Mireille Morisson

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 virus. Further, we show how the ducks defense mechanisms against influenza infection have been optimized through the diversification of its β-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.

Cloning of Ovocalyxin-36, a Novel Chicken Eggshell Protein Related to Lipopolysaccharide-binding Proteins, Bactericidal Permeability-increasing Proteins, and Plunc Family Proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20–25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

ChickRH6: a chicken whole-genome radiation hybrid panel

As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6 000 rads with HPRT-deficient hamster Wg3hCl2 cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs.

Species difference in ANP32A underlies influenza A virus polymerase host restriction

Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals.

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. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 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.

Integrative mapping analysis of chicken microchromosome 16 organization

BackgroundThe chicken karyotype is composed of 39 chromosome pairs, of which 9 still remain totally absent from the current genome sequence assembly, despite international efforts towards complete coverage. Some others are only very partially sequenced, amongst which microchromosome 16 (GGA16), particularly under-represented, with only 433 kb assembled for a full estimated size of 9 to 11 Mb. Besides the obvious need of full genome coverage with genetic markers for QTL (Quantitative Trait Loci) mapping and major genes identification studies, there is a major interest in the detailed study of this chromosome because it carries the two genetically independent MHC complexes B and Y. In addition, GGA16 carries the ribosomal RNA (rRNA) genes cluster, also known as the NOR (nucleolus organizer region). The purpose of the present study is to construct and present high resolution integrated maps of GGA16 to refine its organization and improve its coverage with genetic markers.ResultsWe developed 79 STS (Sequence Tagged Site) markers to build a physical RH (radiation hybrid) map and 34 genetic markers to extend the genetic map of GGA16. We screened a BAC (Bacterial Artificial Chromosome) library with markers for the MHC-B, MHC-Y and rRNA complexes. Selected clones were used to perform high resolution FISH (Fluorescent In Situ Hybridization) mapping on giant meiotic lampbrush chromosomes, allowing meiotic mapping in addition to the confirmation of the order of the three clusters along the chromosome. A region with high recombination rates and containing PO41 repeated elements separates the two MHC complexes.ConclusionsThe three complementary mapping strategies used refine greatly our knowledge of chicken microchromosome 16 organisation. The characterisation of the recombination hotspots separating the two MHC complexes demonstrates the presence of PO41 repetitive sequences both in tandem and inverted orientation. However, this region still needs to be studied in more detail.

Transcriptome-wide investigation of genomic imprinting in chicken

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken.

High resolution physical map of porcine chromosome 7 QTL region and comparative mapping of this region among vertebrate genomes

BackgroundOn porcine chromosome 7, the region surrounding the Major Histocompatibility Complex (MHC) contains several Quantitative Trait Loci (QTL) influencing many traits including growth, back fat thickness and carcass composition. Previous studies highlighted that a fragment of ~3.7 Mb is located within the Swine Leucocyte Antigen (SLA) complex. Internal rearrangements of this fragment were suggested, and partial contigs had been built, but further characterization of this region and identification of all human chromosomal fragments orthologous to this porcine fragment had to be carried out.ResultsA whole physical map of the region was constructed by integrating Radiation Hybrid (RH) mapping, BAC fingerprinting data of the INRA BAC library and anchoring BAC end sequences on the human genome. 17 genes and 2 reference microsatellites were ordered on the high resolution IMNpRH212000rad Radiation Hybrid panel. A 1000:1 framework map covering 550 cR12000 was established and a complete contig of the region was developed. New micro rearrangements were highlighted between the porcine and human genomes. A bovine RH map was also developed in this region by mapping 16 genes. Comparison of the organization of this region in pig, cattle, human, mouse, dog and chicken genomes revealed that 1) the translocation of the fragment described previously is observed only on the bovine and porcine genomes and 2) the new internal micro rearrangements are specific of the porcine genome.ConclusionWe estimate that the region contains several rearrangements and covers 5.2 Mb of the porcine genome. The study of this complete BAC contig showed that human chromosomal fragments homologs of this heavily rearranged QTL region are all located in the region of HSA6 that surrounds the centromere. This work allows us to define a list of all candidate genes that could explain these QTL effects.

The chicken RH map: current state of progress and microchromosome mapping.

The ChickRH6 radiation hybrid panel has been used to construct consensus chromosome radiation hybrid (RH) maps of the chicken genome. Markers genotyped were either from throughout the genome or targeted to specific chromosomes and a large proportion (one third) of data was the result of collaborative efforts. Altogether, 2,531 markers were genotyped, allowing the construction of RH reference maps for 20 chromosomes and linkage groups for four other chromosomes. Amongst the markers, 581 belong to the framework maps, while 1,721 are on the comprehensive maps. Around 800 markers still have to be assigned to linkage groups. Our attempt to assign the supercontigs from the chrun (virtual chromosome containing all the genome sequence that could not be attributed to a chromosome) as well as EST (Expressed Sequence Tag) contigs that do not have a BLAST hit in the genome assembly led to the construction of new maps for microchromosomes either absent or for which very little data is present in the genome assembly. RH data is presented through our ChickRH webserver (http://chickrh.toulouse.inra.fr/), which is a mapping tool as well as the official repository RH database for genotypes. It also displays the RH reference maps and comparison charts with the sequence thus highlighting the possible discrepancies. Future improvements of the RH maps include complete coverage of the sequence assigned to chromosomes, further mapping of the chrun and mapping of EST contigs absent from the assembly. This will help finish the mapping of the smallest gene-rich microchromosomes.

A high-resolution radiation hybrid map of chicken chromosome 5 and comparison with human chromosomes

BackgroundThe resolution of radiation hybrid (RH) maps is intermediate between that of the genetic and BAC (Bacterial Artificial Chromosome) contig maps. Moreover, once framework RH maps of a genome have been constructed, a quick location of markers by simple PCR on the RH panel is possible. The chicken ChickRH6 panel recently produced was used here to construct a high resolution RH map of chicken GGA5. To confirm the validity of the map and to provide valuable comparative mapping information, both markers from the genetic map and a high number of ESTs (Expressed Sequence Tags) were used. Finally, this RH map was used for testing the accuracy of the chicken genome assembly for chromosome 5.ResultsA total of 169 markers (21 microsatellites and 148 ESTs) were typed on the ChickRH6 RH panel, of which 134 were assigned to GGA5. The final map is composed of 73 framework markers extending over a 1315.6 cR distance. The remaining 61 markers were placed alongside the framework markers within confidence intervals.ConclusionThe high resolution framework map obtained in this study has markers covering the entire chicken chromosome 5 and reveals the existence of a high number of rearrangements when compared to the human genome. Only two discrepancies were observed in relation to the sequence assembly recently reported for this chromosome.