Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals. Received: 15 March 2000 / Accepted: 27 July 2000     

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). Received: 22 December 1997 / Accepted: 16 March 1998     

Characterization and radiation hybrid mapping of expressed sequence tags from the canine brain

Abstract Maps of the canine genome are now developing rapidly. Most of the markers on the current integrated canine radiation hybrid/genetic linkage/cytogenetic map are highly polymorphic microsatellite (type II) markers that are very useful for mapping disease loci. However, there is still an urgent need for the mapping of gene-based (type I) markers that are required for comparative mapping, as well as identifying candidate genes for disease loci that have been genetically mapped. We constructed an adult brain cDNA library as a resource to increase the number of gene-based markers on the canine genome map. Eighty-one percent of the 2700 sequenced expressed sequence tags (ESTs) represented unique sequences. The canine brain ESTs were compared with sequences in public databases to identify putative canine orthologs of human genes. One hundred nine of the canine ESTs were mapped on the latest canine radiation hybrid (RH) panel to determine the location of the respective canine gene. The addition of these new gene-based markers revealed three conserved segments (CS) between human and canine genomes previously detected by fluorescence in situ hybridization (FISH), but not by RH mapping. In addition, five new CS between dog and human were identified that had not been detected previously by RH mapping or FISH. This work has increased the number of gene-based markers on the canine RH map by approximately 30% and indicates the benefit to be gained by increasing the gene content of the current canine comparative map.     

Use of flow-sorted canine chromosomes in the assignment of canine linkage, radiation hybrid, and syntenic groups to chromosomes: refinement and verification of the comparative chromosome map for dog and human

The mapping of the canine genome has recently been accelerated by the availability of chromosome-specific reagents and publication of radiation hybrid (RH), genetic linkage, and dog/human comparative maps, but the assignment of mapping groups to chromosomes is incomplete. To assign published radiation hybrid, linkage, and "syntenic" groups to chromosomes, individual markers found within each group have been amplified from canine and vulpine flow-sorted, chromosome-specific DNAs as templates. Here a further 102 type I genetic markers (previously mapped in human) and 21 further type II markers are assigned to canine chromosomes using marker-specific PCR. We have assigned all linkage, RH, and syntenic groups in the two most recently published canine genome maps to chromosomes. This demonstrates directly that there is at least one published mapping group for each of the 38 canine autosomes and thus that the coverage of the canine chromosome map is approaching completion. The dog/human comparative map is one of the most complex so far described, with 90 separate segments of chromosomal homology previously seen in dog-on-human cross-species chromosome-painting studies. The total of 142 type I markers now placed on canine chromosomes using this method of marker mapping has allowed us to confirm the placement of the great majority (83) of the 90 homologous segments. The positions of the remaining homologous segments were confirmed in new cross-species chromosome-painting experiments (dog-on-human, fox-on-human).     

A high-resolution comparative map of canine Chromosome 5q14.3–q33 constructed utilizing the 1.5× canine genomesequence

A high-density map of the region of canine Chromosome 5 (CFA5) surrounding the evolutionary breakpoint between human Chromosomes 1p32 and 17p11 was constructed by integrating a radiation hybrid map including 41 microsatellites, 10 BACs, and 59 genes and a linkage map including 18 markers. A collection of canine genomic survey sequences providing 1.5× coverage was used to identify dog orthologs of human genes, proving instrumental in the development of this map. Of particular interest is the canine BHD gene, within which we have previously described a single nucleotide polymorphism associated with Hereditary Multifocal Renal Cystadenocarcinoma and Nodular Dermatofibrosis (RCND) in German Shepherd dogs. The corresponding region of the human genome is particularly gene rich, containing genes involved in development, metabolism, and cancer that are likely to be of interest in future mapping studies. This current mapping effort on CFA5 expands the degree to which initial findings of linkage in canine families can be followed by successful positional cloning efforts and increases the value of the human genome sequence for defining candidate genes. Moreover, this study demonstrates the utility of genomic survey sequences when combined with accurate genome maps for rapid mapping of disease susceptibility loci.     

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.     

Fifty-four new gene-based canine microsatellite markers

Fifty-four new markers were developed to fill in gaps in the current map of canine microsatellites and to complement existing markers that may not be sufficiently informative in highly inbred canine pedigrees. Canine genes contained on the radiation hybrid map were used to obtain the sequence of the human homolog. A BLAST search versus the canine whole genome shotgun (wgs) sequence resource was used to obtain the sequence of the canine genomic contigs containing the homolog of the corresponding human gene. Canine sequences that contained microsatellites and mapped back to the correct location in the human genome were used to design primers for amplification of the microsatellites from canine genomic DNA. Heterozygosities of the markers were tested by genotyping grandparental DNAs obtained from the Nestle Purina Reference family DNA distribution center plus DNAs from unrelated Bouviers and Irish wolfhounds. Canine map positions of markers on the July 2004 freeze of the canine genome assembly were determined by in silico PCR or BLAST.     

Comparative mapping in the pig: localization of 214 expressed sequence tags

In total, 214 ESTs (Expressed Sequence Tags) were assigned to the porcine gene map by using somatic cell hybrid mapping, radiation hybrid mapping, and FISH. The ESTs were isolated from a porcine small intestine cDNA library on the basis of significant sequence identity with human annotated genes. In total, 390 primer pairs were designed primarily in the 3' UTR of the sequences. Overall, 58.6% of the ESTs were successfully mapped by this approach. In total, 191 of the localizations are in agreement with the human comparative map, strongly indicating that these represent true orthologous genes. The remaining 23 ESTs provide new comparative mapping data, which should be considered as preliminary until confirmed by other studies. Our mapping efforts provide a significant contribution to the porcine map as well as to the comparative map for human and pig.     

Assignment of 60 human ESTs in cattle

As part of the human genome study, large-scale cDNA sequencing has produced thousands of Expressed Sequence Tags (ESTs). Généthon has mapped in human 10,000 of these ESTs and has shown that the primers of about 1000 ESTs could amplify bovine DNA. In this work, we have analyzed 233 primer pairs provided by Genethon, to assign type I sequences to the bovine genome by using a hamster-bovine somatic cell hybrid panel. Among these 233 primer pairs, 109 gave a specific PCR product with bovine genomic DNA, but for 50% the size of the PCR product was the same in cattle and hamster, requiring SSCP analysis. Finally, 60 ESTs were assigned to the bovine genome, and among them 46 were found on the bovine chromosome expected from heterologous painting data between cattle and human. Received: 16 December 1999 / Accepted: 6 May 2000     

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. Received: 10 June 1999 / Accepted: 23 September 1999     

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.     

Digging up the canine genome--a tale to wag about

There is incredible morphological and behavioral diversity among the hundreds of breeds of the domestic dog, CANIS FAMILIARIS. Many of these breeds have come into existence within the last few hundred years. While there are obvious phenotypic differences among breeds, there is marked interbreed genetic homogeneity. Thus, study of canine genetics and genomics is of importance to comparative genomics, evolutionary biology and study of human hereditary diseases. The most recent version of the map of the canine genome is comprised of 3,270 markers mapped to 3,021 unique positions with an average intermarker distance of approximately 1 Mb. The markers include approximately 1,600 microsatellite markers, about 1,000 gene-based markers, and almost 700 bacterial artificial chromosome-end markers. Importantly, integration of radiation hybrid and linkage maps has greatly enhanced the utility of the map. Additionally, mapping the genome has led directly to characterization of microsatellite markers ideal for whole genome linkage scans. Thus, workers are now able to exploit the canine genome for a wide variety of genetic studies. Finally, the decision to sequence the canine genome highlights the dog's evolutionary and physiologic position between the mouse and human and its importance as a model for study of mammalian genetics and human hereditary diseases.     

A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps

Cross-species reciprocal chromosome painting was used to delineate homologous chromosomal segments between domestic dog, red fox, and human. Whole sets of chromosome-specific painting probes for the red fox and dog were made by PCR amplification of flow-sorted chromosomes from established cell cultures. Based on their hybridization patterns, a complete comparative chromosome map of the three species has been built. Thirty-nine of the 44 synteny groups from the published radiation hybrid map and 33 of the 40 linkage groups in the linkage map of the dog have been assigned to specific chromosomes by fluorescence in situ hybridization and PCR-based genotyping. Each canine chromosome has at least one DNA marker assigned to it. The human-canid map shows that the canid karyotypes are among the most extensively rearranged karyotypes in mammals. Twenty-two human autosomal paints delineated 73 homologous regions on 38 canine autosomes, while paints from 38 dog autosomes detected 90 homologous segments in the human genome. Of the 22 human autosomes, only the syntenies of three chromosomes (14, 20, and 21) have been maintained intact in the canid genome. The dog-fox map and DAPI banding comparison demonstrate that the remarkable karyotype differences between fox (2n = 34 + 0-8 Bs) and dog (2n = 78) are due to 26 chromosomal fusion events and 4 fission events. It is proposed that the more easily karyotyped fox chromosomes can be used as a common reference and control system for future gene mapping in the DogMap project and CGH analysis of canine tumor DNA.     

Anchoring of canine linkage groups with chromosome-specific markers

A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest. Received: 23 February 1999 / Accepted: 28 April 1999     

A radiation hybrid map of the X-chromosome of the dog (Canis familiaris)

The dog serves as an animal model for several human diseases including X-chromosome diseases. Although the canine X-chromosome is one of the largest chromosomes in the dog, only a few markers have been mapped to it to date. Using a commercially available canine whole genome radiation hybrid (RH) panel we have localized 14 microsatellite markers, 18 genes and 13 STSs on the canine X-chromosome, extending the total number of mapped markers to 45 covering an estimated 830 cR. Out of these 45 markers, seven distinct groups of markers could be established with an average spacing of 18.8 cR(3000) and ten markers remained unlinked. Using FISH analysis, six markers could be mapped physically to the p- or q-arm of the X-chromosome. Combined with the FISH mapping, three RH groups could be assigned to the p-arm and two RH groups to the q-arm. Comparison with the human X-chromosome map revealed conserved synteny up to 234 cR (TIMP1-ALAS2-AR-IL2RG-XIST). We show here that the similarity of the canine and human X-chromosomes is the largest for any mammalian species beyond the primates.     

Physical and radiation hybrid mapping of canine chromosome 12, in a region corresponding to human chromosome 6p12-q12

The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited.     

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.     

Mapping of EST- and STS-markers in the human genome using a panel fo radiation hybrids

Ten DNA markers were localized in the human genome by a screening procedure against the radiation hybrid somatic cell panel (GeneBridge 4 RH Panel) using polymerase chain reaction (RH mapping method). DNA markers were developed to nucleotide sequences adjacent to NotI sites of human chromosome 3 (NotI-STS markers) and also to nucleotide sequences of human cDNA (EST markers). Three EST markers mapped (B10164, S16R and 18F5R) were localized in the human genome for the first time. Marker B10164 was found to be homologous to the nucleotide sequence of the BASP1 gene coding a major receptor protein. Markers S16R and 18F5R presumably tagged new genes, because no homologies were revealed among the nucleotide sequences presented in the databases. For four NotI-STS, more precise localization on human chromosome 3 was determined. On the basis of the data obtained, the NotI map may be integrated with other types of physical maps of human chromosome 3. RH mapping with a standard commercial panel of radiation hybrid somatic cells provided a chance to integrate the data obtained into international databases and existing integrated human chromosomal maps.     

A high-resolution radiation hybrid map of the proximal region of rat Chromosome 4

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment. Received: 4 December 1998 / Accepted: 19 January 1999     

Cloning, characterization, and physical mapping of the canine Prop-1 gene (PROP1): exclusion as a candidate for combined pituitary hormone deficiency in German shepherd dogs

Abnormalities in the genes encoding Pit-1 and Prop-1 have been reported to cause combined pituitary hormone deficiency (CPHD) in mice and humans. In dogs, a similar phenotype has been described in the German shepherd breed. We have previously reported that the Pit-1 gene (POU1F1) is not mutated in affected German shepherd dogs. In this study, we report the isolation and mapping of the canine Prop-1 gene (PROP1), and we assessed the involvement of PROP1 in German shepherd dog dwarfism. The canine PROP1 gene was found to contain three exons, encoding a 226 amino acid protein. The deduced amino acid sequence was 79% and 84% homologous with the mouse and human Prop-1 protein, respectively. Using fluorescence in situ hybridization, PROP1 was mapped to canine chromosome 11. Further mapping with a canine radiation hybrid panel showed co-localization with the polymorphic DNA marker AHT137. Sequence analysis of genomic DNA from dwarf German shepherd dogs revealed no alterations in the PROP1 gene. Moreover, linkage analysis of AHT137 revealed no co-segregation between the PROP1 locus and the CPHD phenotype, excluding this gene as candidate for canine CPHD and providing a new spontaneous model of hypopituitarism.