Contribution of radiation hybrids to genome mapping in domestic animals.

Radiation hybrid mapping has emerged in the end of the 1990 s as a successful and complementary approach to map genomes, essentially because of its ability to bridge the gaps between genetic and clone-based physical maps, but also using comparative mapping approaches, between 'gene-rich' and 'gene-poor' maps. Since its early development in human, radiation hybrid mapping played a pivotal role in the process of mapping animal genomes, especially mammalian ones. We review here all the different steps involved in radiation hybrid mapping from the constitution of panels to the construction of maps. A description of its contribution to whole genome maps with a special emphasis on domestic animals will also be presented. Finally, current applications of radiation hybrid mapping in the context of whole genome assemblies will be described.

A comparative genome approach to marker ordering.

MOTIVATION: Genome maps are fundamental to the study of an organism and essential in the process of genome sequencing which in turn provides the ultimate map of the genome. The increased number of genomes being sequenced offers new opportunities for the mapping of closely related organisms. We propose here an algorithmic formalization of a genome comparison approach to marker ordering. RESULTS: In order to integrate a comparative mapping approach in the algorithmic process of map construction and selection, we propose to extend the usual statistical model describing the experimental data, here radiation hybrids (RH) data, in a statistical framework that models additionally the evolutionary relationships between a proposed map and a reference map: an existing map of the corresponding orthologous genes or markers in a closely related organism. This has concretely the effect of exploiting, in the process of map selection, the information of marker adjacencies in the related genome when the information provided by the experimental data is not conclusive for the purpose of ordering. In order to compute efficiently the map, we proceed to a reduction of the maximum likelihood estimation to the Traveling Salesman Problem. Experiments on simulated RH datasets as well as on a real RH dataset from the canine RH project show that maps produced using the likelihood defined by the new model are significantly better than maps built using the traditional RH model. AVAILABILITY: The comparative mapping approach is available in the last version of de Givry,S. et al. [(2004) Bioinformatics, 21, 1703-1704, www.inra.fr/mia/T/CarthaGene], a free (the LKH part is free for academic use only) mapping software in C++, including LKH (Helsgaun,K. (2000) Eur. J. Oper. Res., 126, 106-130, www.dat.ruc.dk/keld/research/LKH) for maximum likelihood computation.

Detailed four-way comparative mapping and gene order analysis of the canine ctvm locus reveals evolutionary chromosome rearrangements.

Canine tricuspid valve malformation (CTVM) maps to canine chromosome 9 (CFA9), in a region syntenic with gene-dense human chromosome 17q. To define synteny blocks, we analyzed 148 markers on CFA9 using radiation hybrid mapping and established a four-way comparative map for human, mouse, rat, and dog. We identified a large number of rearrangements, allowing us to reconstruct the evolutionary history of individual synteny blocks and large chromosomal segments. A most parsimonious rearrangement scenario for all four species reveals that human chromosome 17q differs from CFA9 and the syntenic rodent chromosomes through two macroreversals of 9.2 and 23 Mb. Compared to a recovered ancestral gene order, CFA9 has undergone 11 reversals of <3 Mb and 2 reversals of >3 Mb. Interspecies reuse of breakpoints for micro- and macrorearrangements was observed. Gene order and content of the ctvm interval are best extrapolated from murine data, showing that multispecies genome rearrangement scenarios contribute to identifying gene content in canine mapping studies.

Comparison of MultiMap and TSP/CONCORDE for constructing radiation hybrid maps.

Radiation hybrid (RH) map construction allows investigators to locate both type I and type II markers on a given genome map. The process is composed of two steps. The first consists of determining the pattern distribution of a set of markers within the different cell lines of an RH panel. This is mainly done by polymerase chain reaction (PCR) amplification and gel electrophoresis, and results in a series of numbers indicating the presence or the absence of each marker in each cell line. The second step consists of a comparison of these numbers, using various algorithms, to group and then order markers. Because different algorithms may provide (slightly) different orders, we have compared the merits of the MultiMap and TSP/CONCORDE packages using a data set of information currently under analysis for construction of the canine genome RH map.

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.

The LAMMER protein kinase encoded by the Doa locus of Drosophila is required in both somatic and germline cells and is expressed as both nuclear and cytoplasmic isoforms throughout development.

Activity of the Darkener of apricot (Doa) locus of Drosophila melanogaster is required for development of the embryonic nervous system, segmentation, photoreceptor maintenance, normal transcription, and sexual differentiation. The gene encodes a protein kinase, with homologues throughout eukaryotes known as the LAMMER kinases. We show here that DOA is expressed as at least two different protein isoforms of 105 and 55 kD throughout development, which are primarily localized to the cytoplasm and nucleus, respectively. Doa transcripts and protein are expressed in all cell types both during embryogenesis and in imaginal discs. Although it was recently shown that DOA kinase is essential for normal sexual differentiation, levels of both kinase isoforms are equal between the sexes during early pupal development. The presence of the kinase on the cell membrane and in the nuclei of polytene salivary gland cells, as well as exclusion from the nuclei of specific cells, may be indicative of regulated kinase localization. Mosaic analysis in both the soma and germline demonstrates that Doa function is essential for cell viability. Finally, in contrast to results reported in other systems and despite some phenotypic similarities, genetic data demonstrate that the LAMMER kinases do not participate in the ras-MAP kinase signal transduction pathway.

Using the fluorogenic 5' nuclease assay for high-throughput detection of (CA)n repeats in radiation hybrid mapping.

Here, the power of the 5' nuclease assay to detect PCR products containing (CA)n repeats was compared with that of the classical electrophoretic analysis. This assay, which relies on the use of a unique (CA)10 energy transfer-labeled probe and the 5' nuclease activity of Taq DNA polymerase, was used to construct a dog radiation hybrid map consisting of microsatellite markers. Data from over 7000 PCRs were analyzed in parallel by the fluorogenic assay and the conventional ethidium bromide-stained, agarose gel-based assay. We show that the fluorogenic assay provides a sensitive, reliable and specific method for detecting (CA)n amplimers. Moreover, as no processing is required after the PCR, the risk of carryover contamination and the time required for sample analysis are greatly reduced. All radiation hyrid (RH) assays can be performed using a single PCR protocol, and a standard analysis method has been developed that enables numerically automated data processing. On the whole, using this strategy greatly enhanced the rapidity, throughput and accuracy of the RH mapping of microsatellite markers.

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.

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.

A 356-Kb sequence of the subtelomeric part of the MHC Class I region.

The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3. The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence. Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases. Most of the ESTs are clustered in two regions. In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes.

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.