Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes.

We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest.

Identification and characterization of a set of 100 tri- and dinucleotide microsatellites in the canine genome.

A set of 100 canine microsatellite markers--83 dinucleotides and 17 trinucleotides--is reported. A study of their frequency in the dog genome showed that, while the frequency of the CA repeats is one (CA)n every 47 kb, the 10 trinucleotidic frequencies vary from one every 117 kb (AGG)n to one every 875 kb (AGT)n. Polymorphism analysis performed on 16 unrelated mongrel dogs showed that 80% of dinucleotides are polymorphic, while only 30% of the trinucleotides are so. Of this set of 100 markers, 56 have been mapped on the RHDF5000 dog/hamster whole genome radiation hybrid panel. Moreover, through systematic BLAST analogy searches of the microsatellite-containing clone sequence, three new dog genes could be identified, based on their human ortholog. All of the markers presented may prove useful in physical mapping methods, and polymorphic microsatellites in genetic linkage studies or parentage controls in dog.

An integrated linkage-radiation hybrid map of the canine genome.

Purebred dogs are a unique resource for dissecting the molecular basis of simple and complex genetic diseases and traits. As a result of strong selection for physical and behavioral characteristics among the 300 established breeds, modern dogs are characterized by high levels of interbreed variation, complemented by significant intrabreed homogeneity. A high-resolution map of the canine genome is necessary to exploit the mapping power of this unusual resource. We describe here the integration of an expanded canine radiation hybrid map, comprised of 600 markers, with the latest linkage map of 341 markers, to generate a map of 724 markers-the densest map of the canine genome described to date. Through the inclusion of 217 markers on both the linkage and RH maps, the 77 RH groups are reduced to 44 syntenic groups, thus providing comprehensive coverage of most of the canine genome.

Five new linkage groups in the canine linkage map.

Nineteen further polymorphic loci were typed on the DogMap reference panel. Five new linkage groups were identified. Additionally, five markers were added to earlier defined linkage groups. Three of the new linkage groups contain markers mapped earlier to specific dog chromosomes by physical mapping. These results make a further contribution to the canine genome map and provides more linkage groups physically assigned to known chromosomes.

Characterization of 463 type I markers suitable for dog genome mapping.

In total, 463 canine gene markers were identified and characterized to serve as reagents in canine genome map projects. These markers are distributed over 221 canine gene markers, 139 TOASTs (Traced Orthologous Sequence Tags), 27 canine TOASTs, and 76 huESTs (human Expressed Sequence Tags). Out of 310 canine gene markers, 59%-84% were successfully amplified on dog DNA, the highest rates of success being observed when the exon/intron structure is known. Concerning TOASTs and human ESTs, of the 225 and 300 markers analyzed, 62% and 25% respectively were able to produce a dog positive amplification. As part of an ongoing project to map the canine genome using a dog/hamster radiation hybrid panel, these markers were tested for their specificity on dog versus hamster DNA. Thus 61%, 21%, and 12% of dog gene markers, TOASTs, and huESTs met the criteria required for radiation hybrid mapping, respectively. All of these 463 canine gene markers, however, are available and will be of value to any other mapping strategies.

Construction and optimization of a dog whole-genome radiation hybrid panel.

A dog whole-genome radiation hybrid (WGRH) panel including 126 clones was constructed by fusing dog fibroblasts irradiated at 5000 rads with thymidine kinase-deficient hamster cells. The average retention frequency of the panel designated as RHDF5000 is 21%, and its resolution power is estimated at 600 kb. The data provided by typing 400 markers were used to estimate linkage power changes subsequent to panel reduction. These changes were analyzed by recomputing typing data from five reduced panels. From these simulations, the parameters allowing investigation of the evolution of the linkage power in the course of panel reduction were determined. Guidelines for constructing a WGRH panel are proposed.

Toward a dog radiation hybrid map.

Dog fibroblasts grown from a biopsy performed in a male mongrel were fused after gamma irradiation with thymidine kinase-deficient hamster cells and cultivated in selection medium. A total of 148 clones were obtained and screened by means of PCR amplification using primers corresponding to a dog-specific short repetitive element and to dog microsatellites and genes. One hundred seven cell lines were selected and grown in roller bottles and the distribution of 39 markers was analyzed in the extracted DNA. The results clearly indicate that this panel of hybrid cell lines should prove invaluable for constructing a map of the canine genome. In parallel, for more than 500 microsatellites present in the databases or screened from two libraries of short inserts, we have determined PCR conditions favoring dog-specific products even in the presence of hamster DNA. These highly polymorphic microsatellites should be useful in further linkage studies. We have also characterized 254 markers: dog genes, human expressed sequenced tags (huESTs), and traced orthologous amplified sequenced tags (TOASTs). Once mapped, these will constitute powerful tools to detect regions of conserved synteny in human and other mammalian genomes.

Identification and analysis of the dog keratin 9 (KRT9) gene.

We have identified the gene coding for the canine ortholog of the human keratin 9 protein using the inverse-polymerase chain reaction (PCR) strategy. Sequence comparison and structure analysis of the gene show marked similarity with the human gene. This gene spans about 7 kb and spreads over eight exons. In the dog gene, the reading frame is extended by 20 codons, the first in-frame stop codon being in exon 8 in the dog rather than in exon 7 as in humans. Alignment of human and dog predicted amino acid sequences confirms the high analogy, reaching 75% identity and 95% similarity in the rod domain. Interestingly, the glycine-loop motif number in the C-terminal V2 variable subdomain of the protein increases from 19 in human to 43 in dog, generating a size difference of 12 kDa between the two proteins. Due to its restricted expression pattern in mammalian epidermis, dog keratin 9 gene was a good candidate gene for the genetic palmoplantar hyperkeratosis observed in the Dogue de Bordeaux. However, no polymorphism associated with the pathology was detected within an affected Dogue de Bordeaux pedigree ruling out this hypothesis.

[The importance of the canine model in medical genetics]. / Intérêt du modèle canin pour la génétique médicale.

Dog domestication dates back to as early as 100,000 years ago, or 10,000 years depending upon the data used, and nowadays more than 350 breeds are duly registered in the different kennel clubs around the world. Due to intensive selection in the course of breeding, dog presently comes in any shape, size, color one can imagine, in addition to displaying a wide panel of characters, capacities and behaviours. As a consequence of excessive breeding, numerous breeds are plagued by a large variety of genetic diseases, many of them resembling those observed in human. All this makes dog an attractive model to track down genes and alleles responsible for those phenotypic behavioural or pathological traits, provided a genome map with polymorphic markers, and genes is available.

Traced orthologous amplified sequence tags (TOASTs) and mammalian comparative maps.

Progress on mammalian comparative maps could be significantly accelerated by developing reagents defining orthologous landmarks in the genome of many mammalian species. Using the large databases of gene sequences, we designed 225 orthologous gene-specific primer pairs corresponding to 146 functional genes. Of these 225 primer pairs, 155 (68.9%), 182 (80.9%), 126 (56.0%), and 82 (36.4%) produced a single PCR product when tested against human, pig, dog, and hamster genomic DNA, respectively. In addition to the general rules of primer designing, particular factors must be taken into consideration when choosing gene-specific universal primers--for instance, preference for single-exon regions or highly conserved segments among species, avoidance of GC-rich regions. Sequencing all the canine PCR products traced by these primers demonstrated that of 123 traced canine fragments with readable and reliable sequences, 121 (98.4%) were found to match the GenBank orthologous sequences used for designing the primers, after a BLAST search. Comparative characterization of PCR fragments among human, pig, dog, and hamster revealed that the length of a single exon was much conserved among species, with few exceptions. As the fragments were traced with amplification by orthologous gene-specific primers, we suggest they be termed Traced Orthologous Amplified Sequence Tags (TOASTs).

A whole-genome radiation hybrid map of the dog genome.

A whole genome radiation hybrid (RH) map of the canine genome was constructed by typing 400 markers, including 218 genes and 182 microsatellites, on a panel of 126 radiation hybrid cell lines. Fifty-seven RH groups have been determined with lod scores greater than 6, and 180 framework landmarks were ordered with odds greater than 1000:1. Average spacing between adjacent markers is 23 cR5000, an estimated physical distance of 3.8 Mb. Fourteen groups have been assigned to 9 of the canine chromosomes, and a comparison of RH and genetic groups allowed the successful bridging of both types of data on one map composed of 31 RH and 13 syntenic RH groups. Comparison of canine, human, mouse, and pig maps underlined regions of conserved synteny. This integrated map, covering an estimated 80% of the dog genome, should prove a powerful tool for localizing and identifiying genes implicated in pathological and phenotypical traits.