Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes.

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3' end of LINE-1_Cfs (i.e., LINE-1_Cf 3'-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.

PTPN11 mutations in canine and human disseminated histiocytic sarcoma.

In humans, histiocytic sarcoma (HS) is an aggressive cancer involving histiocytes. Its rarity and heterogeneity explain that treatment remains a challenge. Sharing high clinical and histopathological similarities with human HS, the canine HS is conversely frequent in specific breeds and thus constitutes a unique spontaneous model for human HS to decipher the genetic bases and to explore therapeutic options. We identified sequence alterations in the MAPK pathway in at least 63.9% (71/111) of HS cases with mutually exclusive BRAF (0.9%; 1/111), KRAS (7.2%; 8/111) and PTPN11 (56.75%; 63/111) mutations concentrated at hotspots common to human cancers. Recurrent PTPN11 mutations are associated to visceral disseminated HS subtype in dogs, the most aggressive clinical presentation. We then identified PTPN11 mutations in 3/19 (15.7%) human HS patients. Thus, we propose PTPN11 mutations as key events for a specific subset of human and canine HS: the visceral disseminated form. Finally, by testing drugs targeting the MAPK pathway in eight canine HS cell lines, we identified a better anti-proliferation activity of MEK inhibitors than PTPN11 inhibitors in canine HS neoplastic cells. In combination, these results illustrate the relevance of naturally affected dogs in deciphering genetic mechanisms and selecting efficient targeted therapies for such rare and aggressive cancers in humans.

FEELnc: a tool for long non-coding RNA annotation and its application to the dog transcriptome.

Whole transcriptome sequencing (RNA-seq) has become a standard for cataloguing and monitoring RNA populations. One of the main bottlenecks, however, is to correctly identify the different classes of RNAs among the plethora of reconstructed transcripts, particularly those that will be translated (mRNAs) from the class of long non-coding RNAs (lncRNAs). Here, we present FEELnc (FlExible Extraction of LncRNAs), an alignment-free program that accurately annotates lncRNAs based on a Random Forest model trained with general features such as multi k-mer frequencies and relaxed open reading frames. Benchmarking versus five state-of-the-art tools shows that FEELnc achieves similar or better classification performance on GENCODE and NONCODE data sets. The program also provides specific modules that enable the user to fine-tune classification accuracy, to formalize the annotation of lncRNA classes and to identify lncRNAs even in the absence of a training set of non-coding RNAs. We used FEELnc on a real data set comprising 20 canine RNA-seq samples produced by the European LUPA consortium to substantially expand the canine genome annotation to include 10 374 novel lncRNAs and 58 640 mRNA transcripts. FEELnc moves beyond conventional coding potential classifiers by providing a standardized and complete solution for annotating lncRNAs and is freely available at https://github.com/tderrien/FEELnc.

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.

Dogs accompanied humans during the Neolithic expansion into Europe.

Near Eastern Neolithic farmers introduced several species of domestic plants and animals as they dispersed into Europe. Dogs were the only domestic species present in both Europe and the Near East prior to the Neolithic. Here, we assessed whether early Near Eastern dogs possessed a unique mitochondrial lineage that differentiated them from Mesolithic European populations. We then analysed mitochondrial DNA sequences from 99 ancient European and Near Eastern dogs spanning the Upper Palaeolithic to the Bronze Age to assess if incoming farmers brought Near Eastern dogs with them, or instead primarily adopted indigenous European dogs after they arrived. Our results show that European pre-Neolithic dogs all possessed the mitochondrial haplogroup C, and that the Neolithic and Post-Neolithic dogs associated with farmers from Southeastern Europe mainly possessed haplogroup D. Thus, the appearance of haplogroup D most probably resulted from the dissemination of dogs from the Near East into Europe. In Western and Northern Europe, the turnover is incomplete and haplogroup C persists well into the Chalcolithic at least. These results suggest that dogs were an integral component of the Neolithic farming package and a mitochondrial lineage associated with the Near East was introduced into Europe alongside pigs, cows, sheep and goats. It got diluted into the native dog population when reaching the Western and Northern margins of Europe.

Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus.

The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles. We present a complete structural and functional reannotation of the Ectocarpus genome. The large-scale assembly of the Ectocarpus genome was significantly improved and genome-wide gene re-annotation using extensive RNA-seq data improved the structure of 11 108 existing protein-coding genes and added 2030 new loci. A genome-wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNAs. Conservation of lncRNAs between Ectocarpus and another brown alga, the kelp Saccharina japonica, suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism-based markers was developed for genetic analyses. These resources are available through an updated and improved genome database. This study significantly improves the utility of the Ectocarpus genome as a high-quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles.

Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping.

The extraordinary phenotypic diversity of dog breeds has been sculpted by a unique population history accompanied by selection for novel and desirable traits. Here we perform a comprehensive analysis using multiple test statistics to identify regions under selection in 509 dogs from 46 diverse breeds using a newly developed high-density genotyping array consisting of >170,000 evenly spaced SNPs. We first identify 44 genomic regions exhibiting extreme differentiation across multiple breeds. Genetic variation in these regions correlates with variation in several phenotypic traits that vary between breeds, and we identify novel associations with both morphological and behavioral traits. We next scan the genome for signatures of selective sweeps in single breeds, characterized by long regions of reduced heterozygosity and fixation of extended haplotypes. These scans identify hundreds of regions, including 22 blocks of homozygosity longer than one megabase in certain breeds. Candidate selection loci are strongly enriched for developmental genes. We chose one highly differentiated region, associated with body size and ear morphology, and characterized it using high-throughput sequencing to provide a list of variants that may directly affect these traits. This study provides a catalogue of genomic regions showing extreme reduction in genetic variation or population differentiation in dogs, including many linked to phenotypic variation. The many blocks of reduced haplotype diversity observed across the genome in dog breeds are the result of both selection and genetic drift, but extended blocks of homozygosity on a megabase scale appear to be best explained by selection. Further elucidation of the variants under selection will help to uncover the genetic basis of complex traits and disease.

MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers.

Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14-26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition.

A canine Arylsulfatase G (ARSG) mutation leading to a sulfatase deficiency is associated with neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) represent the most common group of inherited progressive encephalopathies in children. They are characterized by progressive loss of vision, mental and motor deterioration, epileptic seizures, and premature death. Rare adult forms of NCL with late onset are known as Kufs' disease. Loci underlying these adult forms remain unknown due to the small number of patients and genetic heterogeneity. Here we confirm that a late-onset form of NCL recessively segregates in US and French pedigrees of American Staffordshire Terrier (AST) dogs. Through combined association, linkage, and haplotype analyses, we mapped the disease locus to a single region of canine chromosome 9. We eventually identified a worldwide breed-specific variant in exon 2 of the Arylsulfatase G (ARSG) gene, which causes a p.R99H substitution in the vicinity of the catalytic domain of the enzyme. In transfected cells or leukocytes from affected dogs, the missense change leads to a 75% decrease in sulfatase activity, providing a functional confirmation that the variant might be the NCL-causing mutation. Our results uncover a protein involved in neuronal homeostasis, identify a family of candidate genes to be screened in patients with Kufs' disease, and suggest that a deficiency in sulfatase is part of the NCL pathogenesis.

Genome sequencing of 2000 canids by the Dog10K consortium advances the understanding of demography, genome function and architecture.

BACKGROUND: The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. RESULTS: We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. CONCLUSIONS: We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

A high-resolution map of the Nile tilapia genome: a resource for studying cichlids and other percomorphs.

BACKGROUND: The Nile tilapia (Oreochromis niloticus) is the second most farmed fish species worldwide. It is also an important model for studies of fish physiology, particularly because of its broad tolerance to an array of environments. It is a good model to study evolutionary mechanisms in vertebrates, because of its close relationship to haplochromine cichlids, which have undergone rapid speciation in East Africa. The existing genomic resources for Nile tilapia include a genetic map, BAC end sequences and ESTs, but comparative genome analysis and maps of quantitative trait loci (QTL) are still limited. RESULTS: We have constructed a high-resolution radiation hybrid (RH) panel for the Nile tilapia and genotyped 1358 markers consisting of 850 genes, 82 markers corresponding to BAC end sequences, 154 microsatellites and 272 single nucleotide polymorphisms (SNPs). From these, 1296 markers could be associated in 81 RH groups, while 62 were not linked. The total size of the RH map is 34,084 cR(3500) and 937,310 kb. It covers 88% of the entire genome with an estimated inter-marker distance of 742 Kb. Mapping of microsatellites enabled integration to the genetic map. We have merged LG8 and LG24 into a single linkage group, and confirmed that LG16-LG21 are also merged. The orientation and association of RH groups to each chromosome and LG was confirmed by chromosomal in situ hybridizations (FISH) of 55 BACs. Fifty RH groups were localized on the 22 chromosomes while 31 remained small orphan groups. Synteny relationships were determined between Nile tilapia, stickleback, medaka and pufferfish. CONCLUSION: The RH map and associated FISH map provide a valuable gene-ordered resource for gene mapping and QTL studies. All genetic linkage groups with their corresponding RH groups now have a corresponding chromosome which can be identified in the karyotype. Placement of conserved segments indicated that multiple inter-chromosomal rearrangements have occurred between Nile tilapia and the other model fishes. These maps represent a valuable resource for organizing the forthcoming genome sequence of Nile tilapia, and provide a foundation for evolutionary studies of East African cichlid fishes.

Annotation of the domestic dog genome sequence: finding the missing genes.

There are over 350 genetically distinct breeds of domestic dog that present considerable variation in morphology, physiology, and disease susceptibility. The genome sequence of the domestic dog was assembled and released in 2005, providing an estimated 20,000 protein-coding genes that are a great asset to the scientific community that uses the dog system as a genetic biomedical model and for comparative and evolutionary studies. Although the canine gene set had been predicted using a combination of ab initio methods, homology studies, motif analysis, and similarity-based programs, it still requires a deep annotation of noncoding genes, alternative splicing, pseudogenes, regulatory regions, and gain and loss events. Such analyses could benefit from new sequencing technologies (RNA-Seq) to better exploit the advantages of the canine genetic system in tracking disease genes. Here, we review the catalog of canine protein-coding genes and the search for missing genes, and we propose rationales for an accurate identification of noncoding genes though next-generation sequencing.

The genome and diet of a 35,000-year-old Canis lupus specimen from the Paleolithic painted cave, Chauvet-Pont d'Arc, France.

The Chauvet-Pont-d'Arc Cave (Ardèche, France) contains some of the oldest Paleolithic paintings recorded to date, as well as thousands of bones of the extinct cave bear, and some remains and footprints of other animals. As part of the interdisciplinary research project devoted to this reference cave site, we analyzed a coprolite collected within the deep cave. AMS radiocarbon dating of bone fragments from the coprolite yielded an age of 30,450 ± 550 RC yr. BP (AAR-19656; 36,150-34,000 cal BP), similar to ages assigned to Paleolithic artwork and cave bear remains from the same cave sector. Using high-throughput shotgun DNA sequencing, we demonstrated a high abundance of canid DNA and lesser amounts of DNA from the extinct cave bear. We interpret the sample as feces from a canid that had consumed cave bear tissue. The high amount of canid DNA allowed us to reconstruct a complete canid mitochondrial genome sequence (average coverage: 83×) belonging to a deeply divergent clade of extinct mitochondrial wolf lineages that are most closely related to coeval (~35 ka) Belgian wolves. Analysis of the nuclear genome yielded a similar coverage for the X chromosome (2.4×) and the autosomes (range: 2.3-3.2×), indicating that the Chauvet canid was a female. Comparing the relationship of the nuclear genome of this specimen with that of a variety of canids, we found it more closely related to gray wolves' genomes than to other wild canid or dog genomes, especially wolf genomes from Europe and the Middle East. We conclude that the coprolite is feces from an animal within an extinct wolf lineage. The consumption of cave bear by this wolf likely explains its intrusion into the dark cave sectors and sheds new light on the paleoecology of a major cave site.

Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets.

Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT, was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4, and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7, GABPB1, and SPPL2A, located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

A radiation hybrid map of the European sea bass (Dicentrarchus labrax) based on 1581 markers: Synteny analysis with model fish genomes.

The selective breeding of fish for aquaculture purposes requires the understanding of the genetic basis of traits such as growth, behaviour, resistance to pathogens and sex determinism. Access to well-developed genomic resources is a prerequisite to improve the knowledge of these traits. Having this aim in mind, a radiation hybrid (RH) panel of European sea bass (Dicentrarchus labrax) was constructed from splenocytes irradiated at 3000 rad, allowing the construction of a 1581 marker RH map. A total of 1440 gene markers providing ~4400 anchors with the genomes of three-spined stickleback, medaka, pufferfish and zebrafish, helped establish synteny relationships with these model species. The identification of Conserved Segments Ordered (CSO) between sea bass and model species allows the anticipation of the position of any sea bass gene from its location in model genomes. Synteny relationships between sea bass and gilthead seabream were addressed by mapping 37 orthologous markers. The sea bass genetic linkage map was integrated in the RH map through the mapping of 141 microsatellites. We are thus able to present the first complete gene map of sea bass. It will facilitate linkage studies and the identification of candidate genes and Quantitative Trait Loci (QTL). The RH map further positions sea bass as a genetic and evolutionary model of Perciformes and supports their ongoing aquaculture expansion.

Dog10K_Boxer_Tasha_1.0: A Long-Read Assembly of the Dog Reference Genome.

The domestic dog has evolved to be an important biomedical model for studies regarding the genetic basis of disease, morphology and behavior. Genetic studies in the dog have relied on a draft reference genome of a purebred female boxer dog named "Tasha" initially published in 2005. Derived from a Sanger whole genome shotgun sequencing approach coupled with limited clone-based sequencing, the initial assembly and subsequent updates have served as the predominant resource for canine genetics for 15 years. While the initial assembly produced a good-quality draft, as with all assemblies produced at the time, it contained gaps, assembly errors and missing sequences, particularly in GC-rich regions, which are found at many promoters and in the first exons of protein-coding genes. Here, we present Dog10K_Boxer_Tasha_1.0, an improved chromosome-level highly contiguous genome assembly of Tasha created with long-read technologies that increases sequence contiguity >100-fold, closes >23,000 gaps of the CanFam3.1 reference assembly and improves gene annotation by identifying >1200 new protein-coding transcripts. The assembly and annotation are available at NCBI under the accession GCF_000002285.5.

Donskoy cats as a new model of oculocutaneous albinism with the identification of a splice-site variant in Hermansky-Pudlak Syndrome 5 gene.

In the feline Donskoy breed, a phenotype that breeders call "pink-eye," with associated light-brown skin, yellow irises and red-eye effect, has been described. Genealogical data indicated an autosomal recessive inheritance pattern. A single candidate region was identified by genome-wide association study and SNP-based homozygosity mapping. Within that region, we further identified HPS5 (HPS5 Biogenesis Of Lysosomal Organelles Complex 2 Subunit 2) as a strong candidate gene, since HPS5 variants have been identified in humans and animals with Hermansky-Pudlak syndrome 5 or oculocutaneous albinism. A homozygous c.2571-1G>A acceptor splice-site variant located in intron 16 of HPS5 was identified in pink-eye cats. Segregation of the variant was 100% consistent with the inheritance pattern. Genotyping of 170 cats from 19 breeds failed to identify a single carrier in non-Donskoy cats. The c.2571-1G>A variant leads to HPS5 exon-16 splicing that is predicted to produce a 52 amino acids in-frame deletion in the protein. These results support an association of the pink-eye phenotype with the c.2571-1G>A variant. The pink-eye Donskoy cat extends the panel of reported HPS5 variants and offers an opportunity for in-depth exploration of the phenotypic consequences of a new HPS5 variant.

Genetic diversity of canine olfactory receptors.

BACKGROUND: Evolution has resulted in large repertoires of olfactory receptor (OR) genes, forming the largest gene families in mammalian genomes. Knowledge of the genetic diversity of olfactory receptors is essential if we are to understand the differences in olfactory sensory capability between individuals. Canine breeds constitute an attractive model system for such investigations. RESULTS: We sequenced 109 OR genes considered representative of the whole OR canine repertoire, which consists of more than 800 genes, in a cohort of 48 dogs of six different breeds. SNP frequency showed the overall level of polymorphism to be high. However, the distribution of SNP was highly heterogeneous among OR genes. More than 50% of OR genes were found to harbour a large number of SNP, whereas the rest were devoid of SNP or only slightly polymorphic. Heterogeneity was also observed across breeds, with 25% of the SNP breed-specific. Linkage disequilibrium within OR genes and OR clusters suggested a gene conversion process, consistent with a mean level of polymorphism higher than that observed for introns and intergenic sequences. A large proportion (47%) of SNP induced amino-acid changes and the Ka/Ks ratio calculated for all alleles with a complete ORF indicated a low selective constraint with respect to the high level of redundancy of the olfactory combinatory code and an ongoing pseudogenisation process, which affects dog breeds differently. CONCLUSION: Our demonstration of a high overall level of polymorphism, likely to modify the ligand-binding capacity of receptors distributed differently within the six breeds tested, is the first step towards understanding why Labrador Retrievers and German Shepherd Dogs have a much greater potential for use as sniffer dogs than Pekingese dogs or Greyhounds. Furthermore, the heterogeneity in OR polymorphism observed raises questions as to why, in a context in which most OR genes are highly polymorphic, a subset of these genes is not? This phenomenon may be related to the nature of their ligands and their importance in everyday life.

Revisiting the missing protein-coding gene catalog of the domestic dog.

BACKGROUND: Among mammals for which there is a high sequence coverage, the whole genome assembly of the dog is unique in that it predicts a low number of protein-coding genes, ~19,000, compared to the over 20,000 reported for other mammalian species. Of particular interest are the more than 400 of genes annotated in primates and rodent genomes, but missing in dog. RESULTS: Using over 14,000 orthologous genes between human, chimpanzee, mouse rat and dog, we built multiple pairwise synteny maps to infer short orthologous intervals that were targeted for characterizing the canine missing genes. Based on gene prediction and a functionality test using the ratio of replacement to silent nucleotide substitution rates (d(N)/d(S)), we provide compelling structural and functional evidence for the identification of 232 new protein-coding genes in the canine genome and 69 gene losses, characterized as undetected gene or pseudogenes. Gene loss phyletic pattern analysis using ten species from chicken to human allowed us to characterize 28 canine-specific gene losses that have functional orthologs continuously from chicken or marsupials through human, and 10 genes that arose specifically in the evolutionary lineage leading to rodent and primates. CONCLUSION: This study demonstrates the central role of comparative genomics for refining gene catalogs and exploring the evolutionary history of gene repertoires, particularly as applied for the characterization of species-specific gene gains and losses.