A genome-wide epistatic network underlies the molecular architecture of continuous color variation of body extremities.

Deciphering the molecular architecture of coat coloration for a better understanding of the biological mechanisms underlying pigmentation still remains a challenge. We took advantage of a rabbit French experimental population in which both a pattern and a gradient of coloration from white to brown segregated within the himalayan phenotype. The whole experimental design was genotyped using the high density Affymetrix® AxiomOrcun™ SNP Array and phenotyped into 6 different groups ordered from the lighter to the darker. Genome-wide association analyses pinpointed an oligogenic determinism, under recessive and additive inheritance, involving genes already known in melanogenesis (ASIP, KIT, MC1R, TYR), and likely processed pseudogenes linked to ribosomal function, RPS20 and RPS14. We also identified (i) gene-gene interactions through ASIP:MC1R affecting light cream/beige phenotypes while KIT:RPS responsible of dark chocolate/brown colors and (ii) a genome-wide epistatic network involving several others coloration genes such as POT1 or HPS5. Finally, we determined the recessive inheritance of the English spotting phenotype likely involving a copy number variation affecting at least the end of the coding sequence of the KIT gene. Our analyses of coloration as a continuous trait allowed us to go beyond much of the established knowledge through the detection of additional genes and gene-gene interactions that may contribute to the molecular architecture of the coloration phenotype.

RNA-Seq Data for Reliable SNP Detection and Genotype Calling: Interest for Coding Variant Characterization and Cis-Regulation Analysis by Allele-Specific Expression in Livestock Species.

In addition to their common usages to study gene expression, RNA-seq data accumulated over the last 10 years are a yet-unexploited resource of SNPs in numerous individuals from different populations. SNP detection by RNA-seq is particularly interesting for livestock species since whole genome sequencing is expensive and exome sequencing tools are unavailable. These SNPs detected in expressed regions can be used to characterize variants affecting protein functions, and to study cis-regulated genes by analyzing allele-specific expression (ASE) in the tissue of interest. However, gene expression can be highly variable, and filters for SNP detection using the popular GATK toolkit are not yet standardized, making SNP detection and genotype calling by RNA-seq a challenging endeavor. We compared SNP calling results using GATK suggested filters, on two chicken populations for which both RNA-seq and DNA-seq data were available for the same samples of the same tissue. We showed, in expressed regions, a RNA-seq precision of 91% (SNPs detected by RNA-seq and shared by DNA-seq) and we characterized the remaining 9% of SNPs. We then studied the genotype (GT) obtained by RNA-seq and the impact of two factors (GT call-rate and read number per GT) on the concordance of GT with DNA-seq; we proposed thresholds for them leading to a 95% concordance. Applying these thresholds to 767 multi-tissue RNA-seq of 382 birds of 11 chicken populations, we found 9.5 M SNPs in total, of which ∼550,000 SNPs per tissue and population with a reliable GT (call rate ≥ 50%) and among them, ∼340,000 with a MAF ≥ 10%. We showed that such RNA-seq data from one tissue can be used to (i) detect SNPs with a strong predicted impact on proteins, despite their scarcity in each population (16,307 SIFT deleterious missenses and 590 stop-gained), (ii) study, on a large scale, cis-regulations of gene expression, with ∼81% of protein-coding and 68% of long non-coding genes (TPM ≥ 1) that can be analyzed for ASE, and with ∼29% of them that were cis-regulated, and (iii) analyze population genetic using such SNPs located in expressed regions. This work shows that RNA-seq data can be used with good confidence to detect SNPs and associated GT within various populations and used them for different analyses as GTEx studies.

Avian Expression Patterns and Genomic Mapping Implicate Leptin in Digestion and TNF in Immunity, Suggesting That Their Interacting Adipokine Role Has Been Acquired Only in Mammals

In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.

Two new structural mutations in the 5' region of the ASIP gene cause diluted feather color phenotypes in Japanese quail.

BACKGROUND: In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification). RESULTS: For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5' region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5' region of the ASIP gene. CONCLUSIONS: We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5' region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5' region of ASIP are absent in the fawn-2/beige mutant.

Correction to: Mapping of leptin and its syntenic genes to chicken chromosome 1p.

CORRECTION: After the publication of this work [1] an error was noticed in one of the author surnames. The author name Leif Anderson should be spelt as Leif Andersson.

Mapping of leptin and its syntenic genes to chicken chromosome 1p.

BACKGROUND: Misidentification of the chicken leptin gene has hampered research of leptin signaling in this species for almost two decades. Recently, the genuine leptin gene with a GC-rich (~70%) repetitive-sequence content was identified in the chicken genome but without indicating its genomic position. This suggests that such GC-rich sequences are difficult to sequence and therefore substantial regions are missing from the current chicken genome assembly. RESULTS: A radiation hybrid panel of chicken-hamster Wg3hCl2 cells was used to map the genome location of the chicken leptin gene. Contrary to our expectations, based on comparative genome mapping and sequence characteristics, the chicken leptin was not located on a microchromosome, which are known to contain GC-rich and repetitive regions, but at the distal tip of the largest chromosome (1p). Following conserved synteny with other vertebrates, we also mapped five additional genes to this genomic region (ARF5, SND1, LRRC4, RBM28, and FLNC), bridging the genomic gap in the current Galgal5 build for this chromosome region. All of the short scaffolds containing these genes were found to consist of GC-rich (54 to 65%) sequences comparing to the average GC-content of 40% on chromosome 1. In this syntenic group, the RNA-binding protein 28 (RBM28) was in closest proximity to leptin. We deduced the full-length of the RBM28 cDNA sequence and profiled its expression patterns detecting a negative correlation (R = - 0.7) between the expression of leptin and of RBM28 across tissues that expressed at least one of the genes above the average level. This observation suggested a local regulatory interaction between these genes. In adipose tissues, we observed a significant increase in RBM28 mRNA expression in breeds with lean phenotypes. CONCLUSION: Mapping chicken leptin together with a cluster of five syntenic genes provided the final proof for its identification as the true chicken ortholog. The high GC-content observed for the chicken leptin syntenic group suggests that other similar clusters of genes in GC-rich genomic regions are missing from the current genome assembly (Galgal5), which should be resolved in future assemblies of the chicken genome.

Accounting for linkage disequilibrium in genome scans for selection without individual genotypes: The local score approach.

Detecting genomic footprints of selection is an important step in the understanding of evolution. Accounting for linkage disequilibrium in genome scans increases detection power, but haplotype-based methods require individual genotypes and are not applicable on pool-sequenced samples. We propose to take advantage of the local score approach to account for linkage disequilibrium in genome scans for selection, cumulating (possibly small) signals from single markers over a genomic segment, to clearly pinpoint a selection signal. Using computer simulations, we demonstrate that this approach detects selection with higher power than several state-of-the-art single-marker, windowing or haplotype-based approaches. We illustrate this on two benchmark data sets including individual genotypes, for which we obtain similar results with the local score and one haplotype-based approach. Finally, we apply the local score approach to Pool-Seq data obtained from a divergent selection experiment on behaviour in quail and obtain precise and biologically coherent selection signals: while competing methods fail to highlight any clear selection signature, our method detects several regions involving genes known to act on social responsiveness or autistic traits. Although we focus here on the detection of positive selection from multiple population data, the local score approach is general and can be applied to other genome scans for selection or other genomewide analyses such as GWAS.

Embryonic environment and transgenerational effects in quail.

BACKGROUND: Environmental exposures, for instance to chemicals, are known to impact plant and animal phenotypes on the long term, sometimes across several generations. Such transgenerational phenotypes were shown to be promoted by epigenetic alterations such as DNA methylation, an epigenetic mark involved in the regulation of gene expression. However, it is yet unknown whether transgenerational epigenetic inheritance of altered phenotypes exists in birds. The purpose of this study was to develop an avian model to investigate whether changes to the embryonic environment had a transgenerational effect that could alter the phenotypes of third-generation offspring. Given its impact on the mammalian epigenome and the reproductive system in birds, genistein was used as an environment stressor. RESULTS: We compared several third-generation phenotypes of two quail "epilines", which were obtained from genistein-injected eggs (Epi+) or from untreated eggs (Epi-) from the same founders. A "mirrored" crossing strategy was used to minimize between-line genetic variability by maintaining similar ancestor contributions across generations in each line. Three generations after genistein treatment, a significant difference in the sexual maturity of the females, which, after three generations, could not be attributed to direct maternal effects, was observed between the lines, with Epi+ females starting to lay eggs later. Adult body weight was significantly affected by genistein treatment applied in a previous generation, and a significant interaction between line and sex was observed for body weight at 3 weeks. Behavioral traits, such as evaluating the birds' reaction to social isolation, were also significantly affected by genistein treatment. Yet, global methylation analyses revealed no significant difference between the epilines. CONCLUSIONS: These findings demonstrate that embryonic environment affects the phenotype of offspring three generations later in quail. While one cannot rule out the existence of some initial genetic variability between the lines, the mirrored animal design should have minimized its effects, and thus, the observed differences in animals of the third generation may be attributed, at least partly, to transgenerational epigenetic phenomena.

The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3.

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors.

Influence of grand-mother diet on offspring performances through the male line in Muscovy duck.

BACKGROUND: In mammals, multigenerational environmental effects have been documented by either epidemiological studies in human or animal experiments in rodents. Whether such phenomena also occur in birds for more than one generation is still an open question. The objective of this study was to investigate if a methionine deficiency experienced by a mother (G0) could affect her grand-offspring phenotypes (G2 hybrid mule ducks and G2 purebred Muscovy ducks), through their Muscovy sons (G1). Muscovy drakes are used for the production of mule ducks, which are sterile offspring of female common duck (Anas platyrhynchos) and Muscovy drakes (Cairina moschata). In France, mule ducks are bred mainly for the production of "foie gras", which stems from hepatic steatosis under two weeks of force-feeding (FF). Two groups of female Muscovy ducks received either a methionine deficient diet or a control diet. Their sons were mated to Muscovy or to common duck females to produce Muscovy or Mule ducks, respectively. Several traits were measured in the G2 progenies, concerning growth, feed efficiency during FF, body composition after FF, and quality of foie gras and magret. RESULTS: In the G2 mule duck progeny, grand-maternal methionine deficiency (GMMD) decreased 4, 8, and 12 week body weights but increased weight gain and feed efficiency during FF, and abdominal fat weight. The plasmatic glucose and triglyceride contents at the end of FF were higher in the methionine deficient group. In the G2 purebred Muscovy progeny, GMMD tended to decrease 4 week body weight in both sexes, and decreased weight gain between the ages of 4 and 12 weeks, 12 week body weight, and body weight at the end of FF in male offspring only. GMMD tended to increase liver weight and increased the carcass proportion of liver in both sexes. CONCLUSION: Altogether, these results show that the mother's diet is able to affect traits linked to growth and to lipid metabolism in the offspring of her sons, in Muscovy ducks. Whether this transmission through the father of information induced in the grand-mother by the environment is epigenetic remains to be demonstrated.

Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates.

RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome.

Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome.

Free fatty acid receptors (FFAR) belong to a family of five G-protein coupled receptors that are involved in the regulation of lipid metabolism, so that their loss of function increases the risk of obesity. The aim of this study was to determine the expansion of genes encoding paralogs of FFAR2 in the chicken, considered as a model organism for developmental biology and biomedical research. By estimating the gene copy number using quantitative polymerase chain reaction, genomic DNA resequencing, and RNA sequencing data, we showed the existence of 23 ± 1.5 genes encoding FFAR2 paralogs in the chicken genome. The FFAR2 paralogs shared an identity from 87.2% up to 99%. Extensive gene conversion was responsible for this high degree of sequence similarities between these genes, and this concerned especially the four amino acids known to be critical for ligand binding. Moreover, elevated nonsynonymous/synonymous substitution ratios on some amino acids within or in close-vicinity of the ligand-binding groove suggest that positive selection may have reduced the effective rate of gene conversion in this region, thus contributing to diversify the function of some FFAR2 paralogs. All the FFAR2 paralogs were located on a microchromosome in a same linkage group. FFAR2 genes were expressed in different tissues and cells such as spleen, peripheral blood mononuclear cells, abdominal adipose tissue, intestine, and lung, with the highest rate of expression in testis. Further investigations are needed to determine whether these chicken-specific events along evolution are the consequence of domestication and may play a role in regulating lipid metabolism in this species.

Combined QTL and selective sweep mappings with coding SNP annotation and cis-eQTL analysis revealed PARK2 and JAG2 as new candidate genes for adiposity regulation.

Very few causal genes have been identified by quantitative trait loci (QTL) mapping because of the large size of QTL, and most of them were identified thanks to functional links already known with the targeted phenotype. Here, we propose to combine selection signature detection, coding SNP annotation, and cis-expression QTL analyses to identify potential causal genes underlying QTL identified in divergent line designs. As a model, we chose experimental chicken lines divergently selected for only one trait, the abdominal fat weight, in which several QTL were previously mapped. Using new haplotype-based statistics exploiting the very high SNP density generated through whole-genome resequencing, we found 129 significant selective sweeps. Most of the QTL colocalized with at least one sweep, which markedly narrowed candidate region size. Some of those sweeps contained only one gene, therefore making them strong positional causal candidates with no presupposed function. We then focused on two of these QTL/sweeps. The absence of nonsynonymous SNPs in their coding regions strongly suggests the existence of causal mutations acting in cis on their expression, confirmed by cis-eQTL identification using either allele-specific expression or genetic mapping analyses. Additional expression analyses of those two genes in the chicken and mice contrasted for adiposity reinforces their link with this phenotype. This study shows for the first time the interest of combining selective sweeps mapping, coding SNP annotation and cis-eQTL analyses for identifying causative genes for a complex trait, in the context of divergent lines selected for this specific trait. Moreover, it highlights two genes, JAG2 and PARK2, as new potential negative and positive key regulators of adiposity in chicken and mice.

A medium density genetic map and QTL for behavioral and production traits in Japanese quail.

BACKGROUND: Behavioral traits such as sociability, emotional reactivity and aggressiveness are major factors in animal adaptation to breeding conditions. In order to investigate the genetic control of these traits as well as their relationships with production traits, a study was undertaken on a large second generation cross (F2) between two lines of Japanese Quail divergently selected on their social reinstatement behavior. All the birds were measured for several social behaviors (social reinstatement, response to social isolation, sexual motivation, aggression), behaviors measuring the emotional reactivity of the birds (reaction to an unknown object, tonic immobility reaction), and production traits (body weight and egg production). RESULTS: We report the results of the first genome-wide QTL detection based on a medium density SNP panel obtained from whole genome sequencing of a pool of individuals from each divergent line. A genetic map was constructed using 2145 markers among which 1479 could be positioned on 28 different linkage groups. The sex-averaged linkage map spanned a total of 3057 cM with an average marker spacing of 2.1 cM. With the exception of a few regions, the marker order was the same in Japanese Quail and the chicken, which confirmed a well conserved synteny between the two species. The linkage analyses performed using QTLMAP software revealed a total of 45 QTLs related either to behavioral (23) or production (22) traits. The most numerous QTLs (15) concerned social motivation traits. Interestingly, our results pinpointed putative pleiotropic regions which controlled emotional reactivity and body-weight of birds (on CJA5 and CJA8) or their social motivation and the onset of egg laying (on CJA19). CONCLUSION: This study identified several QTL regions for social and emotional behaviors in the Quail. Further research will be needed to refine the QTL and confirm or refute the role of candidate genes, which were suggested by bioinformatics analysis. It can be hoped that the identification of genes and polymorphisms related to behavioral traits in the quail will have further applications for other poultry species (especially the chicken) and will contribute to solving animal welfare issues in poultry production.

The loss of adipokine genes in the chicken genome and implications for insulin metabolism.

Gene loss is one of the main drivers in the evolution of genomes and species. The demonstration that a gene has been lost by pseudogenization is truly complete when one finds the pseudogene in the orthologous genomic region with respect to active genes in other species. In some cases, the identification of such orthologous loci is not possible because of chromosomal rearrangements or if the gene of interest has not yet been sequenced. This question is particularly important in the case of birds because the genomes of avian species possess only about 15,000 predicted genes, in comparison with 20,000 in mammals. Yet, gene loss raises the question of which functions are affected by the changes in gene counts. We describe a systematic approach that makes it possible to demonstrate gene loss in the chicken genome even if a pseudogene has not been found. By using phylogenetic and synteny analysis in vertebrates, genome-wide comparisons between the chicken genome and expressed sequence tags, RNAseq data analysis, statistical analysis of the chicken genome, and radiation hybrid mapping, we show that resistin, TNFα, and PAI-1 (SERPINE1), three genes encoding adipokines inhibiting insulin sensitivity, have been lost in chicken and zebra finch genomes. Moreover, omentin, a gene encoding an adipokine that enhances insulin sensitivity, has also been lost in the chicken genome. Overall, only one adipokine inhibiting insulin sensitivity and five adipokines enhancing insulin sensitivity are still present in the chicken genome. These genetic differences between mammals and chicken, given the functions of the genes in mammals, would have dramatic consequences on chicken endocrinology, leading to novel equilibriums especially in the regulation of energy metabolism, insulin sensitivity, as well as appetite and reproduction.

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.

Fine mapping of complex traits in non-model species: using next generation sequencing and advanced intercross lines in Japanese quail.

BACKGROUND: As for other non-model species, genetic analyses in quail will benefit greatly from a higher marker density, now attainable thanks to the evolution of sequencing and genotyping technologies. Our objective was to obtain the first genome wide panel of Japanese quail SNP (Single Nucleotide Polymorphism) and to use it for the fine mapping of a QTL for a fear-related behaviour, namely tonic immobility, previously localized on Coturnix japonica chromosome 1. To this aim, two reduced representations of the genome were analysed through high-throughput 454 sequencing: AFLP (Amplified Fragment Length Polymorphism) fragments as representatives of genomic DNA, and EST (Expressed Sequence Tag) as representatives of the transcriptome. RESULTS: The sequencing runs produced 399,189 and 1,106,762 sequence reads from cDNA and genomic fragments, respectively. They covered over 434 Mb of sequence in total and allowed us to detect 17,433 putative SNP. Among them, 384 were used to genotype two Advanced Intercross Lines (AIL) obtained from three quail lines differing for duration of tonic immobility. Despite the absence of genotyping for founder individuals in the analysis, the previously identified candidate region on chromosome 1 was refined and led to the identification of a candidate gene. CONCLUSIONS: These data confirm the efficiency of transcript and AFLP-sequencing for SNP discovery in a non-model species, and its application to the fine mapping of a complex trait. Our results reveal a significant association of duration of tonic immobility with a genomic region comprising the DMD (dystrophin) gene. Further characterization of this candidate gene is needed to decipher its putative role in tonic immobility in Coturnix.

Detection of a Cis [corrected] eQTL controlling BCMO1 gene expression leads to the identification of a QTG for chicken breast meat color.

Classical quantitative trait loci (QTL) analysis and gene expression QTL (eQTL) were combined to identify the causal gene (or QTG) underlying a highly significant QTL controlling the variation of breast meat color in a F2 cross between divergent high-growth (HG) and low-growth (LG) chicken lines. Within this meat quality QTL, BCMO1 (Accession number GenBank: AJ271386), encoding the ß-carotene 15, 15'-monooxygenase, a key enzyme in the conversion of ß-carotene into colorless retinal, was a good functional candidate. Analysis of the abundance of BCMO1 mRNA in breast muscle of the HG x LG F2 population allowed for the identification of a strong cis eQTL. Moreover, reevaluation of the color QTL taking BCMO1 mRNA levels as a covariate indicated that BCMO1 mRNA levels entirely explained the variations in meat color. Two fully-linked single nucleotide polymorphisms (SNP) located within the proximal promoter of BCMO1 gene were identified. Haplotype substitution resulted in a marked difference in BCMO1 promoter activity in vitro. The association study in the F2 population revealed a three-fold difference in BCMO1 expression leading to a difference of 1 standard deviation in yellow color between the homozygous birds at this haplotype. This difference in meat yellow color was fully consistent with the difference in carotenoid content (i.e. lutein and zeaxanthin) evidenced between the two alternative haplotypes. A significant association between the haplotype, the level of BCMO1 expression and the yellow color of the meat was also recovered in an unrelated commercial broiler population. The mutation could be of economic importance for poultry production by making possible a gene-assisted selection for color, a determining aspect of meat quality. Moreover, this natural genetic diversity constitutes a new model for the study of ß-carotene metabolism which may act upon diverse biological processes as precursor of the vitamin A.

Integrative mapping analysis of chicken microchromosome 16 organization.

BACKGROUND: 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 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. RESULTS: 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 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. CONCLUSIONS: The 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.

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

BACKGROUND: 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. FINDINGS: 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. CONCLUSIONS: Both methods used to obtain reduced representations for high-throughput sequencing were successful after several improvements.The protocols may be used for several sequencing applications, such as de novo sequencing, tagged PCR fragments or long fragment sequencing of cDNA.