Construction of a radiation hybrid map of chicken chromosome 2 and alignment to the chicken draft sequence

BACKGROUND: The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2 (Gallus gallus chromosome 2) linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances. RESULTS: Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cR(6000 )long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths. CONCLUSION: We obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate

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

BACKGROUND: The 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. RESULTS: A 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. CONCLUSION: The 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

Pyroséquençage pour le développement d'EST et de SNP aviaires

Le but du programme est de combler les déficits en marqueurs observés pour trois espèces aviaires : la caille, le canard et la poule. La stratégie choisie est l'obtention, à partir de plusieurs individus de lignées d'intérêt, de SNP (Single Nucleotide Polymorphism, polymorphisme d'un nucléotide) par une nouvelle technologie de séquençage à haut débit (séquenceur 454 GS-FLX, Roche). Nous séquençons des représentations réduites du génome, en sélectionnant d'une part des fragments de restriction d'ADN génomique - les mêmes chez tous les individus - et d'autre part les transcrits qui représentent globalement la partie du génome correspondant aux gènes exprimés. Ces expérimentations sont réalisées à partir d'échantillons d'ADN ou d'ARN issus d'individus de lignées à l'origine de croisements existants, pour chacune des trois espèces. Les données générées par plusieurs "runs" de séquence seront traitées in silico : contigage à haut débit, recherche de SNP, comparaison avec les banques de séquences connues...En plus de l'intérêt que représente la production d'un très grand nombre de SNP nouveaux, cette technologie devrait permettre de mieux séquencer les régions riches en (G+C) correspondant aux plus petits des microchromosomes pour lesquels il n'y a pas de séquence chez la poule. La comparaison des séquences des transcrits obtenues chez la caille et le canard avec la séquence du génome de la poule permettra d'établir une "cartographie virtuelle" des SNP obtenus, grâce à la grande conservation de synténie existant entre ces trois espèces

Non PCR-amplified Transcripts and AFLP fragments as reduced representations of the quail genome for 454 Titanium sequencing

<p>Abstract</p> <p>Background</p> <p>SNP (Single Nucleotide Polymorphism) discovery is now routinely performed using high-throughput sequencing of reduced representation libraries. Our objective was to adapt 454 GS FLX based sequencing methodologies in order to obtain the largest possible dataset from two reduced representations libraries, produced by AFLP (Amplified Fragment Length Polymorphism) for genomic DNA, and EST (Expressed Sequence Tag) for the transcribed fraction of the genome.</p> <p>Findings</p> <p>The expressed fraction was obtained by preparing cDNA libraries without PCR amplification from quail embryo and brain. To optimize the information content for SNP analyses, libraries were prepared from individuals selected in three quail lines and each individual in the AFLP library was tagged. Sequencing runs produced 399,189 sequence reads from cDNA and 373,484 from genomic fragments, covering close to 250 Mb of sequence in total.</p> <p>Conclusions</p> <p>Both methods used to obtain reduced representations for high-throughput sequencing were successful after several improvements.</p> <p>The protocols may be used for several sequencing applications, such as <it>de novo </it>sequencing, tagged PCR fragments or long fragment sequencing of cDNA.</p

Integrative mapping analysis of chicken microchromosome 16 organization

<p>Abstract</p> <p>Background</p> <p>The 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 <it>MHC </it>complexes <it>B </it>and <it>Y</it>. In addition, GGA16 carries the ribosomal RNA (<it>rRNA</it>) genes cluster, also known as the <it>NOR </it>(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.</p> <p>Results</p> <p>We 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 <it>MHC-B</it>, <it>MHC-Y </it>and <it>rRNA </it>complexes. Selected clones were used to perform high resolution FISH (Fluorescent <it>In Situ </it>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 <it>MHC </it>complexes.</p> <p>Conclusions</p> <p>The three complementary mapping strategies used refine greatly our knowledge of chicken microchromosome 16 organisation. The characterisation of the recombination hotspots separating the two <it>MHC </it>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.</p

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

Differentially expressed genes and gene networks involved in pig ovarian follicular atresia

Ovarian folliculogenesis corresponds to the development of follicles leading to either ovulation or degeneration, this latter process being called atresia. Even if atresia involves apoptosis, its mechanism is not well-understood. The objective of this project was to analyse global gene expression in pig granulosa cells of ovarian follicles during atresia. The transcriptome analysis was performed using 9216 cDNAs microarray to identify gene networks and candidate genes involved in pig ovarian follicular atresia. One thousand six hundred and eighty four significantly regulated genes were differentially regulated between small healthy follicles and small atretic follicles. Among them, two hundred and eighty seven genes had a fold-change higher than 2 between the two follicle groups. Eleven genes (DKK3, GADD45A, CAMTA2, CCDC80, DAPK2, ECSIT, MSMB, NUPR1, RUNX2, SAMD4A, and ZNF628) having a fold-change higher than 5 between groups could likely serve as markers of follicular atresia. Moreover, automatic confrontation of deregulated genes with literature data enlightened 93 genes as regulatory candidates of pig granulosa cell atresia. Among these genes known to be inhibitors of apoptosis, stimulators of apoptosis or tumor suppressors INHBB, HNF4, CLU, different interleukins (IL5, IL24), TNF-associated receptor (TNFR1), and cytochrome-c oxidase (COX) were suggested as playing an important role in porcine atresia. Present study also enlists key upstream regulators in follicle atresia based on our results and on a literature review. The novel gene candidates and gene networks identified in the current study lead to a better understanding of the molecular regulation of ovarian follicular atresia

Derivation of induced pluripotent stem cells from an infertile boar carrying a reciprocal translocation

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