.001), dry fat-free carcass mass (5.51 +/- 0.085 vs. 5.08 +/- 0.092 g; P <0.001) and myofibrillar protein mass (1.62 +/- 0.045 vs. 1.49 +/- 0.048 g; P <0.05), although the fractional content of fat in the carcass was lower (167 +/- 7.0 vs. 197 +/- 7.7 g/kg wet weight) in transgenic animals. There was no evidence of muscle hypertrophy and no change in the proportion of slow type I myofibres in the limb muscles of Rskalpha-actin/hIGF-I transgenic mice at 3 or 6 months of age. Phenotypic changes in Rskalpha-actin/hIGF-I mice are likely to be due to systemic as well as autocrine/paracrine effects of overproduction of IGF-I due to expression of the human IGF-I transgene. The effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was tested on: (i) muscle regeneration in auto-transplanted whole muscle grafts; (ii) myofibre atrophy following sciatic nerve transection; and (iii) sarolemmal damage and myofibre necrosis in dystrophic mdx muscle. No beneficial effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was seen in these three experimental models. DOI
- 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. DOI
- Chicken genome: current status and future opportunities
The chicken genome sequence is important for several reasons. First, the chicken shared a common ancestor with mammals approximately 310 million years ago (Mya) at a phylogenetic distance not previously covered by other genome sequences. It therefore fills a gap in our knowledge and understanding of the evolution and conservation of genes, regulatory sequences, genomes, and karyotypes. The chicken is also a major source of protein in the world, with billions of birds used in meat and egg production each year. It is the first livestock species to be sequenced and so leads the way for others. The sequence and the 2.8 million genetic polymorphisms defined in a parallel project are expected to benefit agriculture and cast new light on animal domestication. Also, as the first bird to be sequenced, it is a model for the 9600 avian species thought to exist today. Many of the features of the chicken genome and its biology make it an ideal organism for studies in development and evolution, along with applications in agriculture and medicine. DOI
- Mapping of quantitative trait loci affecting organ weights and blood variables in a broiler layer cross
1. A genome scan was performed to locate genomic regions associated with traits that are known to vary in birds ( most commonly broilers) suffering from heart, lung or muscular dysfunction and for weight of the dressed carcass and some internal organs.
2. The F-2 population studied was derived from a cross between a broiler and a layer line and consisted of over 460 birds that were genotyped for 101 markers.
3. There was strong support for segregation of quantitative trait loci (QTL) for carcass and organ weights and blood variables. We identified 11 genome-wide significant QTL ( most of them for dressed carcass weight) and several genome-wide suggestive QTL.
4. The results point to some genome regions that may be associated with health-related traits and merit further study, with the final aim of identifying linked genetic markers that could be used in commercial breeding programmes to decrease the incidence of muscular and metabolic disorders in broiler populations.DOI
- Poultry genomics puts meat on the table
- Development of a cDNA array for chicken gene expression analysis
The application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes. DOI
- Second report on chicken genes and chromosomes 2005
- ChickVD: a sequence variation database for the chicken genome
Working in parallel with the efforts to sequence the chicken (Gallus gallus) genome, the Beijing Genomics Institute led an international team of scientists from China, USA, UK, Sweden, The Netherlands and Germany to map extensive DNA sequence variation throughout the chicken genome by sampling DNA from domestic breeds. Using the Red Jungle Fowl genome sequence as a reference, we identified 3.1 million non-redundant DNA sequence variants. To facilitate the application of our data to avian genetics and to provide a foundation for functional and evolutionary studies, we created the 'Chicken Variation Database' (ChickVD). A graphical MapView shows variants mapped onto the chicken genome in the context of gene annotations and other features, including genetic markers, trait loci, cDNAs, chicken orthologs of human disease genes and raw sequence traces. ChickVD also stores information on quantitative trait loci using data from collaborating institutions and public resources. Our data can be queried by search engine and homology-based BLAST searches. ChickVD is publicly accessible at http://chicken.genomics.org.cn. DOI
- Comparison of the chicken and turkey genomes reveals a higher rate of nucleotide divergence on microchromosomes than macrochromosomes
A distinctive feature of the avian genome is the large heterogeneity in the size of chromosomes, which are usually classified into a small number of macrochromosomes and numerous microchromosomes. These chromosome classes show characteristic differences in a number of interrelated features that could potentially affect the rate of sequence evolution, such as GC content, gene density, and recombination rate. We studied the effects of these factors by analyzing patterns of nucleotide substitution in two sets of chicken-turkey sequence alignments. First, in a set of 67 orthologous introns, divergence was significantly higher in microchromosomes (chromosomes 11-38; 11.7% divergence) than in both macrochromosomes (chromosomes 1-5; 9.9% divergence; P = 0.016) and intermediate-sized chromosomes (chromosomes 6-10; 9.5% divergence; P = 0.026). At least part of this difference was due to the higher incidence of CpG sites on microchromosomes. Second, using 155 orthologous coding sequences we noted a similar pattern, in which synonymous substitution rates on microchromosomes (13.1%) were significantly higher than were rates on macrochromosomes (10.3%; P = 0.024). Broadly assuming neutrality of introns and synonymous sites, or constraints on such sequences do not differ between chromosomal classes, these observations imply that microchromosomal genes are exposed to more germ line mutations than those on other chromosomes. We also find that dN/dS ratios for genes located on microchromosomes (average, 0.094) are significantly lower than those of macrochromosomes (average, 0.185; P = 0.025), suggesting that the proteins of genes on microchromosomes are under greater evolutionary constraint. DOI
- Isolation and mapping the chicken zona pellucida genes: an insight into the evolution of orthologous genes in different species
The avian oocyte is surrounded by a specialized extracellular glycoproteinaceous matrix, the perivitelline membrane, which is equivalent to the zona pellucida (ZP) in mammals and the chorion in teleosts. A number of related ZP genes encode the proteins that make up this matrix. These proteins play an important role in the sperm/egg interaction and may be involved in speciation. The human genome is known to contain ZP1, ZP2, ZP3, and ZPB genes, while a ZPAX gene has also been identified in Xenopus. The rapid evolution of these genes has confused the nomenclature and thus orthologous relationships across species. In order to clarify these homologies, we have identified ZP1, ZP2, ZPC, ZPB, and ZPAX genes in the chicken and mapped them to chromosomes 5, 14, 10, 6, and 3, respectively, establishing conserved synteny with human and mouse. The amino acid sequences of these genes were compared to the orthologous genes in human, mouse, and Xenopus, and have given us an insight into the evolution of these genes in a variety of different species. The presence of the ZPAX gene in the chicken has highlighted a pattern of probable gene loss by deletion in mouse and gene inactivation by deletion, and base substitution in human. DOI
- Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution
We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome - composed of approximately one billion base pairs of sequence and an estimated 20,000 - 23,000 genes - provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.DOI
- The chicken genome and the developmental biologist
Recently the initial draft sequence of the chicken genome was released. The reasons for sequencing the chicken were to boost research and applications in agriculture and medicine, through its use as a model of vertebrate development. In addition, the sequence of the chicken would provide an important anchor species in the phylogenetic study of genome evolution. The chicken genome project has its roots in a decade of map building by genetic and physical mapping methods. Chicken genetic markers for map building have generally depended on labour intensive screening procedures. In recent years this has all changed with the availability of over 450,000 EST sequences, a draft sequence of the entire chicken genome and a map of over 1 million SNPs. Clearly, the future for the chicken genome and developmental biology is an exciting one. Through the integration of these resources, it will be possible to solve challenging scientific questions exploiting the power of a chicken model. In this paper we review progress in chicken genomics and discuss how the new tools and information on the chicken genome can help the developmental biologists now and in the future. DOI
- Segregation of QTL for production traits in commercial meat-type chickens
This study investigated whether quantitative trait loci (QTL) identified in experimental crosses of chickens provide a short cut to the identification of QTL in commercial populations. A commercial population of broilers was targeted for chromosomal regions in which QTL for traits associated with meat production have previously been detected in extreme crosses. A three-generation design, consisting of 15 grandsires, 608 half-sib hens and over 15 000 third-generation offspring, was implemented within the existing breeding scheme of a broiler breeding company. The first two generations were typed for 52 microsatellite markers spanning regions of nine chicken chromosomes and covering a total of 730 cM, approximately one-fifth of the chicken genome. Using half-sib analyses with a multiple QTL model, linkage was studied between these regions and 17 growth and carcass traits. Out of 153 trait x region comparisons, 53 QTL exceeded the threshold for genome-wide significance while an additional 23 QTL were significant at the nominal 1% level. Many of the QTL affect the carcass proportions and feed intake, for which there are few published studies. Given intensive selection for efficient growth in broilers for more than 50 generations it is surprising that many QTL affecting these traits are still segregating. Future fine-mapping efforts could elucidate whether ancestral mutations are still segregating as a result of pleiotropic effects on fitness traits or whether this variation is due to new mutations.
- 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.
- In-silico identification of chicken immune-related genes
In order to increase the resources available in chicken, a large-scale expressed sequence tag (EST) project was recently undertaken, resulting in the addition of more than 330,000 sequences to the databases. With the sequencing of further EST collections, there are now more than 460,000 chicken EST sequences publicly available (http://www.ncbi.nlm.nih.gov/). Previous analyses of the EST data estimate that the chicken genome may contain up to 35,000 genes. However, human data indicate that there may only be around 25,000, although there may be many more transcripts than actual genes. Here we describe how we used a bioinformatics approach with this large EST collection in order to identify immune-related genes, many of which were previously unreported in the chicken. The ESTs include cytokines, chemokines, antigens, cell surface proteins, receptors and MHC-associated genes. The identification of these kinds of genes will allow further study of avian immunology and will pave the way for large-scale immune-related microarray experiments, giving new insight into functional and evolutionary studies. DOI
- Chicken genomics charts a path to the genome sequence
In this paper, the current status of chicken genomics is reviewed. This is timely given the current intense activity centred on sequencing the complete genome of this model species. The genome project is based on a decade of map building by genetic linkage and cytogenetic methods, which are now being replaced by high-resolution radiation hybrid and bacterial artificial chromosome (BAC) contig maps. Markers for map building have generally depended on labour-intensive screening procedures, but in recent years this has changed with the availability of almost 500,000 chicken expressed sequence tags (ESTs). These resources and tools will be critical in the coming months when the chicken genome sequence is being assembled (eg cross-checked with other maps) and annotated (eg gene structures based on ESTs). The future for chicken genome and biological research is an exciting one, through the integration of these resources. For example, through the proposed chicken Ensembl database, it will be possible to solve challenging scientific questions by exploiting the power of a chicken model. One area of interest is the study of developmental mechanisms and the discovery of regulatory networks throughout the genome. Another is the study of the molecular nature of quantitative genetic variation. No other animal species have been phenotyped and selected so intensively as agricultural animals and thus there is much to be learned in basic and medical biology from this research.
- On reassessment of the chicken TGFB4 gene as TGFB1
Three TGFB isoforms, TGFB1-3, are present in mammalian cells. The presence of four TGFB isoforms has been reported in avian species, though the sequence of TGFB4 was not conclusively determined. Our previously published data show that TGFB4 is actually the chicken ortholog of TGFB1. We mapped TGFB1 but not TGFB4 to linkage group E25C31 on GGA32 next to RYR1 forming a conserved segment with human chromosome HSA19q13.1-q13.2 and, therefore, being definitely the ortholog of human TGFB1. We, therefore, suggest that what was once called chicken "TGFB4" is actually TGFB1. Thus, in the evolutionary lineages of birds and mammals there are only three TGFB isoforms.
- Craniofacial development in the talpid3 chicken mutant
The talpid(3) chicken mutant has a pleiotropic phenotype including polydactyly and craniofacial abnormalities. Limb polydactyly in talpid(3) suggests a gain of Hedgehog (Hh) signaling, whereas, paradoxically, absence of midline facial structures suggests a loss of Hh function. Here we analyze the status of Shh signaling in the talpid(3) mutant head. We show that Shh expression domains are lost from the talpid(3) head--in hindbrain, midbrain, zona limitans intrathalamica, and stomodeal ectoderm--and that direct targets of Hedgehog signaling, Ptc1, Ptc2, and Gli1, are also absent even in areas associated with primary Shh expression. These data suggest that the talpid(3) mutation leads to defective activation of the Shh pathway and, furthermore, that tissue-to-tissue transduction of Shh expression in the developing head depends on Hh pathway activation. Failure to activate the Shh pathway can also explain absence of floor plate and Hnf-3beta and Netrin-1 expression in midbrain and hindbrain and absence of Fgf-8 expression in commissural plate. Other aspects of gene expression in the talpid(3) head, however, suggest misspecification, such as maintenance of floor plate-like gene expression in telencephalon. In branchial arches and lower jaw, where Shh is expressed, changes in expression of genes involved in patterning and mesodermal specification suggest both gain and loss of Hedgehog function. Thus, analysis of gene expression in talpid(3) head shows that, as in talpid(3) limb, expression of some genes is lost, while others are ectopically expressed. Unlike the limb, many head regions depend on Hh induction of a secondary domain of Shh expression, and failure of this induction in talpid(3), together with the inability to activate the Shh pathway, explain the loss-of-function head phenotype. This gene expression analysis in the talpid(3) head also confirms and extends knowledge of the importance of Shh signaling and the balance between activation and repression of Shh targets in many aspects of craniofacial morphogenesis. DOI
- 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.
- A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms
We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms (SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds (a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines--in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases. DOI
- Quantitative trait loci for meat yield and muscle distribution in a broiler layer cross
An F-2 cross of male and female chickens from broiler and layer lines was used to detect and map quantitative trait loci (QTL) affecting muscle yields and the relative weights of different muscles. Phenotypic data from 442 individuals in 30 families were analysed by within-family regression analyses using 102 microsatellite markers in 27 linkage groups with genome-wide significance thresholds. Interactions of the QTL with sex or family were unimportant and, for each trait, there was no evidence for imprinting or of multiple QTL on any chromosome. There were 30 significant QTL on 12 chromosomes (chromosomes 1 to 9, 13, 27 and Z) for 11 traits. Significant dominance effects were detected for 10 of the QTL and several were of relatively large effect. The magnitude of each QTL accounted for 3.2-5.7% of the residual phenotypic variation and the additive effects for 0.2-0.8 phenotypic standard deviations (S.D.). The results suggest that there are QTL affecting relative yield of carcass parts and of breast muscle in particular. (C) 2003 Elsevier B.V All fights reserved. DOI
- Genetic, ophthalmic, morphometric and histopathological analysis of the Retinopathy Globe Enlarged (rge) chicken
Purpose: To identify the locus responsible for rge (retinopathy globe enlarged) in chickens and further characterise the rge phenotype.
Methods: A colony of chickens carrying the rge mutation was rederived from a single heterozygous animal of the original line. The eyes of blind, heterozygous and normal birds were subjected to ophthalmic, morphometric and histopathological examination to confirm and extend published observations. DNA samples were obtained and subjected to a whole genome linkage search.
Results: From 138 classified backcross progeny, 56 birds were blind and 82 sighted. Heterozygous birds were indistinguishable from wild type, but homozygotes had sluggish or unresponsive pupils, posterior sub-capsular lens opacities and an atrophic pecten. The fundus appeared normal with no significant pigmentary disturbance, but axial length and eye weight were increased. Pathology revealed focal retinal lesions. Linkage analysis placed the rge locus in a small region of chicken chromosome 1.
Conclusions: rge is a severe recessive retinal dystrophy in chickens, with associated globe enlargement. Linkage mapping has highlighted chicken chromosome 1 in a region most probably homologous to human chromosomes 7q31-35, 21q21 or 22q12-21. Candidate disease loci include RP10 (IMPDH1) and uncharacterised Ushers (USH1E) and glaucoma (GLC1F) loci.
- Genetics. Chicken genome--science nuggets to come soon
- Quantitative trait locus detection in commercial broiler lines using candidate regions
A QTL that explained a large proportion of the phenotypic difference between broiler and layer chickens in an experimental cross was evaluated in a commercial broiler line. A three-generation design, consisting of 15 grandsires, 608 half-sib hens, and more than 50,000 third-generation offspring, was implemented within the existing breeding scheme of a broiler breeding company. Four markers from a candidate region on chicken chromosome 4 were selected for their informativeness in the grandsires and used to genotype the first two generations. Using half-sib analyses, linkage was studied between these markers and 13 growth and carcass traits. The QTL analyses confirmed the presence of significant QTL for body weight (P <0.01) and residual feed intake (P <0.05) on chicken chromosome 4. Furthermore, evidence was found for QTL affecting the relative weight of bone and muscle in the thigh. Four more markers were added to increase resolution of the QTL positions. This increased the significance of the QTL for body weight (P <0.001) and residual feed intake (P <0.01) and showed evidence (P <0.05) for additional QTL affecting carcass weight and conformation score. This study showed for the first time that a QTL that explains differences between broilers and layers was segregating in lines that have been selected for body weight over 50 generations. A possible explanation could be a pleiotropic or closely linked effect on fitness-related traits that are not part of the present study. The results demonstrate the feasibility of QTL detection and the potential for marker-assisted selection within a commercial broiler line without altering the existing breeding scheme.
- Chicken syndecan-4 (SDC4) maps to linkage group E32E47W24
- Mapping the ABCA4, IMPDH2 and TIMP3 genes in chicken
- Preliminary linkage map of the turkey (Meleagris gallopavo) based on microsatellite markers
The turkey is an agriculturally important species for which, until now, there is no published genetic linkage map based on microsatellite markers--still the markers most used in the chicken and other farm animals. In order to increase the number of markers on a turkey genetic linkage map we decided to map new microsatellite sequences obtained from a GT-enriched turkey genomic library. In different chicken populations more than 35-55% of microsatellites are polymorphic. In the turkey populations tested here, 43% of all turkey primers tested were found to be polymorphic, in both commercial and wild type turkeys. Twenty linkage groups (including the Z chromosome) containing 74 markers have been established, along with 37 other unassigned markers. This map will lay the foundations for further genetic mapping and the identification of genes and quantitative trait loci in this economically important species. Genome comparisons, based on genetic maps, with related species such as the chicken would then also be possible. All primer information, polymerase chain reaction (PCR) conditions, allele sizes and genetic linkage maps can be viewed at http://roslin.thearkdb.org/. The DNA is also available on request through the Roslin Institute.
- Analysis of the rdd locus in chicken: a model for human retinitis pigmentosa
To identify the locus responsible for the blind mutation rdd (retinal dysplasia and degeneration) in chickens and to further characterise the rdd phenotype.
- Mapping quantitative trait loci and identification of genes that control fatness in poultry
Chicken genomics has benefited from the rapid technological advances in the genomics of model organisms and man. A number of resources and approaches are now well established, in the chicken, including genetic markers and maps (both genetic and physical), quantitative trait loci mapping, comparative mapping, expressed sequence tag and bacterial artificial chromosome resources, and physical mapping. In addition, the next phase of gene discovery, functional genomics, is underway. Progress in mapping quantitative trait loci for growth and fatness traits will be discussed, as an application of these new technologies and approaches in the study of avian physiology and genetics.
- A comprehensive review on the analysis of QTL in animals
- Origin and evolution of avian microchromosomes
The origin of avian microchromosomes has long been the subject of much speculation and debate. Microchromosomes are a universal characteristic of all avian species and many reptilian karyotypes. The typical avian karyotype contains about 40 pairs of chromosomes and usually 30 pairs of small to tiny microchromosomes. This characteristic karyotype probably evolved 100-250 million years ago. Once the microchromosomes were thought to be a non-essential component of the avian genome. Recent work has shown that even though these chromosomes represent only 25% of the genome; they encode 50% of the genes. Contrary to popular belief, microchromosomes are present in a wide range of vertebrate classes, spanning 400-450 million years of evolutionary history. In this paper, comparative gene mapping between the genomes of chicken, human, mouse and zebrafish, has been used to investigate the origin and evolution of avian microchromosomes during this period. This analysis reveals evidence for four ancient syntenies conserved in fish, birds and mammals for over 400 million years. More than half, if not all, microchromosomes may represent ancestral syntenies and at least ten avian microchromosomes are the product of chromosome fission. Birds have one of the smallest genomes of any terrestrial vertebrate. This is likely to be the product of an evolutionary process that minimizes the DNA content (mostly through the number of repeats) and maximizes the recombination rate of microchromosomes. Through this process the properties (GC content, DNA and repeat content, gene density and recombination rate) of microchromosomes and macrochromosomes have diverged to create distinct chromosome types. An ancestral genome for birds likely had a small genome, low in repeats and a karyotype with microchromosomes. A "Fission-Fusion Model" of microchromosome evolution based on chromosome rearrangement and minimization of repeat content is discussed. DOI
- Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes
Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X. DOI
- Mapping of quantitative trait loci for body weight at three, six, and nine weeks of age in a broiler layer cross
An F2 chicken population was established from a cross of a broiler sire-line and an egg laying (White Leghorn) line. There were two males and two females from both lines in the base population. The F1 progeny consisted of 8 males and 32 females. Over 500 F2 offspring from five hatches were reared to slaughter at a live weight of 2 kg at 9 wk of age. Body weights at 3, 6, and 9 wk were recorded. The DNA was extracted from blood samples, and genotypes for 101 microsatellite markers were determined. Data of 466 individuals from 30 families were available for analysis. Interval mapping QTL analyses were carried out. The QTL significant at the genome wide level that affected body weight at two ages were identified on chromosomes 1, 2, 4, 7, and 8 and a QTL on Chromosome 13 influenced body weight at all three ages. Genetic effects were generally additive, and the broiler allele increased body weight in all cases. The effects for significant individual QTL accounted for between 0.2 and 1.0 phenotypic standard deviations and the sum of the additive effects accounted for approximately 0.75 of the line difference in body weight at 6 wk of age. The largest single additive effect was on chromosome 4, and the effect of substituting one copy of the gene was an increase in weight of 249 g. Interactions of the QTL with sex or family were unimportant. There was no evidence for imprinting or of two or more QTL at the same location for any of the traits.
- Chromosomal localization of the chicken and mammalian orthologues of the orphan phosphatase PHOSPHO1 gene
PHOSPHO1 is a recently identified phosphatase expressed at high levels in the chicken growth plate and which may be involved in generating inorganic phosphate for skeletal matrix mineralization. Using a degenerate RT-PCR approach a fragment of human PHOSPHO1 was cloned. This enabled the identification of the human orthologue on HSA17q21, and the mouse orthologue on a region of MMU11 that exhibits conservation of synteny with HSA17q21. Chicken PHOSPHO1 was mapped by SSCP analysis to position 44 cM on GGA27, adjacent to the HOXB@ (44 cM) and COL1A1 (36 cM) loci. Comparison of genes on GGA27 with their orthologues on the preliminary draft of the human genome identifies regions of conserved synteny equivalent to 25 Mb on HSA17q21.2-23.3 and approximately 20 Mb on GGA27 in which the gene order appears to be conserved. Mapping of the PHOSPHO1 genes to regions of HSA17q21.3, MMU11 and GGA27 that exhibit conservation of synteny provides strong evidence that they are orthologous.
- P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid
- Comparative mapping in farm animals
This paper summarises the current status of comparative mapping in farm animals. For most of the major farm animal species, a wide range of genomic tools are now available to create high-resolution genetic and physical maps of the genome. For many farm animals, the use of radiation hybrid panels and sequence data from expressed sequence tag (EST) projects has accelerated the development of high-resolution comparative maps, with human--the model species for farm animals. These tools and comparative maps are being used to map and identify the genes at the loci for simple and complex traits. The development of detailed physical maps in farm animals based on radiation hybrid panels and bacterial artificial chromosome (BAC) contigs provides a direct link between the 'information-poor' maps of farm animals and the 'information-rich' genomes of human and other model organisms.
- A comprehensive collection of chicken cDNAs
Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates. DOI
- Comparative mapping of human Chromosome 19 with the chicken shows conserved synteny and gives an insight into chromosomal evolution
Human Chromosome 19 (HSA19) is virtually completely sequenced. A complete physical contig map made up of BACs and cosmids is also available for this chromosome. It is, therefore, a rich source of information that we have used as the basis for a comparative mapping study with the chicken. Various orthologs of genes known to map to HSA19 have been mapped in the chicken. Five chicken microchromosomes (two of which were previously undefined) are seen to show conserved synteny with this chromosome, along with individual gene homologs on Chr 1 and another tiny microchromosome. Compared with the mouse, which has 12 chromosomal regions homologous to HSA19, the chicken genotype displays fewer evolutionary rearrangements. The ancestral nature of the chicken karyotype is demonstrated and may prove to be an excellent tool for studying genome evolution. DOI
- Quantitative trait loci affecting fatness in the chicken
An F-2 chicken population of 442 individuals from 30 families, obtained by crossing a broiler line with a layer line, was used for detecting and mapping Quantitative Trait Loci (QTL) affecting abdominal fat weight, skin fat weight and fat distribution. Within-family regression analyses using 102 microsatellite markers in 27 linkage groups were carried out with genome-wide significance thresholds. The QTL for abdominal fat weight were found on chromosomes 3, 7, 15 and 28; abdominal fat weight adjusted for carcass weight on chromosomes 1, 5, 7 and 28; skin and subcutaneous fat on chromosomes 3, 7 and 13; skin fat weight adjusted for carcass weight on chromosomes 3 and 28; and skin fat weight adjusted for abdominal fat weight on chromosomes 5, 7 and 15. Interactions of the QTL with sex or family were unimportant and, for each trait, there was no evidence for imprinting or of multiple QTL on any chromosome. Significant dominance effects were obtained for all but one of the significant locations for QTL affecting the weight of abdominal fat, none for skin fat and one of the three QTL affecting fat distribution. The magnitude of each QTL ranged from 3.0 to 5.2% of the residual phenotypic variation or 0.2-0.8 phenotypic standard deviations. The largest additive QTL (on chromosome 7) accounted for more than 20% of the mean weight of abdominal fat. Significant positive and negative QTL were identified from both lines.
- The ARKdb: genome databases for farmed and other animals
The ARKdb genome databases provide comprehensive public repositories for genome mapping data from farmed species and other animals (http://www.thearkdb.org) providing a resource similar in function to that offered by GDB or MGD for human or mouse genome mapping data, respectively. Because we have attempted to build a generic mapping database, the system has wide utility, particularly for those species for which development of a specific resource would be prohibitive. The ARKdb genome database model has been implemented for 10 species to date. These are pig, chicken, sheep, cattle, horse, deer, tilapia, cat, turkey and salmon. Access to the ARKdb databases is effected via the World Wide Web using the ARKdb browser and Anubis map viewer. The information stored includes details of loci, maps, experimental methods and the source references. Links to other information sources such as PubMed and EMBL/GenBank are provided. Responsibility for data entry and curation is shared amongst scientists active in genome research in the species of interest. Mirror sites in the United States are maintained in addition to the central genome server at Roslin. DOI
- Mapping of the luteinizing hormone/choriogonadotropin receptor gene (LHCGR) to chicken chromosome 3
- A consensus linkage map of the chicken genome
A consensus linkage map has been developed in the chicken that combines all of the genotyping data from the three available chicken mapping populations. Genotyping data were contributed by the laboratories that have been using the East Lansing and Compton reference populations and from the Animal Breeding and Genetics Group of the Wageningen University using the Wageningen/Euribrid population. The resulting linkage map of the chicken genome contains 1889 loci. A framework map is presented that contains 480 loci ordered on 50 linkage groups. Framework loci are defined as loci whose order relative to one another is supported by odds greater then 3. The possible positions of the remaining 1409 loci are indicated relative to these framework loci. The total map spans 3800 cM, which is considerably larger than previous estimates for the chicken genome. Furthermore, although the physical size of the chicken genome is threefold smaller then that of mammals, its genetic map is comparable in size to that of most mammals. The map contains 350 markers within expressed sequences, 235 of which represent identified genes or sequences that have significant sequence identity to known genes. This improves the contribution of the chicken linkage map to comparative gene mapping considerably and clearly shows the conservation of large syntenic regions between the human and chicken genomes. The compact physical size of the chicken genome, combined with the large size of its genetic map and the observed degree of conserved synteny, makes the chicken a valuable model organism in the genomics as well as the postgenomics era. The linkage maps, the two-point lod scores, and additional information about the loci are available at web sites in Wageningen (http://www.zod.wau.nl/vf/ research/chicken/frame_chicken.html) and East Lansing (http://poultry.mph.msu.edu/).
- Conserved synteny between the chicken Z sex chromosome and human chromosome 9 includes the male regulatory gene DMRT1: a comparative (re)view on avian sex determination
Sex-determination mechanisms in birds and mammals evolved independently for more than 300 million years. Unlike mammals, sex determination in birds operates through a ZZ/ZW sex chromosome system, in which the female is the heterogametic sex. However, the molecular mechanism remains to be elucidated. Comparative gene mapping revealed that several genes on human chromosome 9 (HSA 9) have homologs on the chicken Z chromosome (GGA Z), indicating the common ancestry of large parts of GGA Z and HSA 9. Based on chromosome homology maps, we isolated a Z-linked chicken ortholog of DMRT1, which has been implicated in XY sex reversal in humans. Its location on the avian Z and within the sex-reversal region on HSA 9p suggests that DMRT1 represents an ancestral dosage-sensitive gene for vertebrate sex-determination. Z dosage may be crucial for male sexual differentiation/determination in birds. DOI
- Human chromosomes 3 and 21 are the products of an ancestral gene arrangement that is at least 300 million years old
- First report on chicken genes and chromosomes 2000
- Erratum: A chromosome-based model for estimating the number of conserved segments between pairs of species from comparative genetic maps
- A chromosome-based model for estimating the number of conserved segments between pairs of species from comparative genetic maps
Comparative genetic maps of two species allow insights into the rearrangements of their genomes since divergence from a common ancestor. When the map details the positions of genes (or any set of orthologous DNA sequences) on chromosomes, syntenic blocks of one or more genes may be identified and used, with appropriate models, to estimate the number of chromosomal segments with conserved content conserved between species. We propose a model for the distribution of the lengths of unobserved segments on each chromosome that allows for widely differing chromosome lengths. The model uses as data either the counts of genes in a syntenic block or the distance between extreme members of a block, or both. The parameters of the proposed segment length distribution, estimated by maximum likelihood, give predictions of the number of conserved segments per chromosome. The model is applied to data from two comparative maps for the chicken, one with human and one with mouse.
- Integration of the genetic and physical maps of the chicken macrochromosomes
A large amount of genetic mapping information has been obtained in the chicken from the East Lansing, Compton and Wageningen reference populations. Physical mapping information has however, been more limited. We have mapped 14 new clones, both genetically and physically, and all 14 have been assigned to macrochromosomes. The orientation of linkage groups E01C01C11W01 (Chr 1), E06C02W02 (Chr 2), E02C03W03 (Chr 3), E05C04W04 (Chr 4), E07E34C05W05 (Chr 5), E11C10W06 (Chr 6), E45C07W07 (Chr 7) and E43C12W11 (Chr 8) has been established. Here we present integrated maps of the eight macrochromosomes and the Z chromosome of the chicken and correlate genetic with physical distances for chromosomes 1-3 and the Z sex chromosome. DOI
- Differences in gene density on chicken macrochromosomes and microchromosomes
The chicken karyotype comprises six pairs of large macrochromosomes and 33 pairs of smaller microchromosomes. Cytogenetic evidence suggests that microchromosomes may be more gene-dense than macrochromosomes. In this paper, we compare the gene densities on macrochromosomes and microchromosomes based on sequence sampling of cloned genomic DNA, and from the distribution of genes mapped by genetic linkage and physical mapping. From these different approaches we estimate that microchromosomes are twice as gene-dense as macrochromosomes and show that sequence sampling is an effective means of gene discovery in the chicken. Using this method we have also detected a conserved linkage between the genes for serotonin 1D receptor (HTR1D) and the platelet-activating factor receptor protein gene (PTAFR) on chicken chromosome 5 and human chromosome 1p34.3. Taken together with its advantages as an experimental animal, and public access to genetic and physical mapping resources, the chicken is a useful model genome for studies on the structure, function and evolution of the vertebrate genome. DOI
- Mapping the RAIDD gene of chicken (Gallus gallus): identification of a region homologous to the mouse high-growth region
- Mapping of the leptin receptor gene (LEPR) to chicken chromosome 8
- Genetic Nomenclature Guide - chick
- Turkey sperm mobility influences paternity in the context of competitive fertilization
We have devised a novel means of investigating competitive fertilization in turkeys, using microsatellite genotyping to identify male parentage. Our results demonstrate that sperm mobility is a mechanism responsible in part for paternity efficiency in turkeys. Sperm mobility is composed of several parameters in which sperm motility is a component. Differences between ejaculates in the number of sperm penetrating into a dense, insert, nontoxic solution were measured and used to classify males into high, average, or low sperm mobility phenotypes. Microsatellite genotyping was used to determine parentage of poults after equal numbers of sperm from 10 males (either high or average phenotype, n = 5, mixed with low phenotype, n = 5) were inseminated simultaneously. In a separate study, the numbers of sperm hydrolyzing the perivitelline layer of eggs were compared between hens inseminated with sperm from high-, average-, or low-phenotype males. Overall, heterospermic inseminations resulted in consistently fewer offspring produced by low-mobility phenotype males. This correlated with physiological data in which semen from the low-mobility males had reduced numbers of sperm at the fertilization site as determined by sperm hole counts in the perivitelline layer of eggs. This is the first illustration of a measurable sperm trait predictive of paternity success in a competitive fertilization trial in turkeys, a species that is predominately reproduced by artificial insemination of multiple-sire pools.
- Micro- and macrochromosome paints generated by flow cytometry and microdissection: tools for mapping the chicken genome
Despite the chicken being one of the most genetically mapped of all animals, its karyotype remains poorly defined. This is primarily due to microchromosomes that belie assignment by conventional methods. To address this problem, we have developed chromosome-specific paints using flow cytometry and microdissection. For the microchromosomes it was necessary to amplify and label DNA from single microdissected chromosomes. DOI
- Expression of transcription factor c-Rel and apoptosis occurrence in polydactylous and syndactylous limb buds of the talpid3 mutant chick embryo
The chicken proto-oncogene c-rel encodes a transcription factor of the Rel/NF-kappa B family. We have previously shown that c-rel mRNAs accumulate in different types of apoptotic cells of the chick embryo, especially in mesenchymal cells within the four cell death areas of the limb bud: the anterior and posterior necrotic zones, the opaque patch and the interdigital necrotic zones. This study aimed to further establish the involvement of c-Rel in apoptosis of the developing limb by investigating its expression in the talpid3 mutant which was originally shown to be defective in apoptosis. However, our preliminary examinations highlighted the apparent presence of apoptotic cells in talpid3 embryos. Hence, we performed a systematic study of the occurrence of apoptosis in mutant and control embryos by the TUNEL method. The results revealed that apoptosis does occur in talpid3 embryos but with altered spatial and temporal patterns. This suggests that the talpid3 mutation does not affect a gene involved in apoptosis per se but rather in the determination of the pattern of apoptosis. Neither the expression of c-Rel nor that of its I kappa B alpha inhibitor are grossly modified in talpid3 limb buds, suggesting that the talpid3 mutation does not affect any of these genes. They are mostly expressed in epidermal, endodermal and striated muscle cells in control and in talpid3 limb buds as well. C-Rel was also detected in some scarce mesenchymal cells that could be apoptotic, in both control and mutant embryos. The only slight difference between control and talpid3 limbs lies in the perichondrium which is not fully differentiated in talpid3 embryos: c-Rel and I kappa B alpha are only faintly expressed in talpid3 perichondrial cells, whereas they are both detected in control perichondrial cells. Taken together, these results suggest that c-Rel could participate in several developmental processes, especially in the differentiation of perichondrial cells, besides its already documented involvement in apoptosis and haematopoeisis. DOI
- 300 million years of conserved synteny between chicken Z and human chromosome 9
- A novel integral membrane protein is differentially expressed in the chick growth plate and maps to chromosome 1
The growth plate is a specialised region of cartilage located at the growing ends of long bones in higher vertebrates. It is responsible for longitudinal bone growth and is under the control of many local and systemic factors. The growth plate consists of an orderly arrangement of small proliferative and larger mature hypertrophic chondrocytes. This paper describes the isolation by differential display of a 988-bp cDNA fragment derived from a transcript that is more highly expressed in proliferating rather than hypertrophic chondrocytes of the chick growth plate. Using 3' RACE, a further 939 bp of cDNA sequence was obtained. The 1.9 kb sequence contains a 924-bp open reading frame encoding an unknown 308 amino acid protein. This protein has a putative transmembrane domain near its N-terminus and three dileucine motifs at its carboxy tail. This gene was expressed in all other tissues examined. A polymorphism was identified by SSCP analysis and the gene was mapped to the centromeric region of the short arm of chicken chromosome 1, close to the locus for autosomal dwarfism.
- The dynamics of chromosome evolution in birds and mammals
Comparative mapping, which compares the location of homologous genes in different species, is a powerful tool for studying genome evolution. Comparative maps suggest that rates of chromosomal change in mammals can vary from one to ten rearrangements per million years. On the basis of these rates we would expect 84 to 600 conserved segments in a chicken comparison with human or mouse. Here we build comparative maps between these species and estimate that numbers of conserved segments are in the lower part of this range. We conclude that the organization of the human genome is closer to that of the chicken than the mouse and by adding comparative mapping results from a range of vertebrates, we identify three possible phases of chromosome evolution. The relative stability of genomes such as those of the chicken and human will enable the reconstruction of maps of ancestral vertebrates. DOI
- Mapping of the prolactin gene to chicken chromosome 2
- Expression of ptc and gli genes in talpid3 suggests bifurcation in Shh pathway
talpid3 is an embryonic-lethal chicken mutation in a molecularly un-characterised autosomal gene. The recessive, pleiotropic phenotype includes polydactylous limbs with morphologically similar digits. Previous analysis established that hox-D and bmp genes, that are normally expressed posteriorly in the limb bud in response to a localised, posterior source of Sonic Hedgehog (Shh) are expressed symmetrically across the entire anteroposterior axis in talpid3 limb buds. In contrast, Shh expression itself is unaffected. Here we examine expression of patched (ptc), which encodes a component of the Shh receptor, and is probably itself a direct target of Shh signalling, to establish whether talpid3 acts in the Shh pathway. We find that ptc expression is significantly reduced in talpid3 embryos. We also demonstrate that talpid3 function is not required for Shh signal production but is required for normal response to Shh signals, implicating talpid3 in transduction of Shh signals in responding cells. Our analysis of expression of putative components of the Shh pathway, gli1, gli3 and coupTFII shows that genes regulated by Shh are either ectopically expressed or no longer responsive to Shh signals in talpid3 limbs, suggesting possible bifurcation in the Shh pathway. We also describe genetic mapping of gli1, ptc, shh and smoothened in chickens and confirm by co-segregation analysis that none of these genes correspond to talpid3.
- Parameters of the chicken genome (Gallus gallus)
As more information on the chicken genome is gathered, it is becoming increasingly more important to be able to correlate genetic and physical maps. Quantitation of the chicken karyotype is important in establishing parameters which define the genome. Here we report on the physical lengths of the chicken macrochromosomes and establish the DNA content of each, thus identifying implicitly how much of the genome is represented by the microchromosomal component. For the first time, genetic and physical data on the chicken karyotype are presented in relation to one another.
- Characterization of whole genome radiation hybrid mapping resources for non-mammalian vertebrates
Radiation hybrid panels are already available for genome mapping in human and mouse. In this study we have used two model organisms (chicken and zebrafish) to show that hybrid panels that contain a full complement of the donor genome can be generated by fusion to hamster cells. The quality of the resulting hybrids has been assessed using PCR and FISH. We confirmed the utility of our panels by establishing the percentage of donor DNA present in the hybrids. Our hybrid resources will allow inexpensive gene mapping and we expect that this technology can be transferred to many other species. Such successes are providing the basis for a new era of mapping tools, in the form of whole genome radiation hybrid panels, and are opening new possibilities for systematic genome analysis in the animal genetics community. DOI
- The chicken CP49 gene contains an extra exon compared to the human CP49 gene which identifies an important step in the evolution of the eye lens intermediate filament proteins
The gene structure for chicken CP49 gene is presented. It differs from the human CP49 gene with the presence of an extra exon in helix IB and the apparent loss of an intron, intron H. The CP49 gene localises to chromosome 2 in the chicken genome where it is flanked by homologues that map to human chromosome 10p13 (VIM) 6p24-p23 (BMP6). Two transcripts, CP49 and CP49ins, are produced from the single chicken CP49 gene. The difference is a 49-amino-acid insertion in helix IB of CP49 that is encoded by a novel exon found in the chicken CP49 gene. An extended helix IB is believed to be a characteristic of the ancestral intermediate filament protein as it is found in many invertebrate intermediate filament proteins but has been lost from all vertebrate intermediate filament proteins except the nuclear lamins. Although the intron position and length of the helix IB insert sequences in CP49ins differ to those found both in the invertebrate intermediate filament proteins and the vertebrate lamins, the CP49 gene is the first vertebrate cytoplasmic intermediate filament protein to be described with an extended helix IB. The chicken CP49 gene is also the first where differential splicing can remove such a feature. Human and bovine CP49 appear to have lost the helix IB insert sequences, and so the avian CP49 gene provides an interesting evolutionary link between the eye lens proteins and the ancestral intermediate filament protein. DOI
- Genetic mapping of the chicken prolactin receptor gene: a candidate gene for the control of broodiness
- Cloning, expression and map assignment of chicken prosaposin
Prosaposin is the precursor of four small glycoproteins, saposins A-D, that activate lysosomal sphingolipid hydrolysis. A full-length cDNA encoding prosaposin from chicken brain was isolated by PCR. The deduced amino acid sequence predicted that, similarly to human and other mammalian species studied, chicken prosaposin contains 518 residues, including four domains that correspond to saposins A-D. There was 59% identity and 76% similarity of human and chicken prosaposin amino acid sequences. The basic three-dimensional structures of these saposins is predicted to be similar on the basis of the conservation of six cysteine residues and an N-glycosylation site. Identity of amino acid sequences was higher among saposins A, B and D than in saposin C. The predicted amino acid sequence of saposin B matched exactly that of purified chicken saposin B protein. The chicken prosaposin gene was mapped to a single locus, PSAP, in chicken linkage group E11C10 and is closely linked to the ACTA2 locus. This confirms the homology between chicken and human prosaposins and defines a new conserved segment with human chromosome 10q21-q24.
- The Chicken Gene Map
- Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance
Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are 'permissive' for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein-Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use. DOI
- Two applications to facilitate the viewing of database search result files on the Macintosh
MOTIVATION: MacBOB (Macintosh BLAST Output Browser) and MacBOB Filter are two Macintosh-based applications that greatly simplify the viewing of BLAST and FASTA search results files. AVAILABILITY: The programs can be obtained via anonymous ftp from ftp.ri.bbsrc.ac.uk from the directory/pub/software/MacBOB, or via WWW from the Roslin Institute Home Page (http://www.ri.bbsrc.ac.uk/).
- Gene homologs on human chromosome 15q21-q26 and a chicken microchromosome identify a new conserved segment
The genes for insulin-like growth factor 1 receptor (IGF1R), aggrecan (AGC1), beta2-microglobulin (B2M), and an H6-related gene have been mapped to a single chicken microchromosome by genetic linkage analysis. In addition, a second H6-related gene was mapped to chicken macrochromosome 3. The Igf1r and Agc1 loci are syntenic on mouse Chr 7, together with Hmx3, an H6-like locus. This suggests that the H6-related locus, which maps to the chicken microchromosome in this study, is the homolog of mouse Hmx3. The IGF1R, AGC1, and B2M loci are located on human Chr 15, probably in the same order as found for this chicken microchromosome. This conserved segment, however, is not entirely conserved in the mouse and is split between Chr 7 (Igf1r-Agc) and 2 (B2m). This comparison also predicts that the HMX3 locus may map to the short arm of human Chr 15. The conserved segment defined by the IGF1R-AGC1-HMX3-B2M loci is approximately 21-35 Mb in length and probably covers the entire chicken microchromosome. These results suggest that a segment of human Chr 15 has been conserved as a chicken microchromosome. The significance of this result is discussed with reference to the evolution of the avian and mammalian genomes. DOI
- Introduction to veterinary genetics
- Nomenclature for naming loci, alleles, linkage groups and chromosomes to be used in poultry genome publications and databases
- Comparative genome organization of vertebrates. The First International Workshop on Comparative Genome Organization
- Expression and regulation of Cek-8, a cell to cell signalling receptor in developing chick limb buds
The Eph-related receptor tyrosine kinase gene, Cek-8, is expressed in mesenchyme at the tip of chick limb buds, with high levels of transcripts posteriorly and apically but fading out anteriorly. Expression of Cek-8 in distal mesenchyme is regulated by apical ridge- and FGF-polarising signals and retinoic acid, and is uniform across the anteroposterior axis in talpid3 mutants. These data indicate that Cek-8 expression responds to regulatory signals during limb patterning and suggest that this receptor tyrosine kinase may have a role in coordinating responses to signals in the progress zone of early buds. Later on in limb development, Cek-8 expression is associated with cell condensations that form tendons and their attachments to cartilage rudiments and then in developing feather buds.
- Genetic and physical mapping of the chicken IGF1 gene to chromosome 1 and conservation of synteny with other vertebrate genomes
The chicken insulin-like growth factor 1 gene has been assigned to the short arm of chromosome 1 near the centromere by fluorescence in situ hybridization and genetic linkage analysis. Comparison of physical and genetic linkage maps locates the centromere between the IGF1 and GAPD loci. Comparison of the genetic maps of chicken and other vertebrates reveals a highly conserved syntenic group, including the GAPD-IGF1 loci.
- Expression of the gene for transforming growth factor-beta in avian dyschondroplasia
Previous immunolocalisation studies of dyschondroplasia have indicated that there is a reduction in the number of growth plate chondrocytes containing the protein transforming growth factor beta 3 (TGF-beta 3). The reduction in TGF-beta 3 in dyschondroplasia is likely to be a direct result of a reduction in the expression of the TGF-beta 3 gene. mRNA was extracted from small (0.09 g) samples of growth cartilage from the proximal tibiotarsus of three-week-old broiler chicks. The cartilage samples contained cells from all three zones of the growth plate (proliferative, transitional and upper hypertrophic) and were collected from normal and dyschondroplastic growth plates. The dyschondroplastic growth plates were identified by an accumulation of transitional chondrocytes which were considered to be a result of a failure to differentiate to the hypertrophic phenotype. A semi-quantitative polymerase chain reaction (PCR) was used to estimate the quantity of mRNA specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for each of the three isoforms of TGF beta (TGF-beta 1, TGF-beta 2 and TGF-beta 3) in each of the cartilage samples. The levels of expression of mRNA for GAPDH, TGF-beta 1 and TGF-beta 2 were similar in the two groups, but the expression of TGF-beta 3 mRNA was significantly reduced in the samples from the dyschondroplastic growth plates. The reduction in TGF-beta 3 levels is thought to be associated with the failure of chondrocyte hypertrophy in dyschondroplasia, and provides in vivo evidence that TGF-beta 3 is part of the cascade of events associated with the differentiation of chondrocytes during endochondral ossification in the chick. DOI
- qValue--a program to calculate comparative measures of genomic reorganisation from cytogenetic and/or linkage information
A program qValue, which calculates a measure of the genomic reorganisation that has occurred between pairs of species since their divergence from a common ancestor, is described. The program takes a tab-delimited text file containing data describing the location of various genetic loci in multiple species and generates an output text file, also in tab-delimited format, that lists various parameters of genomic reorganisation between all possible pairs of species considered. This provides a useful tool for the developing field of comparative genome mapping, particularly in the study of the evolution of the vertebrate genome.
- The expression of transforming growth factor-beta by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P <0.05) and 10(-10) M (20.7%; P <0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P <0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.
- Genetic Nomenclature Guide - Chick
- Chicken genome mapping: a new era in avian genetics
More than 460 loci representing either expressed or anonymous sequences have been mapped on to the first comprehensive molecular genetic linkage map of the chicken genome. Here, we review the current status of poultry genome mapping and discuss some of the new opportunities this provides. DOI
- A SstI RFLP at the chicken transforming growth factor-beta 2 locus (TGFB2)
- Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: their relationships in the signalling cascade involved in limb patterning
The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds. DOI
- Expression of transforming growth factor-beta mRNA in chicken ovarian follicular tissue
RNA was isolated from chicken thecal tissue from the largest (F1), third largest (F3), and fifth largest (F5) preovulatory follicles, from small yolky follicles, and from granulosa tissue from F1 follicles. Transforming growth factor-beta (TGF-beta) gene expression was measured using reverse transcription polymerase chain reaction. Thecal cells from all follicle sizes expressed all three isoforms of TGF-beta. TGF-beta 1 mRNA was detected in granulosa cells at levels comparable to those seen in thecal cells. However, TGF-beta 2 and TGF-beta 3 mRNA was expressed at significantly lower levels in granulosa than in thecal tissue. This is the first demonstration of TGF-beta gene expression in the ovary of a nonmammalian species. The similarities between mammalian and avian TGF-beta gene expression are remarkable, especially in light of the distinctive patterns of avian follicular development and the differing steroidogenic capacities of ovarian cell types of the two classes of vertebrates. DOI
- The chicken transforming growth factor-beta 3 gene: genomic structure, transcriptional analysis, and chromosomal location
In this paper, we report the isolation, characterization, and mapping of the chicken transforming growth factor-beta 3 (TGF-beta 3) gene. The gene contains seven exons and six introns spanning 16-kb of the chicken genome. A comparison of the 5'-flanking regions of human and chicken TGF-beta 3 genes reveals two regions of sequence conservation. The first contains ATF/CRE and TBP/TATA sequence motifs within an 87-bp region. The second is a 162-bp region with no known sequence motifs. Identification of transcription start sites using chicken RNA isolated from various embryonic and adult tissues reveals two sites of initiation, P1 and P2, which map to these two conserved regions. Comparison of 3'-flanking regions of chicken and mammalian TGF-beta 3 genes also revealed conserved sequences. The most significant homologies were found in the 3'-most end of the transcribed region. DNA sequence analysis of chicken TGF-beta 3 cDNAs isolated by 3'-RACE revealed multiple polyadenylation sites unusually distant from a poly(A) signal motif. A Msc I restriction fragment length polymorphism (RFLP) marker was used to map the TGFB3 locus to linkage group E7 on the East Lansing reference backcross. Linkage to the TH locus showed that the TGFB3 locus was physically located on chicken chromosome 5. DOI
- Manipulation of the avian genome
- Dinucleotide repeat polymorphisms at the chicken ACTAB locus
- Mapping the chicken GAPD locus using heteroduplex polymorphisms
- Dinucleotide repeat polymorphisms at the chicken ACTA2 locus
- Mapping the chicken ACTA2 locus using heteroduplex polymorphisms
- Molecular cloning and expression of bone morphogenetic protein-7 in the chick epiphyseal growth plate
Longitudinal bone growth occurs in the epiphyseal growth plate and is regulated by a network of paracrine and autocrine interactions. Bone morphogenetic proteins (BMPs) are a family of growth factors whose potent osteogenic properties suggest that they may play an important role within this network, but direct evidence for this is lacking. To address this question, a cDNA encoding chick BMP-7 was cloned from a chick embryo cDNA library. Sequence homology and evolutionary arguments strongly suggested that we had cloned the chicken BMP-7 homologue. Using a reverse transcription-PCR assay, BMP-7 expression was readily detected in bone, growth plate cartilage, brain and heart, and was just detectable in liver, skeletal muscle and adipose tissue. In contrast to the pattern of BMP-7 expression in the rat and mouse, no BMP-7 expression was detected in the chick kidney. In situ hybridization was used to locate the site of BMP-7 expression more precisely within the growth plate. BMP-7 expression was confined to hypertrophic chondrocytes adjacent to and at the tips of the metaphyseal vessels. No expression was detected in the reserve zone or in proliferating chondrocytes. These results point to a specific role for BMP-7 in the growth plate, possibly in osteoblast activation or as a chemotactic agent for the metaphyseal vessels.
- A MspI RFLP at the chicken tyrosine hydroxylase locus (TH)
- Evolution of the transforming growth factor-beta superfamily
Transforming growth factor beta 1 (TGF-beta 1) is the prototype of an increasingly complex superfamily of growth and differentiation factors. To date, a total of 74 TGF-beta-like sequences have been published, probably representing 23 distinct genes. These sequences were obtained from mammalian, avian, amphibian and insect species, thus emphasising the ancient nature of the TGF-beta superfamily peptides. This article summarises current hypotheses concerning the evolutionary history of this protein superfamily, based on the molecular phylogeny of the published sequences. Comparison of the deduced amino acid sequences leads to the definition of five main groups within the superfamily (TGF-beta, Bone Morphogenetic Proteins [BMP], Anti-Müllerian Hormone [AMH], Inhibin alpha [INH alpha] and GDF-9) and six subgroups within the BMPs (60A, Decapentaplegic [dpp], Vg1, BMP-3, Inhibin beta [INH beta A/B] and nodal). This classification predicts possible phylogenetic and functional relationships among these proteins.
- Tissue specific expression of an alpha-skeletal actin-lacZ fusion gene during development in transgenic mice
Transgenic mice carrying a chimaeric transgene containing 730 bp of the 5'-flanking sequences and the entire first intron of the rat alpha-skeletal actin gene fused to the lacZ reporter gene have been produced by microinjection. The lacZ reporter gene was used to verify the suitability of using the rat alpha-actin promoter elements to target expression of genes of agricultural and therapeutic value exclusively to skeletal and heart muscle cells and fibres of transgenic mice. Expression of the transgene indicates a tightly regulated developmental and muscle specific control of the rat alpha-skeletal actin gene, making it a useful promoter for gene targeting to muscle tissues. The cells destined to form muscle tissues in these transgenic mice are readily visualized in intact embryos by staining for beta-galactosidase activity, making them a suitable animal model for studying the origin and development of skeletal and cardiac muscle tissues.
- Insulin-like growth factor-I in the ovary of the laying hen: gene expression and biological actions on granulosa and thecal cells
Concentrations of insulin-like growth factor-I (IGF-I) were measured in granulosa and thecal tissue dissected from the three largest follicles in the ovaries of laying hens. The higher concentration was found in extracts of granulosa (0.82 +/- 0.01 pmol/g wet wt) and theca (0.36 +/- 0.02), both of which were greater than that in liver extracts (0.25 +/- 0.01). RNA was extracted from these tissues, and by using reverse transcription and the polymerase chain reaction with primers specific for chicken IGF-I, both granulosa and thecal tissue were shown to express chicken IGF-I mRNA. Granulosa and thecal cell cultures were established and used to measure IGF binding sites and the response to exogenous IGF peptides in terms of DNA synthesis. Both cell types bound [125I]IGF-I, which was displaced by IGF-I, IGF-II, and insulin in descending order of potency, characteristic of a type-I IGF receptor. Treatment of granulosa and thecal cell cultures with IGF-I resulted in a dose-dependent increase in [3H]thymidine incorporation into DNA by both cell types. LH, but not FSH, stimulated DNA synthesis in cultured granulosa cells but not in cultured thecal cells. This effect was enhanced in granulosa cells by the addition of IGF-I to the culture medium. These data are consistent with an autocrine or paracrine role for IGF-I within the developing ovarian follicle of the domestic hen. DOI
- Evolutionary grouping of the transforming growth factor-beta superfamily
TGF beta 1 is the prototype of a superfamily of differentiation factors with at least 18 distinct members. This classification is based on amino acid homology in the C-terminus of these polypeptides. The rates of amino acid substitution for several family members were estimated from a comparison of homologous sequences derived from different species. These rates were approximately constant for any given protein, but values varied greatly between different groups. This variation is a reflection of the great functional diversity found within the TGF beta superfamily. Maximum parsimony analysis allowed us to classify members of the TGF beta superfamily into five main groups (INH alpha, MIS, TGF beta s, INH beta s and BMPs) and various subgroups. This classification predicts possible phylogenetic relationships among these proteins. In the future, it is hoped that this method of classification will be adopted by all our colleagues, as an aid in deciding whether newly discovered proteins are the product of duplicated or homologous genes. This would suggest proteins with either similar or identical functions.
- Estimation of restriction maps with known site order using a generalized linear model
A generalized linear model with Gamma errors is used to estimate the coordinates of a restriction map when the site order is known. This can be conveniently programmed in a wide range of statistical packages (e.g. Genstat 5, Minitab, SAS), and gives maximum likelihood estimates with their associated optimal properties. Regression diagnostics allow the checking of assumptions and help to identify mis-specified, influential or discordant fragment lengths. A specific diagnostic for identifying fragment lengths causing reversal of restriction site order is derived. Exact 'fragment' lengths from DNA sequencing can be conveniently included in an approximate manner by giving them a larger weight than observed restriction fragment lengths. Two examples and the Genstat 5 codes used in their analysis are presented.
- Correction: a new interpretation of a chicken transforming growth factor-beta 4 complementary DNA
- Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells
We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.
- Evolutionary origins of the transforming growth factor-beta gene family
A molecular phylogeny for the transforming growth factor-beta (TGF-beta) gene family based on a comparison of nucleotide sequences is proposed. A phylogenetic tree constructed from these sequences shows that the family evolved from a common ancestral gene that came into existence at about the time of arthropod and chordate divergence. This model suggests that the present day TGF-beta gene family consists of four members: TGF-beta 1 (= TGF-beta 4), TGF-beta 2, TGF-beta 3, and TGF-beta 5. The molecular phylogeny and Southern hybridization data also suggest that the proteins for mammalian TGF-beta 1 and chicken TGF-beta 4 are the products of homologous rather than duplicated genes. If the gene duplication event that produced the ancestral gene for TGF-beta 1 occurred before the divergence of birds and mammals, then sufficient time would have elapsed to generate these quite distinct avian and mammalian TGF-beta 1 proteins. Therefore, the TGF-beta family contains four distinct proteins, TGF-beta 1, 2, 3, and 5.
- Tissue specificity of renin promoter activity and regulation in mice
Certain mouse strains (e.g., DBA/2) contain two renin genes (termed Ren-1 and Ren-2) and express higher renin levels in nonkidney tissues than strains with a single renin gene. The 5'-flanking regions of the Ren-1 and Ren-2 genes contain several TATA boxes preceding putative transcriptional start sites. These initiators are termed P1a, P1, P2 (from 5' to 3'), and their function (with the exception of P2) is largely unknown. In this study, we mapped the renin transcriptional start sites in renal and extrarenal tissues [adrenal, brain, testis, heart, and submandibular gland (SMG)] and examined the effect of adenosine 3',5'-cyclic monophosphate (cAMP) on tissue specific promoter usage. Our results showed that, in the unstimulated state, P2 (the predicted initiator) is active in all DBA/2 mouse tissues. Additional transcriptional start sites were detected in the adrenal and testis (originated by P1a and P2) and the SMG (originated by P1a, P1, and P2). The administration of 8-bromoadenosine 3',5'-cyclic monophosphate led to selective stimulation of P1a in the adrenal but did not affect the selective usage of initiation sites in other organs. A locus-specific ddNTP primer extension assay was used to verify which renin gene is induced by cAMP. Results indicated that both Ren-1 and Ren-2 responded to cAMP treatment in identical fashion. Taken together, these data indicate that more than one form of renin transcript is present in several mouse tissues. There is tissue specificity in promoter usage in the unstimulated state and in response to cAMP.
- Cell-dependent posttranslational processing and secretion of recombinant mouse renin-2
In the DBA/2 mouse submandibular gland (SMG), renin is predominantly the expression product of the renin gene Ren-2d. Prorenin is synthesized and rapidly converted to a constitutively secreted single-chain intermediate, which is then processed to and stored as the mature two-chain (2C) form, which is released by regulated secretion. To evaluate whether the mode of renin processing is cell dependent, renin (Ren-2d) complementary DNA was stably integrated in the genome of Chinese hamster ovary (CHO) cells and a mouse pituitary cell line (AtT-20) by transfection, and renin processing and secretion were examined. Transfected CHO cells secreted exclusively prorenin, whereas transfected AtT-20 cells secreted both prorenin and active renin. AtT-20 cells processed prorenin to the single-chain polypeptide (1C-renin) that was the main storage form and was not further processed to the 2C form of correct size, whose site of generation or function is uncertain at this time. In addition, the conversion of prorenin to 1C-renin was much slower in AtT-20 cells than in the SMG. Thus the patterns of renin biosynthesis and secretion in AtT-20 cells show major differences when compared with these processes in the native SMG, suggesting that cell-dependent characteristics, e.g., the presence of specific processing enzymes, are important factors influencing mouse renin processing.
- Molecular cloning and primary structure of the chicken transforming growth factor-beta 2 gene
The chicken transforming growth factor-beta 2 (TGF-beta 2) gene and its flanking regions were cloned and characterized. The gene contains 7 exons and 6 introns spanning about 70 kb. Primer extension analysis identified one major and two minor starts of transcription. A comparison of the 5'-flanking regions for human and chicken TGF-beta 2 genes revealed limited sequence homology around the start of transcription, including conserved TATA-box, CRE, and AP-2 sequence motifs. A species comparison of the 5' untranslated region did not reveal any sequence homology beyond the coding region. In contrast, the 3' untranslated region was highly conserved, suggesting that this region may play an important role in the expression of the TGF-beta 2 gene.
- Comparative analysis of human and chicken transforming growth factor-beta 2 and -beta 3 promoters
The promoters for chicken transforming growth factor-beta 2 (TGF-beta 2) and TGF-beta 3 were cloned and sequenced to study the regulation of these genes. The promoters are GC-rich and lie within CpG islands. Several putative DNA regulatory sequence motifs were identified in the 5'-flanking regions, including matches to particular recognition sequences for several nuclear factors found in other genes. A comparison of chicken and human TGF-beta 2 promoters revealed a 111 bp conserved sequence surrounding the major transcription start site. Two regions of sequence homology were detected in the 5'-flanking regions of chicken and human TGF-beta 3 genes: an 86 bp sequence surrounding the major transcription start and a 156 bp sequence in the 5'-untranslated region. No DNA sequence homology was detected between TGF-beta 1, -beta 2 or -beta 3 promoters. The conserved region near the major transcription start sites in both the TGF-beta 2 and TGF-beta 3 genes, however, does show some structural homology; both promoters contain short conserved sequences that resemble TATA box, cyclic AMP-responsive element and AP-2 sequence motifs, cis-acting elements we believe may be important for promoter activity.
- A lack of genetic linkage of renin gene restriction fragment length polymorphisms with human hypertension
Because renin is an important enzyme in blood pressure regulation, we studied the possibility that an alteration in the structure of the human renin gene is genetically linked to human essential hypertension or associated with levels of plasma renin activity or blood pressure. By using specific DNA probes, we have identified four polymorphisms in the human renin gene with the restriction enzymes Taq I, HindIII, Bgl I, and Bgl II. The gene location of all of these polymorphisms except for the Bgl II polymorphism has been determined, and their frequencies were initially estimated in a population of 50 random subjects. To test the clinical significance of these polymorphisms, we studied 68 persons from a large Utah pedigree with a high incidence of hypertension. Among nine relatives with hypertension, genetic linkage without recombination was ruled out by observing several obligate recombinants. We also found no significant association of the restriction fragment length polymorphisms with quantitative measurements of sitting or standing, systolic or diastolic blood pressures, or plasma renin activity in 59 untreated members of this pedigree. Although we found no genetic linkage in this set of study subjects, the characterization of the restriction fragment length polymorphisms for the renin gene may be useful in future studies of other selected pedigrees for the presence of one or more of these to be a genetic marker in hypertension.
- Negative control elements and cAMP responsive sequences in the tissue-specific expression of mouse renin genes
The 5' flanking regions of the mouse renin genes (Ren1d and Ren2d) contain putative negative control and cAMP responsive elements. Sequence analysis shows additionally that these putative control elements in the Ren2d gene are interrupted by a 160-base-pair insertion. To document the functions of these elements, we isolated these regions and fused them to the reporter gene chloramphenicol acetyltransferase (CAT), which was linked upstream to a thymidine kinase (TK) promoter (pUTKAT1). The chimeric constructs were transfected into mouse pituitary tumor AtT-20 and human choriocarcinoma JEG-3 cells. At the basal unstimulated condition, Ren1d 5' flanking sequence in the sense orientation inhibited basal CAT expression from the TK promoter of pUTKAT1, whereas the same sequence in the antisense orientation did not. The 5' flanking region of Ren2d had no inhibitory effect on basal CAT expression. These data demonstrate that the negative control element is functional in Ren1d but is nonfunctional in Ren2d, suggesting that the 160-base-pair insertion in Ren2d interferes with the function of the negative control elements. In response to 8-bromo-cAMP, both renin genes increased transcription 3-fold, suggesting a functional cis action of the cAMP responsive element in both genes. These data may be important in the understanding of the regulation of the tissue-specific expression of mouse renin genes. DOI
- Identification of negative and positive regulatory elements in the human renin gene
Renin gene expression is tissue-specific and under complex hormonal control. To investigate which DNA elements are involved in the control of human renin gene expression, we performed transient DNA transfer experiments with renin-chloramphenicol acetyltransferase fusions. We have mapped a complex arrangement of positive and negative control sequences in the 5' flanking region of the human renin gene. One positive control element is active in either orientation and defines a renin gene enhancer. The negative element is also active in either orientation and defines a renin gene silencer. Mapping in the same region as the silencer is a cAMP-responsive element, a sequence conserved in mouse, rat, and human renin genes.
- The nucleotide sequence of a mouse renin-encoding gene, Ren-1d, and its upstream region
The renin-encoding genes have been cloned from high (Ren-1d, Ren-2d)- and low (Ren-1c)-renin-producing strains of mice (DBA/2J and C57BL/10). Each of the genes is approx. 9.6 kb in length and consists of nine exons and eight introns. The entire nucleotide sequence of the Ren-1d gene has been determined and the 5'-flanking regions of the three genes, Ren-1c, Ren-1d and Ren-2d, have been compared. The significance of several potential regulatory signals found in the DNA is discussed. DOI
- Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kappaB-like transcription factor
We have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). We found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, we placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by R. Sen and D. Baltimore (Cell 46:705-716, 1986) as the binding site for the nuclear factor NF kappa B. In a cotransfection competition experiment, we could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF kappa B-binding sequence. The same long-terminal-repeat DNA containing mutant NF kappa B-binding sequences (G. Nabel and D. Baltimore, Nature [London] 326:711-713, 1987) did not affect CAT expression, which suggested that binding by an NF kappa B-like factor is required for increased SAA transcription.
- Molecular biology of the renin-angiotensin system
This paper reviews the molecular biology of the renin-angiotensin system. The renin gene structure is analyzed in detail, including an examination of the putative regulatory regions. The combined action of these regulatory sequences would result in the complex, tissue-specific expression and regulation observed in vivo. The expression of the tissue renin-angiotensin systems, which may have important physiological functions, is also described. In addition, the pathway of renin biosynthesis and secretion is reviewed. This includes speculation on the fate of circulating prorenin and the physiological role of multiple renin forms and secretory pathways. The molecular approaches described in this paper have greatly advanced our knowledge of the biology of the renin-angiotensin system. Future studies using these and other approaches should provide further insight into this complex system.
- Functional human renin promoter in transfected cells: evidence for cell-specific expression
Expression of the human renin gene is regulated in a tissue-specific manner, but study of this regulation has been limited by a lack of suitable cell lines that simulate endogenous control. In order to characterize the regulation of renin gene expression, the 5' flanking region (892 base pairs) from the human renin gene was linked to the chloramphenicol acetyltransferase gene and was introduced into multiple human cell lines by calcium phosphate precipitation or electroporation methods to assess transcriptional control. The human renin promoter was active when transfected into cultured human choriocarcinoma cells (JEG-3) and rat vascular smooth muscle cells, but it was not active in many other cloned cell types. These results suggest that selective cell lines contain the specific trans-acting factors necessary for human renin gene expression, and support the concept of cell-specific expression of this gene.
- Identification of potential regulatory regions in the renin gene
- Mapping of the human renin transcription start site: evidence for a single functional promoter
The 5' flanking region of the human renin gene contains two putative promoter sequences (TATA boxes), named P1 and P2. These are located in positions -77 to -71 and -29 to -23 respectively, each followed by a possible translational start site (AUG). In order to identify whether these sequences are functional in the kidney, we employed the RNAse protection assay. Total RNA extracted from human kidney cortex was hybridized with labeled cRNA probes complementary to the 5' flanking region of the human renin gene. After digestion with RNAse, protected hybrids were identified by electrophoresis on polyacrylamide/urea gels and autoradiography. Results showed that only P2 is active in human kidney resulting in a single transcript. This suggests that human kidney renin is synthesized as a single preproform.
- Novel and enhanced IL-1 gene expression in autoimmune mice with lupus
IL-1 is a pleiotropic factor encoded for by at least two genes, alpha and beta, and capable of eliciting a broad set of immunologic and inflammatory events. MRL/MP-lpr (MRL-lpr) mice are an appealing model for studies of renal injury inasmuch as disease in this strain is spontaneous, rapid, predictable, and regulated by the lpr gene. Infiltration of macrophages and the proliferation of the glomerular mesangial cells are prominent features of renal disease. Because both mesangial cells and macrophages can synthesize IL-1, the purpose of this study was to determine whether enhanced IL-1 gene expression is associated with lupus nephritis in the MRL-lpr mouse model. Glomerular macrophages, abundant in the kidneys of MRL-lpr mice but rarely present in the kidney of congenic MRL/MP-++(MRL-++) mice, were isolated and cultured and found to express a 10-fold increase in both IL-1 alpha and IL-1 beta mRNA transcripts as compared with MRL-++ and MRL-lpr mesangial cells. IL-1 alpha was not detected in the total RNA extracted from freshly excised kidney, whereas IL-1 beta transcripts were detected in both the renal cortex of MRL-lpr as well as MRL-++ animals. A previously undetected truncated 1200 nucleotide IL-1 beta transcript together with the conventional 1600 nucleotide IL-1 beta transcript was found in kidneys from MRL-lpr and was abundantly expressed in glomeruli of MRL-lpr mice with lupus nephritis. Isolated glomeruli from MRL-lpr mice with nephritis produce IL-1, whereas in normal glomeruli from MRL-++ and C3H/FeJ mice this cytokine was not detected. Glomerular macrophages and mesangial cells cultured from MRL-lpr mice with nephritis both secrete IL-1. These studies indicate that IL-1 beta gene expression and IL-1 protein are increased in the kidneys of autoimmune mice with lupus nephritis and is generated, at least in part, by glomerular macrophages. We speculate that an alteration in IL-1 beta gene expression may be responsible for causing a cascade of events leading to acute and chronic renal injury.
- Increased tumor necrosis factor and IL-1 beta gene expression in the kidneys of mice with lupus nephritis
Because TNF and IL-1 can initiate immunologic and inflammatory events alone or synergistically, a local increase in the levels of one or both of these cytokines in vivo may cause irreparable tissue damage. The purpose of this study was to evaluate local TNF and IL-1 beta gene expression in vivo in the kidneys of MRL-Ipr mice with autoimmune lupus nephritis. TNF mRNA was detected in the renal cortex of MRL-Ipr mice but was not present in the cortex of normal congenic MRL-++ or C3H/FeJ mice. MRL-Ipr mice with lupus nephritis expressed higher amounts of TNF mRNA compared with MRL-Ipr mice prior to disease. In addition, freshly isolated, unstimulated glomeruli from MRL-Ipr mice with nephritis were found to secrete detectable levels of TNF, whereas glomeruli from MRL-++ mice did not. IL-1 beta mRNA, present in the renal cortex of C3H/FeJ, MRL-++, and young MRL-Ipr mice with normal kidneys, was also more abundantly expressed in MRL-Ipr mice with nephritis. Cultured macrophages from glomeruli of mice with nephritis were found to express TNF and IL-1 beta mRNA and product. These macrophages are prominent only in MRL-Ipr mice with renal disease and are the likely source of increased gene expression for both cytokines.
- A retroviral provirus closely associated with the Ren-2 gene of DBA/2 mice
We have determined the entire nucleotide sequence of an intra-cisternal A particle (IAP) genome, associated with the Ren-2 gene of DBA/2 mice. This genome (MIARN) displays features common to other IAP retroviral-like genomes. Long terminal repeats (LTRs) are approximately 430 base pairs (bp) in length and show typical retroviral U3-R-U5 organisation, though the R-region, at 120 bp, is much larger than the average IAP. This difference probably arose by the amplification of a pyrimidine-rich sequence, by a slippage-mispairing mechanism. Flanking the 5' LTR is a sequence complementary to a phenylalanine tRNA, strongly conserved in all rodent IAP genomes and probably required to prime the initiation of (-) strand synthesis. Flanking the 3' LTR, is a purine-rich sequence probably required for (+) strand synthesis. The tRNA binding site (TBS) is flanked by six tandem copies of a sequence homologous to the TBS. The relationship of the MIARN element to other IAP genomes and the significance of its association with the highly expressed Ren-2 is discussed. DOI
- The cis-specificity of the Q-gene product of bacteriophage lambda
A trp/lacW205 substitution, fused to the late region of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q+ and Q- phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ in only significantly active when supplied in cis to its site of action.
- Transcriptional termination sites in the b2 region of bacteriophage lambda that are unresponsive to antitermination
A bacteriophage lambda cloning vector carrying the trp/lacW205 substitution is described. The vector facilitates the fusion in vitro of genetic control signals to the lacZ structural gene of Escherichia coli. This system was used to define transcriptional termination sites in the lambda b2 region. This region contains termination sites that are unresponsive to the lambda antiterminating proteins pQ and pN. DOI
- Molecular cloning of two distinct renin genes from the DBA/2 mouse
We report the molecular cloning of cDNA copies of DBA/2 mouse submaxillary gland (SMG) renin mRNA, which were used to probe Southern transfers of mouse genomic DNA. The results suggested either that there is a single renin gene containing a large intron in that part of the gene corresponding to the probe, or that there are two distinct renin genes. We have shown that the latter is the case by cloning and isolating two similar but distinct renin genes from DBA/2 mouse DNA. Restriction maps of the regions containing the two renin genes are presented, together with nucleotide sequence data locating a complete exon coding for amino acids 268-315 of mouse SMG renin.
- Deciphering chicken fatness trait with integrative genetic and genomic approaches.
- Leukocyte protein Trojan, as a candidate for apoptotic regulatory role
- Trojan, a Possible Regulator of Apoptosis, Belongs to a Novel Protein Family
- 3D analysis of gene expression during limb development in the Chick
Shh signalling in the polarizing region at the posterior edge of the limb bud is pivotal in controlling digit number and pattern in the developing limb. Using microarrays we have compared gene expression levels between anterior and posterior thirds of wing buds from wild-type and talpid mutant chicken embryos, in which both Gli activator and repressor function fail, and identified 1070 differentially expressed genes. These were put into 16 clusters, one of which contains Hoxd13, a gene known to be involved in digit formation. Using optical projection tomography (OPT) we have performed a 3D analysis of the expression patterns of genes in the Hoxd13-like cluster. Through this analysis we aim to identify novel genes that are modulated as a consequence of Shh signaling and therefore play a role in chicken wing development. The 3D gene expression patterns were then mapped onto a digital reference limb using AMIRA software and computational analysis was used to compare the 3D spatial patterns of gene expression, which may possibly identify syn-expression groups suggesting potential functional relationships in limb development. DOI
- Analysis of quatitative trait loci for age and weight at the onset of lay in a broiler x layer cross
- Patterns of selective constraint on bovine toll-like receptor genes
Toll-like receptors (TLRs) are pattern-recognition molecules expressed by cells of the innate immune system and cells at the interface between the host and environment. They recognise the presence of pathogens and signal alarm to the host immune system. Polymorphisms in these molecules may explain a proportion of the variability in disease resistance seen in most species from Drosophila where they were first discovered to mammals such as livestock and man. Thus TLRs have the potential to be candidates for selectable markers for disease resistance that could be exploited by the breeding industry. Our consortium aims to directly link single nucleotide polymorphisms (SNPs) with functional consequences in the innate immune cells, dendritic cells. We are undertaking a SNP discovery approach for all 10 TLRs in cattle in relevant breeds by direct sequencing. By combining this with sequencing of TLRs in related bovid Species including sheep, goats and ibex and other more exotic Species, we aim to undertake a phylogenetic analysis to identify signatures of selection on the nucleotide sequences of bovid TLRs. We will then test their functional relevance by transfecting polymorphic variants and measuring the down-stream effects of ligand binding. We will present phylogenetic evidence that the evolutionary pressures on bovine TLR2 and TLR4 appear to have differed and will speculate on the possible reasons for this, and the consequences for the host. Our overall approach aims to identify SNPs with the most likely potential as markers for selecting animals with enhanced disease resistance. DOI
- Molecular immunotyping of lungs in naive and vaccinated chickens early after pulmonary avian influenxa A (H9N2) virus infection
In a respiratory infection model we study immune reactions in chickens infected with the low pathogenic avian influenza A H9N2 virus strain. For molecular immune response profiling we employed a recently developed chicken immuno-microarray containing approximately 5000 cDNA elements in duplicate (Smith et al., 2006 J. Smith, D. Speed, P.M. Hocking, R.T. Talbot, W.G. Degen, V.E. Schijns, E.J. Glass and D.W. Burt, Development of a chicken 5 K microarray targeted towards immune function, BMC Genomics 7 (2006), p. 49. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (0)Smith et al., 2006). In a first experiment, broiler-type chickens were either mock-immunized (referred to as non-immune), vaccinated with inactivated viral antigen only (immune), or with viral antigen in distinct Th1 or Th2 polarizing immunopotentiators (immune potentiated). Three weeks after vaccination all animals were given a respiratory infection with H9N2 via the oculo-nasal-intratracheal route. From these animals lungs and spleens were removed, RNA was isolated and subsequently used for microarray analysis. In general, we noted less host gene expression in immune potentiated, i.e. adjuvanted, birds when compared to non-immune, infected chickens. Immune potentiated birds showed reduced innate responses, mainly restricted to heat shock proteins, which, likely, are sufficient to control the pulmonary infection together with induced antibodies and T cells. Evaluation of the microarray data suggested gene pathways unique to or common amongst the differently immune groups (Degen et al., 2006). In subsequent experiments we have analyzed, and still are analyzing, the influence of age and type (broiler versus layer) of the chickens, challenge route (oculo-nasal-intratracheal versus spray), and the use of other immune response polarizing immunopotentiators on the host gene expression profiles by using the microarray and quantitative RT-PCR (Q-RT-PCR), as well as classical immune parameters. In this presentation we will highlight our findings. The data collected in this natural influenza A virus target Species, i.e. chicken, are of interest for the design of future human pandemic and seasonal vaccines containing immune response modifying immunopotentiators. DOI
- Novel approaches to enhance disease resistance in ruminants?-Breeding for geographically important TLR SNPs: The ruminant TLR consortium
Opportunistic infections resulting from intensive husbandry of livestock have become one of the major problems in modern animal production. As bacterial resistance to antibiotics is expected to escalate, novel approaches to disease prevention will need to be established. One approach includes breeding for disease resistance by selecting the 'fittest' innate immune system. The innate immune system recognises pathogens by means of pattern recognition receptors, such as Toll-like receptors (TLRs). These interact with various microbial components and induce a specific innate immune response. Several polymorphisms in TLR genes have been described for the human and murine system that influence the abilities of affected TLRs to recognise pathogen-derived molecules-rendering individuals more or less susceptible to infection. The first nucleotide polymorphisms (SNPs) in ruminant TLRs were characterised in bovine TLR4, the receptor for gram-negative bacteria. Analysis of codon-based models of selection identifies many sites in TLR4 under positive selection in the region 261-375 residues. This region is located in the middle of the extracellular domain between clusters of leucine-rich-repeats (LRRs) and is predicted to be the ligand binding domain. We would expect a higher frequency of non-synonymous SNPs to map to his domain. Indeed, the first non-synonymous SNPs map to this region. We have cloned and mapped ten bovine TLRs, and are currently in the progress of functionally characterising these TLRs. In addition, TLR genes from sheep are also being cloned and characterised. TLRs from different cattle and sheep breeds are currently been analysed for the presence of synonymous and non-synonymous SNPs. Our data suggest a heterogeneity in extracellular regions of TLR genes, which may be advantageous to promote a specific disease resistance, and represent a new approach to select disease resistance-based on a geographical distribution of TLR SNPs. Breeding of ruminants with TLRs that confer a 'fitter' innate immune system resulting in disease resistance could play a major role in the future of the farming industry both in the UK and worldwide. DOI
- ChickATLAS: A three-dimensional atlas of gene expression during chick development
The control of gene expression is important in embryonic development. Quantitative assays, such as microarrays, provide gene expression levels for large numbers of genes, but with low spatial resolution. In contrast, in situ methods, such as in situ hybridisation and immunohistochemistry, provide high spatial resolution, but poorer quantification and low genome coverage. Furthermore, crucial 3D data is lost with planar samples. Optical projection tomography (OPT) can capture the full 3D expression pattern in a whole embryo at a reasonably high resolution and at moderately high throughput. A large database containing spatio-temporal patterns of expression for the mouse (EMAGE) has been created and is proving to be a valuable resource. Recently, the chick has become an important model for spatially and temporally controlled gain- and loss-of-function approaches. To date, a well-established gene expression database for the chick does not exist. Thus, the aim of this project is to produce a 3D anatomical atlas and ontology of the chick embryo with a database of gene expression patterns during chick development. This involves a major collaboration between groups in Edinburgh, Dublin, Bath and London. This database will be based on EMAGE and cross-referenced to the mouse through orthologous gene pairs ( http://www.emouseatlas.org/testemage/home.php ). Throughout this project, the data and framework will be used to identify groups of genes that are co-expressed in important signalling regions. Conservation of these genes will be examined in the chick and mouse. This database will be made publicly available ( http://www.echickatlas.org/ ) and will be a valuable resource to the developmental community. DOI
- Allelic imbalance of Shh expression and polydactyly are linked to a SNP in the ZRS region of Lmbr1 in Silkie chickens
The Silkie chicken breed exhibits dominant preaxial polydactyly in the leg and associated ectopic Shh expression during development. Using a White leghorn/Silkie cross we identified a QTL highly associated with polydactyly and have mapped this locus to a SNP within the 'ZRS' region of intron 5 in Lmbr1, a known limb specific cis-regulatory element which controls expression of Shh in other species (Lettice et al., 2002). We have found that expression of Shh is abnormal in both the anterior and the posterior of the developing Silkie leg; expression is stronger, prolonged and there is allelic imbalance in expression of Shh from Silkie and White leghorn Shh alleles. Limb manipulation experiments provide evidence that the abnormal regulation of posterior Shh expression plays a role in the formation of preaxial polydactyly although ultimately polydactyly is dependent on anterior tissue containing the Silkie ZRS SNP. DOI
- The developmental mutant talpid 3 lacks primary cilia
The chicken talpid 3 mutant, with polydactyly and defects in other embryonic regions which depend on hedgehog (Hh) signalling, has a mutation in KIAA0586 . We have shown that cells of talpid 3 mutant embryos lack primary cilia and that the Talpid3 protein is localised to the centrosome. We have identified a highly conserved region of the Talpid3 protein, which is required to rescue primary cilia formation in talpid 3 mutant embryos and is sufficient for centrosomal localisation. Recently we have generated a transgenic mouse in which this highly conserved region is flanked with LoxP sites. We are now analysing the developmental consequences of deleting this conserved region of the gene either throughout the entire mouse embryo or in the limb. DOI
- Quantitative trait loci for feather growth in an F2 broiler x layer cross
- Discovery of a novel GNB3 mutation that causes the retinopathy globe enlarged phenotype and possibly hypertension in chickens
- Understanding the functional consequence of selectively constrained regions of the chicken genome
Access to new genome assemblies of birds made it possible to identify regions in bird genomes which are of functional relevance. Regions which show lower level of divergence relative to an assumed neutral standard can indicate the presence of protein-coding genes and exons, regulatory RNAs, transcription factor binding sites as well as enhancer and silencer sequences. Here we present case studies based on our high-resolution map of selectively constrained elements for the chicken genome and how we can potentially link these with functional information.
In our study we created whole genome multiple sequence alignment of 48 birds and using the software GERP++ we called selectively constrained elements. This constraint map was then compared with the current chicken genome annotation and was integrated with 20, tissue specific, RNA-seq dataset.
Currently, less than 4% of the chicken genome is annotated as functional (Ensembl 75) and code for protein-coding and non-coding genes. In comparison we predicted 1.5 million selectively constrained elements, covering approximately 15% of the genome. Relative to the 18,000 transcripts and 17,000 genes present in Ensembl, we predicted over 200,000 transcripts and 50,000 genes based on our RNA-seq study. Several thousands of the new genes are predicted to be protein coding, based on their phylogenetic signatures, while the rest is potentially non-coding and may play a part in regulation. Our study clearly indicates that the functional complexity of the chicken genome is at present poorly understood, but potentially is on par with the mammalian genomes.
- Identification of molecular circuits involved in epigenetic re-modelling in the ovine pars tuberalis using next generation sequencing data
Seasonally breeding mammals use photoperiod as a critical cue to drive hormone rhythms and synchronize reproduction to the optimal time of year. Photoperiod is encoded by the nocturnal secretion of melatonin (MEL). MEL acts on the pars tuberalis (PT) of the pituitary, regulating PT-specific expression of thyrotropin, controlling hypothalamic thyroid hormone metabolism in adjacent ependymal cells, which drives reproductive changes.
We employed Next Generation Sequencing(NGS) data to define the full repertoire of transcriptional changes within the PT following exposure to long photoperiod(LP). Sheep were housed for 12 weeks on a short photoperiod(SP) and cohorts were exposed to LP for 1, 7 or 28 days. RNASeq reads using the Illumina platform were mapped to the sheep genome. The number of reads mapping to each gene was calculated and normalised to the total number of reads generated. Using edgeR a statistical analysis was performed to identify genes differentially expressed at LP compared to SP. Further to this, an analysis using Biolayout was performed, this takes into account small changes in expression and identifies genes exhibiting similar patterns of expression, for subsequent cluster analysis. The goal is to examine the data as a whole and identify groups of genes that are co-expressed, and may have similar functions.
Our results suggest there may be dramatic tissue/vasculature remodelling of the PT in response to photoperiod. Furthermore, we hypothesise that the observed epigenetic changes may play a role in these seasonal tissue re-modelling responses. To sum up, using bioinformatics analysis based on NGS data we have identified dynamic changes of circuits involved in epigenetic changes, and possible tissue/vasculature remodelling of the PT in response to photoperiod.
- Clocks For All Seasons: Unravelling the Genetic Circadian and Interval Timing Mechanisms in the Mammalian Hypothalamus and Pituitary
There is powerful evidence that photoperiodism in seasonal organisms is driven by the circadian clockwork. The external co-incidence hypothesis of Erwin Bunning proposed that the circadian clock sets a photosensitive phase, which when exposed to light at a critical phase generates a long-photoperiod (LP) response. The molecular circuitry underpinning this model has been extensively explored in plants, but only recently have genetic mechanisms involved in mammals become clear. Thyroid hormones are crucial for driving seasonal reproduction, mediated by photoperiodic control of the de-iodinase enzyme (Dio2) in the ventral hypothalamus. Dio2 is elevated on LPs, leading to conversion of T4 to T3, and a seasonally-dependent T3-driven re-modelling of neuroendocrine circuits. Remarkably, the Dio2 gene is cAMP activated via hypothalamic TSH-receptors, which in turn are controlled by TSH hormone from the adjacent pituitary pars tuberalis (PT), the prime site of melatonin (MEL) action. In the PT, the TSHb gene is LP-activated. The up-stream molecular switch driving TSHb is Eya3, a member of the retinal determining gene family. Rhythmic MEL signals are proposed to drive a circadian oscillation in the PT of Eya3 via E-box mediated mechanism, leading to elevation of Eya3 12h after MEL onset. This only occurs in the absence of continued MEL-signalling, and is de-repressed on LPs (i.e. external co-incidence). Using a sheep-specific antibody, we have shown increased expression of EYA3 on LPs that is co-localised with TSHb in PT TSH-expressing cells. We propose a binary switching model is driving the response in the PT to LP; gradually switching individual cells from an inactive to active state. Finally, using EM, we describe major photoperiodic re-modelling of cells in the PT, driven by MEL signalling. The design principles of the mammalian photoperiodic clock are remarkably similar to those proposed for plants, where an analogous gene, Constans, fulfils an identical role to Eya3, and where epigenetic-regulated re-modelling drives photoperiodic responses.
- Genetic differences at the cholecystokinin A receptor locus result in changes in Agouti related protein expression in the hypothalamus and increased growth rate.
- Genetic differences at the cholecystokinin A receptor locus result in changes in Agouti related protein expression in the hypothalamus and increased growth rate.
- Genetic differences at the cholecystokinin A receptor locus result in changes in Agouti related protein expression in the hypothalamus and increased growth rate.
- Mapping disease resistance genes in the chicken using the 620K SNP Chip
- A comparison of immune responses to avian influenza infection in ducks and chickens: a focus on the interferon-induced transmembrane (IFITM) proteins
- Bioinformatics Analysis to identify Molecular Circuits involved in Epigenetic Re-modelling in the Ovine Pars Tuberalis using Next Generation Sequencing data
- Bioinformatics Technologies for Next Generation Sequencing Data in Animal Science Research
Nowadays the Next Generation Sequencing (NGS) as a novel high-throughput sequencing technology has developed significantly. The vast amount of data generated by these technologies requires proper methods for the extraction of robust quantitative measures and processes. This poster includes the bioinformatics technologies and statistical methods applied to NGS data in animal science research. It also introduces some examples of results from our NGS based research projects since 2009.
Three projects are presented in this poster.
1) Activation of novel molecular circuits involved in epigenetic re-modelling in the Ovine Pars Tuberalis in response to long photoperiods revealed by RNASeq
2) A comparison of immune responses to avian influenza infection in ducks and chickens: a focus on the interferon-induced transmembrane proteins
3) Development of a high density 600K SNP genotyping array for chicken
The analysis is based on RNASeq data, miRNASeq data, SNP data, MeDIPSeq data and BisulfiteSeq data for chicken, sheep, duck and quail. Methods include gene annotation, sequence alignment, transcriptome assembly, alternative splicing detection, transcript factor analysis, microRNA target prediction, genetic variation detection, differential expressed gene identification, epigenetic changes classification, gene clustering and genome visualisation etc.
The results show that NGS data has been widely used in the animal science research. Using bioinformatics and statistical analysis based on these data we identified a large number of dynamic changes of DNA and mRNA involved in molecular controls, immune response, genetic variation and epigenetic remodelling. Many of them have been validated in the lab experiments.
- Activation of Novel Molecular Circuits involved in Epigenetic Re-modelling in the Ovine Pars Tuberalis in Response to Long Photoperiods Revealed by RNA-seq
- Dynamic changes of the ovine pars tuberalis transcriptome in response to long photoperiods
- Npas4, an immediate early gene induced by melatonin in the sheep PT
- Identification of Melatonin and Photoperiodic regulated genes in Sheep pituitary using ISGC sheep genome assembly
- Identification of Melatonin and Photoperiodic regulated genes in Sheep pituitary using ISGC sheep genome assembly
- Development of High Density (600K) Chicken Genotyping Array
- Characterisation of the chicken lung immune system in an infection model
- The evolution of avian colony stimulating factor 1 (CSF-1), interleukin 34 (IL-34) and CSF-1 receptor: a functional and structural-based analysis
- Technologies to identify Melatonin regulated genes in the Sheep Pars Tuberalis using Next Generation Transcriptome Sequencing
- ChickATLAS: A three-dimensional atlas of gene expression during chick development
The control of gene expression is important in embryonic development. Quantitative assays, such as microarrays, provide gene expression levels for large numbers of genes, but with low spatial resolution. In contrast, in situ methods, such as in situ hybridisation and immunohistochemistry, provide high spatial resolution, but poorer quantification and low genome coverage. Furthermore, crucial 3D data is lost with planar samples. Optical projection tomography (OPT) can capture the full 3D expression pattern in a whole embryo at a reasonably high resolution and at moderately high throughput. A large database containing spatio-temporal patterns of expression for the mouse (EMAGE) has been created and is proving to be a valuable resource. Recently, the chick has become an important model for spatially and temporally controlled gain- and loss-of-function approaches. To date, a well-established gene expression database for the chick does not exist. Thus, the aim of this project is to produce a 3D anatomical atlas and ontology of the chick embryo with a database of gene expression patterns during chick development. This involves a major collaboration between groups in Edinburgh, Dublin, Bath and London. This database will be based on EMAGE and cross-referenced to the mouse through orthologous gene pairs (http://www.emouseatlas.org/testemage/home.php). Throughout this project, the data and framework will be used to identify groups of genes that are co-expressed in important signalling regions. Conservation of these genes will be examined in the chick and mouse. This database will be made publicly available (http://www.echickatlas.org/) and will be a valuable resource to the developmental community.
- The variance composition of broiler traits estimated on a large commercial population
- Development of high density chicken genotyping array: 1. Validation of SNPs for final array design
- Next generation sequencing: current status and prospects
- Development of high density chicken genotyping array: 1. Detection of SNPs and criteria for SNP selection
- A three-dimensional atlas of gene expression during chick embryo development
- Global variation in copy number variation in the chicken genome
- Evolutionary analysis of the CSF1R and its ligands in vertebrates
- Detection and selection of SNPs from chicken whole genome sequencing for the design of high throughput genotyping arrays
- CASI [cell autonomous sex identity] and development of the sexual phenotype in birds
- Evolution of avian genomes
- From detection to array design: Exploring the characteristics of the chicken single nucleotide polymorphisms at different phases of their selection process
- The search for new organisers
- Mapping resistance to bacteria and viruses in the chicken
Salmonella and Campylobacter are major zoonotic bacterial pathogens. The vast majority of human cases arise from the consumption of infected poultry. One sustainable solution would be to reduce contamination of poultry by identifying disease resistance genes. Resistance to colonisation with either Salmonella or Campylobacter is partly genetically determined. Inbred lines of chickens, 61 and N, are respectively resistant and susceptible to colonisation with either Salmonella or Campylobacter. Using a backcross and a high density genome-wide SNP panel, we have identified 4 resistance QTL for each colonisation model, one of which is in common. Marek's disease virus is a highly contagious, cell-associated, oncogenic -herpesvirus. We have carried out the most comprehensive gene expression study to date on MDV, using Affymetrix chicken whole genome arrays which also contain 684 transcripts from 17 avian viruses. As well as analyzing the host response to infection in both resistant (61) and susceptible (72) lines, and inherent expression differences between the two lines, we also analysed alterations in viral gene expression following infection. Our assumption was that genes controlling disease resistance would be involved during the initial stages of infection, during the induced innate response to infection, and thus we concentrated on responses on 2, 3 and 4 dpi. Comparison of our gene expression data with previous QTL data allowed us to identify genes as candidates for involvement in resistance to MD, and these were then analysed for SNPs which correlated with resistance - in other words, potential causative mutations for MD resistance/susceptibility.
- The variance composition of broiler traits on a large commercial population
- Whole-genome de novo sequencing of Quail and Grey Partridge
The development in sequencing methods has made it possible to perform whole genome de novo sequencing of species without large commercial interests. Within the EU-financed QUANTOMICS project (KBBE-2A-222664), we have performed de novo sequencing of quail ( Coturnix coturnix ) and grey partridge ( Perdix perdix ) on a Genome Analyzer GAII (Illumina) using paired-end sequencing. The amount of generated sequences amounts to 8 to 9 Gb for each species. The analysis and assembly of the generated sequences is ongoing. Access to the whole genome sequence from these two species will enable enhanced comparative studies towards the chicken genome and will aid in identifying evolutionarily conserved sequences within the Galliformes. The obtained sequences from quail and partridge represent a beginning of generating the whole genome sequence for these species. The continuation of establishing the genome sequence of these species will require generation of more sequences from more diverse libraries. The QUANTOMICS group encourages other research groups to participate in a joint effort to establish the genome sequence of these two Galliformes species.
- The duck genome sequence and the evolution of avian genomes
The mallard is the principal natural reservoir for influenza A viruses. Influenza is a major threat to human health, responsible annually for 500,000 deaths. It is also a potential economic threat; where a pandemic of influenza could cost 800 billion USD. Influenza type A viruses are asymptomatic in ducks, even with most strains of highly pathogenic strains, which can kill other poultry within days. We analysed the genome sequences of ducks and other birds to identify genes that may explain this fundamental difference. We used a phylogenetic approach to compare the intensity of selection (dN/dS ratio) on genes between birds and mammals or ducks and other birds. These comparisons defined genes that evolve at a higher rate within specific clades or subject to positive selection within specific branches. Simple 1:1 orthologs were downloaded from Ensembl for birds and mammals or defined between chicken/duck by reciprocal best hits. Amino acid sequences were aligned using Clustalw and back translated to codon sequences using Pal2nal. Gblocks was used to eliminate regions of poor alignment. The maximum likelihood method Codeml was used to estimate dN/dS ratios. Likelihood ratio tests were performed to identify specific branches evolving at a higher rate than others (branch models) or to identify genes with amino acid sites subject to positive selection (branch-site models). From these and other tests we defined genes that correlate with life histories of birds, and innate immune system of the duck that may protect it from pathological effects of infections by influenza infections.
- Transcriptional Analysis of Melatonin regulated genes in the Sheep Pars Tuberalis using Next Generation Transcriptome Sequencing
- Technologies to identify Melatonin regulated genes in the Sheep Pars Tuberalis using Next Generation Transcriptome Sequencing
- Technologies to identify Melatonin regulated genes in the Sheep Pars Tuberalis using Next Generation Transcriptome Sequencing
- ChickATLAS: A three-dimensional atlas of gene expression during chick development
- Null Mutation in the Chicken Mpdz/mupp1 Gene Causes Retinal Dysplasia and Degeneration: A New Candidate Gene for Human Retinitis Pigmentosa
- The Chicken Gene Nomenclature Committee report
Comparative genomics is an essential component of the post-genomic era. The chicken genome is the first avian genome to be sequenced and it will serve as a model for other avian species. Moreover, due to its unique evolutionary niche, the chicken genome can be used to understand evolution of functional elements and gene regulation in mammalian species. However comparative biology both within avian species and within amniotes is hampered due to the difficulty of recognising functional orthologs. This problem is compounded as different databases and sequence repositories proliferate and the names they assign to functional elements proliferate along with them. Currently, genes can be published under more than one name and one name sometimes refers to unrelated genes. Standardized gene nomenclature is necessary to facilitate communication between scientists and genomic resources. Moreover, it is important that this nomenclature be based on existing nomenclature efforts where possible to truly facilitate studies between different species. We report here the formation of the Chicken Gene Nomenclature Committee (CGNC), an international and centralized effort to provide standardized nomenclature for chicken genes. The CGNC works in conjunction with public resources such as NCBI and Ensembl and in consultation with existing nomenclature committees for human and mouse. The CGNC will develop standardized nomenclature in consultation with the research community and relies on the support of the research community to ensure that the nomenclature facilitates comparative and genomic studies. DOI
- Transcriptomic analysis of the Chicken Anaemia Virus (CAV)-induced immunosuppression
Chicken anaemia virus (CAV) is an unusually small virus that causes severe anaemia and immunosuppression in young chickens. Such effects reduce the efficiency of routine vaccinations while aggravating the effects of other pathogens in chicken populations, constituting a serious economic threat to poultry industry. The replication/pathogenicity of CAV relies on the expression of only a single structural protein VP1 and two non-structural proteins VP2 and VP3 (apoptin). The function of each individual protein is as yet unclear. This study has used transcriptional profiling to identify pathways that are dramatically modulated after 48 hr of infection in an in vitro model (MDCC-MSB1 cells) and after 4 days of experimental infection (1-day-old chickens in vivo model), using Affymetrix chicken oligonucleotide arrays. Changes in transcript levels between infected and uninfected cells indicate a dysfunction in the Mitogen Activated Protein kinases (MAPK) and T cell receptor signalling cascades implying that CAV might subvert host signalling responses to its own benefit. Two highly passaged attenuated cloned isolates and three VP3 CAV mutant viruses were also used in this study with the aim of elucidating the interactions of each of these proteins with the host cell. VP3 mutations resulted in lower levels of transcripts encoding signalling, transcription and mitosis related proteins. Overall, these data extend our understanding of how broadly CAV alters the regulation of host gene products and highlight the virus' 'need' to utilise the host cell machinery to replicate itself. DOI
- Integrated immunogenics in the chicken: deciphering the immune response to identify disease resistance genes
Resistance to infection takes place at many levels, and involves both non-specific and specific immune mechanisms. The chicken has a different repertoire of immune genes, molecules, cells and organs compared to mammals. To understand the role of any disease resistance gene(s), it is therefore important to understand these different repertoires, and the bird's response to a particular pathogen. Our studies focus on the innate immune response, as responses of macrophages from inbred lines of chickens, and heterophils from commercial birds, correlate with resistance or susceptibility to Salmonella infection with a variety of Salmonella serovars and infection models. To map disease resistance genes, we are using a combination of expression quantitative trait loci (eQTLs) from microarray studies, allied with whole genome SNP arrays (WGA) and a candidate gene approach. There are over 500 human genes with the Gene Ontology term "innate immunity. "We have identified over 400 of these genes in the chicken genome, and are actively identifying informative SNPs in them. The segregation of 6,000 WGA SNPs across all of our inbred lines was also assessed, which should yield approximately 900 informative SNPs for a cross between any two lines. The initial focus of these studies is on mapping resistance genes in our inbred lines, but the studies will be extended to commercial flocks. DOI
- Analysis of the host response to viral infection in the chicken and identification of genes involved in disease resistance
- Chicken Gene-Nomenclature / Ortholog Database
- MIQAS - Minimal Information For QTL And Association Studies
- Mapping QTL for performance and carcass traits in chicken chromosomes 6, 7, 8, 11 and 13
A Brazilian F2 resource population, derived from a broiler layer cross, was used to map QTL for performance, carcass traits and organs weight on chicken chromosomes 6 to 8, 11 and 13. A total of 356 F2 chickens from four full-sib families were genotyped with 26 microsatellite markers. Regression methods were applied to a line cross model in the QTL interval mapping analyses. A suggestive QTL affecting five growth-related traits and feed intake was identified on chromosome 7. QTLs associated with feet weight were detected on chromosomes 6 and 11, with gizzard weight on chromosomes 8 and 11, and with heart weight on chromosome 13.
- Chicken genomics: progress and Prospects
- Quantitative trait loci (QTL) affecting fat traits in chickens
- Mouse renin gene structure, evolution and function
- Chicken Genome: current status and future opportunities
- Comparative Genomics
- Chicken Genome
- Applications of biotechnology in the poultry industry
Progress in the selection of production traits in the poultry industry has been
dramatic over the past 75 years. Genetic progress has its biological limits and these may be reached in the next 20 years. The poultry industry will need to adapt with more emphasis on the needs of the consumer (e.g. product quality, health and animal welfare), as well as the needs of the breeders/producers (e.g. lower feed costs and higher fertility). These new traits are difficult and costly to measure by conventional genetic selection methods - the application of genomics is a possible solution. Poultry genomics has benefited from the rapid technological advances in the genetics of model organisms and human. A number of resources and approaches are now well established, including genetic markers and maps (both genetic and physical), QTL mapping, comparative mapping, EST and BAC resources. In addition, the next phase of gene discovery - Functional Genomics is underway. How the poultry industry can benefit and get access to these new technologies is discussed.
- The human genome: a model organism for poultry
- Chicken microchromosomes, W and Z chromosomes
- Chromosome rearrangement in evolution
- Gene mapping in farm animals and birds: an overview
- Physical Map of the chicken: an update
- Bacteriophage Lambda as a vector
- AVIAN COLONY STIMULATING FACTOR 1 RECEPTOR BINDING PROTEINS
- Mapping the chicken genome