Radiation mapping of the short arm of swine chromosome 2

In recent years, maps of mammalian genomes have been acquiring increasingly higher resolution. Integration of maps of different types has become possible. As a tool in integrating maps of mammalian genomes of different types, high-resolution mapping with radiation-induced hybrids (RH) is used. Here, we present an RH6000 map of the short arm of porcine chromosome 2. The map contains 15 microsatellites and five genes (for parathyroid hormone, lactate dehydrogenase A, myogenic factor, follicle-stimulating hormone beta, and calpain I). The RH panel was obtained on the basis of a hybrid cell line bearing the single porcine chromosome 2 against the background of mink chromosomes. The mean frequency of preserving markers examined in the panel was 18.3%. Integration of four genes in the panel and a comparison of gene order in homologous regions of human and porcine chromosomes are presented.     

Evidence for abundant transcription of non-coding regions in the Saccharomyces cerevisiae genome

Background

The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2 (Gallus gallus chromosome 2) linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances.

Results

Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cR₆₀₀₀ long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths.

Conclusion

We obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate.     

Comparative mapping of human chromosome 10 to pig chromosomes 10 and 14

Identification of predictive markers in QTL regions that impact production traits in commercial populations of swine is dependent on construction of dense comparative maps with human and mouse genomes. Chromosomal painting in swine suggests that large genomic blocks are conserved between pig and human, while mapping of individual genes reveals that gene order can be quite divergent. High-resolution comparative maps in regions affecting traits of interest are necessary for selection of positional candidate genes to evaluate nucleotide variation causing phenotypic differences. The objective of this study was to construct an ordered comparative map of human chromosome 10 and pig chromosomes 10 and 14. As a large portion of both pig chromosomes are represented by HSA10, genes at regularly spaced intervals along this chromosome were targeted for placement in the porcine genome. A total of 29 genes from human chromosome 10 were mapped to porcine chromosomes 10 (SSC10) and 14 (SSC14) averaging about 5 Mb distance of human DNA per marker. Eighteen genes were assigned by linkage in the MARC mapping population, five genes were physically assigned with the IMpRH mapping panel and seven genes were assigned on both maps. Seventeen genes from human 10p mapped to SSC10, and 12 genes from human 10q mapped to SSC14. Comparative maps of mammalian species indicate that chromosomal segments are conserved across several species and represent syntenic blocks with distinct breakpoints. Development of comparative maps containing several species should reveal conserved syntenic blocks that will allow us to better define QTL regions in livestock.     

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.     

A high-resolution comparative radiation hybrid map of ovine chromosomal regions that are homologous to human chromosome 6 (HSA6)

In this study, we constructed high-resolution radiation hybrid (RH) and comparative maps of ovine chromosomes or chromosomal segments that are homologous to human chromosome 6 (HSA6). A total of 251 markers were successfully genotyped across the recently developed USUoRH5000 whole-genome panel; 208 of these markers were assigned to five RH linkage groups distributed on three ovine chromosomes (OAR8, 9 and 20). The RH maps have good correspondence with previous chromosome painting data, although a small centromeric region on OAR9 that is homologous to HSA6 had not been previously detected using human chromosome paints on ovine chromosomal spreads. High percentages of the ovine markers were identified as orthologues in the bovine (86.3%), dog (85.8%), horse (69.3%) and human (88.7%) genomes. These maps contribute to investigations in mammalian chromosome evolution and the search for economic trait loci in sheep.     

Mapping of 443 porcine EST improves the comparative maps for SSC1 and SSC7 with the human genome

Numerous mapping studies of complex traits in the pig have resulted in quantitative trait loci (QTL) intervals of 10-20 cM. To improve the chances to identify the genes located in such intervals, increased expressed sequence tags (EST)-based marker density, coupled with comparative mapping with species whose genomes have been sequenced such as human and mouse, is the most efficient tool. In this study, we mapped 443 porcine EST with a radiation hybrid (RH) panel (384 had LOD > 6.0) and a somatic cell hybrid panel. Requiring no discrepancy between two-point and multipoint RH data allowed robust assignment of 309 EST, of which most were located on porcine chromosomes (SSC) 1, 4, 7, 8 and X. Moreover, we built framework maps for two chromosomes, SSC1 and SSC7, with mapped QTL in regions with known rearrangement between pig and human genomes. Using the Blast tool, we found orthologies between 407 of the 443 pig cDNA sequences and human genes, or to existing pig genes. Our porcine/human comparative mapping results reveal possible new homologies for SSC1, SSC3, SSC5, SSC6, SSC12 and SSC14 and add markers in synteny breakpoints for chromosome 7.     

Construction of a 5000<Subscript>rad</Subscript> whole-genome radiation hybrid panel in the horse and generation of a comprehensive and comparative map for ECA11

A 5000_rad whole-genome radiation hybrid (RH) panel was created for the horse. The usefulness of the panel for generating physically ordered maps of individual equine chromosomes was tested by typing 24 markers on horse Chromosome 11 (ECA11). The overall retention of markers on this chromosome was 43.6%. Almost complete retention of two of the typed markers—CA062 and AHT44—clearly indicated the location of thymidine kinase gene on the short arm of ECA11. Seven of the typed markers were FISH mapped to align the RH and cytogenetic maps. With the RH-MAPPER approach, a physically ordered map comprising four linkage groups and incorporating all the markers was obtained. The study provides the first comprehensive map for a horse chromosome that integrates all available mapping data and adds new information that spans the entire length of the equine chromosome. The map clearly underlines the resolving power and utility of the panel and emphasizes the need to have uniformly distributed cytogenetic markers for appropriate alignment of RH map with the chromosome. A comparative status of the ECA11 map in relation to the corresponding human/mouse chromosome is presented. Received: 7 June 2001 / Accepted: 4 October 2001     

A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.     

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.     

Comparative mapping of Homo sapiens chromosome 4 (HSA4) and Sus scrofa chromosome 8 (SSC8) using orthologous genes representing different cytogenetic bands as landmarks.

The recently published draft sequence of the human genome will provide a basic reference for the comparative mapping of genomes among mammals. In this study, we selected 214 genes with complete coding sequences on Homo sapiens chromosome 4 (HSA4) to search for orthologs and expressed sequence tag (EST) sequences in eight other mammalian species (cattle, pig, sheep, goat, horse, dog, cat, and rabbit). In particular, 46 of these genes were used as landmarks for comparative mapping of HSA4 and Sus scrofa chromosome 8 (SSC8); most of HSA4 is homologous to SSC8, which is of particular interest because of its association with genes affecting the reproductive performance of pigs. As a reference framework, the 46 genes were selected to represent different cytogenetic bands on HSA4. Polymerase chain reaction (PCR) products amplified from pig DNA were directly sequenced and their orthologous status was confirmed by a BLAST search. These 46 genes, plus 11 microsatellite markers for SSC8, were typed against DNA from a pig-mouse radiation hybrid (RH) panel with 110 lines. RHMAP analysis assigned these 57 markers to 3 linkage groups in the porcine genome, 52 to SSC8, 4 to SSC15, and 1 to SSC17. By comparing the order and orientation of orthologous landmark genes on the porcine RH maps with those on the human sequence map, HSA4 was recognized as being split into nine conserved segments with respect to the porcine genome, seven with SSC8, one with SSC15, and one with SSC17. With 41 orthologous gene loci mapped, this report provides the largest functional gene map of SSC8, with 30 of these loci representing new single-gene assignments to SSC8.     

Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum

Background

On porcine chromosome 7, the region surrounding the Major Histocompatibility Complex (MHC) contains several Quantitative Trait Loci (QTL) influencing many traits including growth, back fat thickness and carcass composition. Previous studies highlighted that a fragment of ~3.7 Mb is located within the Swine Leucocyte Antigen (SLA) complex. Internal rearrangements of this fragment were suggested, and partial contigs had been built, but further characterization of this region and identification of all human chromosomal fragments orthologous to this porcine fragment had to be carried out.

Results

A whole physical map of the region was constructed by integrating Radiation Hybrid (RH) mapping, BAC fingerprinting data of the INRA BAC library and anchoring BAC end sequences on the human genome. 17 genes and 2 reference microsatellites were ordered on the high resolution IMNpRH2_12000rad Radiation Hybrid panel. A 1000:1 framework map covering 550 cR₁₂₀₀₀ was established and a complete contig of the region was developed. New micro rearrangements were highlighted between the porcine and human genomes. A bovine RH map was also developed in this region by mapping 16 genes. Comparison of the organization of this region in pig, cattle, human, mouse, dog and chicken genomes revealed that 1) the translocation of the fragment described previously is observed only on the bovine and porcine genomes and 2) the new internal micro rearrangements are specific of the porcine genome.

Conclusion

We estimate that the region contains several rearrangements and covers 5.2 Mb of the porcine genome. The study of this complete BAC contig showed that human chromosomal fragments homologs of this heavily rearranged QTL region are all located in the region of HSA6 that surrounds the centromere. This work allows us to define a list of all candidate genes that could explain these QTL effects.     

High-resolution comprehensive radiation hybrid maps of the porcine chromosomes 2p and 9p compared with the human chromosome 11

We are constructing high-resolution, chromosomal 'test' maps for the entire pig genome using a 12,000-rad WG-RH panel (IMNpRH2(12,000-rad))to provide a scaffold for the rapid assembly of the porcine genome sequence. Here we present an initial, comparative map of human chromosome (HSA) 11 with pig chromosomes (SSC) 2p and 9p. Two sets of RH mapping vectors were used to construct the RH framework (FW) maps for SSC2p and SSC9p. One set of 590 markers, including 131 microsatellites (MSs), 364 genes/ESTs, and 95 BAC end sequences (BESs) was typed on the IMNpRH2(12,000-rad) panel. A second set of 271 markers (28 MSs, 138 genes/ESTs, and 105 BESs) was typed on the IMpRH(7,000-rad) panel. The two data sets were merged into a single data-set of 655 markers of which 206 markers were typed on both panels. Two large linkage groups of 72 and 194 markers were assigned to SSC2p, and two linkage groups of 84 and 168 markers to SSC9p at a two-point LOD score of 10. A total of 126 and 114 FW markers were ordered with a likelihood ratio of 1000:1 to the SSC2p and SSC9p RH(12,000-rad) FW maps, respectively, with an accumulated map distance of 4046.5 cR(12,000 )and 1355.2 cR(7,000 )for SSC2p, and 4244.1 cR(12,000) and 1802.5 cR(7,000) for SSC9p. The kb/cR ratio in the IMNpRH2(12,000-rad) FW maps was 15.8 for SSC2p, and 15.4 for SSC9p, while the ratio in the IMpRH(7,000-rad) FW maps was 47.1 and 36.3, respectively, or an approximately 3.0-fold increase in map resolution in the IMNpRH(12,000-rad) panel over the IMpRH(7,000-rad) panel. The integrated IMNpRH(12,000-rad) andIMpRH(7,000-rad) maps as well as the genetic and BAC FPC maps provide an inclusive comparative map between SSC2p, SSC9p and HSA11 to close potential gaps between contigs prior to sequencing, and to identify regions where potential problems may arise in sequence assembly.     

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine x Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.     

A 12,000-rad porcine radiation hybrid (IMNpRH2) panel refines the conserved synteny between SSC12 and HSA17

Reverse or bidirectional Zoo-FISH suggests that synteny between porcine chromosome 12 (SSC12) and human chromosome 17 (HSA17) is completely conserved. The construction of a high-resolution radiation hybrid (RH) map for SSC12 provides a unique opportunity to determine whether chromosomal synteny is reflected at the molecular level by comparative gene mapping of SSC12 and HSA17. We report an initial, high-resolution RH map of SSC12 on the 12,000-rad IMNpRH2 panel using CarthaGene software. This map contains a total of 320 markers, including 20 microsatellites and 300 ESTs/genes, covering approximately 4836.9 cR12,000. The markers were ordered in 16 linkage groups at LOD 6.0 using framework markers previously mapped on the IMpRH7000-rad SSC12 and porcine genetic maps. Ten linkage groups ordered more than 10 markers, with the largest containing 101 STSs. The resolution of the current RH map is approximately 15.3 kb/cR on SSC12, a significant improvement over the second-generation EST SSC12 RH7000-rad map of 103 ESTs and 15 framework markers covering approximately 2287.2 cR7000. Compared to HSA17, six distinct segments were identified, revealing macro-rearrangements within the apparently complete synteny between SSC12 and HSA17. Further analysis of the order of 245 genes (ESTs) on HSA17 and SSC12 also revealed several micro-rearrangements within a synteny segment. A high-resolution SSC12 RH12,000-rad map will be useful in fine-mapping QTL and as a scaffold for sequencing this chromosome.     

An integrated comparative map of the porcine X chromosome

The objectives of this study were to assign both microsatellite and gene-based markers on porcine chromosome X to two radiation hybrid (RH) panels and to develop a more extensive integrated map of SSC-X. Thirty-five microsatellite and 20 gene-based markers were assigned to T43RH, and 16 previously unreported microsatellite and 15 gene-based markers were added to IMpRH map. Of these, 30 microsatellite and 12 gene-based markers were common to both RH maps. Twenty-two gene-based markers were submitted to BLASTN analysis for identification of orthologues of genes on HSA-X. Single nucleotide polymorphisms (SNPs) were detected for 12 gene-based markers, and nine of these were placed on the genetic map. A total of 92 known loci are present on at least one porcine chromosome X map. Thirty-seven loci are present on all three maps; 31 loci are found on only one map. Location of 33 gene-based markers on the comprehensive map translates into an integrated comparative map that supports conservation of gene order between SSC-X and HSA-X. This integrated map will be valuable for selection of candidate genes for porcine quantitative trait loci (QTLs) that map to SSC-X.     

A Radiation Hybrid Map of Mouse Chromosome 13

A mouse radiation hybrid (RH) panel was used to make a framework map for the entire length of mouse chromosome (Chr) 13. Forty-one loci were typed, and while most used primers flanking simple sequence repeats, some genes were included. The most proximal and distal loci are D13Mit132 and D13Mit35. The estimate of map length for Chr 13 is 1328 cR. The map is compared with the same set of loci from the consensus map for Chr 13, which is 70 cM in length, and also with a recombinational map derived from an intraspecies cross typed for many of the same loci. The mouse RH panel gave good resolution for Chr 13 and at the distal end allowed separation of previously nonrecombinant markers that are present on a single 620-kb YAC clone. Data analysis was performed using the RH option for Map Manager QT. This framework RH map of Chr 13 is the second of a series of RH maps for mouse chromosomes.     

A comparative map of interstitial bovine chromosome 5 with human chromosomes 12 and 22

We have constructed a high-density comparative radiation hybrid map of the interstitial region of bovine chromosome 5 (BTA5) using a recently constructed 12,000-rad, whole-genome, cattle-hamster radiation hybrid (WGRH) panel. Sixty-two bovine EST markers were selected which have orthologous sequences on human chromosomes 12 and 22 (HSA12 and HSA22). Sixty markers were included in the multi-point framework map at LOD 3.0. Our comprehensive RH map contains more than twice as many markers (88) than previous generation maps. Because of a higher marker density and increased resolution of the RH(12,000) panel, all markers were placed into a single linkage group based on two-point analysis at a LOD score 6.0. As a result, this new comparative map reveals new blocks of synteny and extensive gene order alterations between species. Breakpoints of synteny are located with high accuracy. Overall, this work reveals widespread chromosomal rearrangements between bovine, human and mouse genomes.     

Conservation of gene order between horse and human X chromosomes as evidenced through radiation hybrid mapping

A radiation hybrid (RH) map of the equine X chromosome (ECAX) was obtained using the recently produced 5000(rad) horse x hamster hybrid panel. The map comprises 34 markers (16 genes and 18 microsatellites) and spans a total of 676 cR(5000), covering almost the entire length of ECAX. Cytogenetic alignment of the RH map was improved by fluorescent in situ hybridization mapping of six of the markers. The map integrates and refines the currently available genetic linkage, syntenic, and cytogenetic maps, and adds new loci. Comparison of the physical location of the 16 genes mapped in this study with the human genome reveals similarity in the order of the genes along the entire length of the two X chromosomes. This degree of gene order conservation across evolutionarily distantly related species has up to now been reported only between human and cat. The ECAX RH map provides a framework for the generation of a high-density map for this chromosome. The map will serve as an important tool for positional cloning of X-linked diseases/conditions in the horse.