Whole-genome Radiation Hybrid Research Articles

The first radiation hybrid map of a perch-like fish: The gilthead seabream ( Sparus aurata L)

Among Teleosts, Perciformes are the largest order of fishes and include numerous species of commercial importance. Perciformes also comprise species of primary interest for evolutionary studies and analysis of the sex determination systems and sex chromosome plasticity. Unfortunately, genomics tools and resources for Perciformes remain to be developed. Here, we report the production of a seabream whole-genome radiation hybrid (RH) panel in which quality was ascertained by the construction of a 2-Mb-resolution RH map. The map encompasses 440 markers (288 microsatellites, 82 gene-based markers, and 70 STS) suitable for linkage analysis and comparative mapping studies. Achievement of a RH panel and a whole-genome RH map should contribute to establishing seabream as a fish model among the Perciformes and should be of importance in aquaculture for marker-assisted selection, improvement of growth performance, and disease management. Development of RH maps in a cost-effective manner for other fishes with the described methodology will offer a powerful approach in aquaculture and will provide extended capabilities for comparing vertebrate genome evolution.

Development of a deer mouse whole-genome radiation hybrid panel and comparative mapping of Mus chromosome 11 loci

A 5000-rad whole-genome radiation hybrid cell panel (BW5000) was developed for mapping the deer mouse (Peromyscus maniculatus bairdii) genome. The panel consists of 103 cell lines and has an estimated marker retention frequency of 63.9% (range, 28%-88%) based on PCR typing of 30 Type I (coding gene) and 25 Type II (microsatellite) markers. Using the composite Mus map, Type I markers were selected from six Mus chromosomes, 22 of which are on Mus Chr 11. Fifteen of the Mus Chr 11 markers were simultaneously mapped on an interspecific (P. maniculatus x P. polionotus) backcross panel to test the utility of the radiation hybrid panel, create a framework map, and help establish gene order. The radiation hybrids have effectively detected linkage in the deer mouse genome between markers as far apart as 6.7 cM and resolved markers that are, in the Mus genome, as close as 0.2 Mb. Combined results from both panels have indicated a high degree of gene order conservation of the telomeric 64 cM of Mus Chr 11 in the deer mouse genome. The remaining centromeric portion also shows gene order conservation with the deer mouse but as a separate linkage group. This indicates a translocation of that portion of Mus Chr 11 in P. maniculatus and is consistent with rearrangement breakpoints observed between Mus and other mammalian genomes, including rat and human. Furthermore, this separate linkage group is likely to reside in a chromosomal region of inversion polymorphism between P. maniculatus and P. polionotus.

Wide-cross whole-genome radiation hybrid mapping of the cotton (Gossypium barbadense L.) genome

Whole-genome radiation hybrid mapping has been applied extensively to human and certain animal species, but little to plants. We recently demonstrated an alternative mapping approach in cotton (Gossypium hirsutum L.), based on segmentation by 5-krad gamma-irradiation and derivation of wide-cross whole-genome radiation hybrids (WWRHs). However, limitations observed at the 5-krad level suggested that higher doses might be advantageous. Here, we describe the development of an improved second-generation WWRH panel after higher dose irradiation and compare the resulting map to the 5-krad map. The genome of G. hirsutum (n = 26) was used to rescue the radiation-segmented genome of G. barbadense (n = 26) introduced via 8- and 12-krad gamma-irradiated pollen. Viable seedlings were not recovered after 12-krad irradiation, but 8-krad irradiation permitted plant recovery and construction of a 92-member WWRH mapping panel. Assessment of 31 SSR marker loci from four chromosomes revealed that the 8-krad panel has a marker retention frequency of ca. 76%, which is approximately equivalent to the rate of loss in a low-dose animal radiation hybrid panel. Retention frequencies of loci did not depart significantly from independence when compared between the A and D subgenomes, or according to positions along individual chromosomes. WWRH maps of chromosomes 10 and 17 were generated by the maximum likelihood RHMAP program and the general retention model. The resulting maps bolster evidence that WWRH mapping complements traditional linkage mapping and works in cotton, and that the 8-krad panel complements the 5-krad panel by offering higher rates of chromosome breakages, lower marker retention frequency, and more retention patterns.

Assignment of non-informative turkey genetic markers through comparative approaches

Molecular markers such as microsatellites, provide genetic signposts for navigating genomes. In general, genetic markers that are monomorphic or non-informative in mapping populations typically remain unmapped and as such are less likely to be included in future studies. The use of hybrid cell panels and in silico mapping via whole genome sequences allow for positional mapping of non-segregating markers. This study utilizes the INRA ChickRH6 whole-genome radiation hybrid panel and chicken whole-genome shotgun sequence to map microsatellite markers from the turkey (Meleagris gallopavo). Thirty-three of the 41 markers typed on the RH panel had significant linkage to at least one other marker and 83 of 100 sequences returned significant BLAST similarities. Positioning of these markers provides additional sequence tagged sites in the turkey genome and increases the potential use of these markers for future genetic studies.

Characterization and RH mapping of bovine microsatellites generated from a microdissected BTA20‐specific DNA library

Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.

Assignment of SRY, ANT3, and CSF2RA to the Bovine Y Chromosome by FISH and RH Mapping

Three genes, SRY, ANT3, and CSF2RA, were mapped to the bovine Y chromosome (BTAY) by fluorescence in situ hybridization (FISH) and/or radiation hybrid (RH) mapping. FISH analysis indicated that the bovine SRY gene maps to the distal region of BTAYq, while ANT3 and CSF2RA are located in the pseudoautosomal region (PAR) of BTAYp and BTAXq. RH mapping with a 7000-rad cattle hamster whole-genome radiation hybrid panel further defined the ANT3 and CSF2RA position in relationship to previously mapped 12 PAR markers, and resulted in a relatively high resolution RH map for the PAR of BTAY.

Assignment of SRY, ANT3, and CSF2RA to the Bovine Y Chromosome by FISH and RH Mapping

Abstract Three genes, SRY, ANT3, and CSF2RA, were mapped to the bovine Y chromosome (BTAY) by fluorescence in situ hybridization (FISH) and/or radiation hybrid (RH) mapping. FISH analysis indicated that the bovine SRY gene maps to the distal region of BTAYq, while ANT3 and CSF2RA are located in the pseudoautosomal region (PAR) of BTAYp and BTAXq. RH mapping with a 7000-rad cattle hamster whole-genome radiation hybrid panel further defined the ANT3 and CSF2RA position in relationship to previously mapped 12 PAR markers, and resulted in a relatively high resolution RH map for the PAR of BTAY.

A 1-Mb resolution radiation hybrid map of the canine genome.

The purebred dog population consists of >300 partially inbred genetic isolates or breeds. Restriction of gene flow between breeds, together with strong selection for traits, has led to the establishment of a unique resource for dissecting the genetic basis of simple and complex mammalian traits. Toward this end, we present a comprehensive radiation hybrid map of the canine genome composed of 3,270 markers including 1,596 microsatellite-based markers, 900 cloned gene sequences and ESTs, 668 canine-specific bacterial artificial chromosome (BAC) ends, and 106 sequence-tagged sites. The map was constructed by using the RHDF5000-2 whole-genome radiation hybrid panel and computed by using MULTIMAP and TSP/CONCORDE. The 3,270 markers map to 3,021 unique positions and define an average intermarker distance corresponding to 1 Mb. We also define a minimal screening set of 325 highly informative well spaced markers, to be used in the initiation of genome-wide scans. The well defined synteny between the dog and human genomes, established in part as a function of this work by the identification of 85 conserved fragments, will allow follow-up of initial findings of linkage by selection of candidate genes from the human genome sequence. This work continues to define the canine system as the method of choice in the pursuit of the genes causing mammalian variation and disease.

Construction and integration of radiation-hybrid and cytogenetic maps of dog Chromosome X.

Chromosome (chr) X is under-represented in current maps of the genome of the domestic dog ( Canis familiaris). To address this problem, we have constructed a small-insert, genomic DNA library in pBluescript from flow-sorted canine Chr X DNA. Fluorescence in situ hybridization (FISH) studies confirmed that the library was highly enriched for Chr X. Clones containing microsatellites were identified and sequenced. Database searches detected significant sequence identity between four X-derived clones and genes previously characterized in other species. Thirty-seven markers derived from these clones were mapped on Chr X by FISH, and of these, 28 were mapped by using the female-derived T72 whole-genome radiation hybrid (RH) panel (Research Genetics). Four X-linked canine genes from publicly available data were also mapped. Eight RH linkage groups with LOD >4.0 were identified, and FISH data were used to locate the groups on the chromosome; four groups could be unambiguously orientated by FISH data. In each case, the FISH and RH data were mutually consistent. The data suggest strongly conserved synteny between canine and human X Chrs. The pseudoautosomal region has been further characterized, and the putative or actual locations of nine genes of clinical relevance have been suggested.

Comparative fine maps of bovine toll-like receptor 4 and toll-like receptor 2 regions.

Toll-like receptors are cell-surface receptors that activate innate and adaptive immune responses. We have used a 5000-rad, whole-genome radiation hybrid panel to map Toll-like receptor 4 (TLR4) to the distal end of bovine Chromosome (Chr) 8, and Toll-like receptor 2 (TLR2) to the proximal end of bovine Chr 17. To facilitate comparative mapping and contig construction, we have also used 5000- and 12,000-rad, whole-genome radiation hybrid panels to produce fine maps of the regions surrounding these genes in cattle. These fine maps triple the number of available markers in the TLR4 region and more than double the number of available markers in the TLR2 region. Comparative analyses show gene order conservation between the bovine Chr 8 region and human Chr 9, and between the bovine Chr 17 region and human Chr 4. In addition, the bovine Chr 8 region refines an evolutionary chromosomal breakpoint from a 10-megabase region to a 2.5-megabase region, and the bovine Chr 17 map suggests a new evolutionary chromosomal breakpoint.

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.

A bovine whole-genome radiation hybrid panel and outline map.

A 3000-rad radiation hybrid panel was constructed for cattle and used to build outline RH maps for all 29 autosomes and the X and Y chromosomes. These outline maps contain about 1200 markers, most of which are anonymous microsatellite loci. Comparisons between the RH chromosome maps, other published RH maps, and linkage maps allow regions of chromosomes that are poorly mapped or that have sparse marker coverage to be identified. In some cases, mapping ambiguities can be resolved. The RH maps presented here are the starting point for mapping additional loci, in particular genes and ESTs that will allow detailed comparative maps between cattle and other species to be constructed. Radiation hybrid cell panels allow high-density genetic maps to be constructed, with the advantage over linkage mapping that markers do not need to be polymorphic. A large quantity of DNA has been prepared from the cells forming the RH panel reported here and is publicly available for mapping large numbers of loci.

ChickRH6: a chicken whole-genome radiation hybrid panel.

As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6 000 rads with HPRT-deficient hamster Wg3hCl2 cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs.

ChickRH6: a chicken whole-genome radiation hybrid panel

ChickRH6: a chicken whole-genome radiation hybrid panel

A higher resolution radiation hybrid map of bovine chromosome 13

In this paper, we present a radiation hybrid framework map of BTA13 composed of nine microsatellite loci, six genes and one EST. The map has been developed using a recently constructed 12'000 rad bovine-hamster whole-genome radiation hybrid panel. Moreover, we present a comprehensive map of BTA13 comprising 72 loci, of which 45 are microsatellites, 20 are genes and seven are ESTs. The map has an estimated length of 2694.7 cR12'000. The proposed order is in general agreement with published maps of BTA13. Our results only partially support previously published information of five blocks of conserved gene order between cattle and man. We found no evidence for the existence of an HSA20 homologous segment of coding DNA on BTA13 located centromeric of a confirmed HSA10 homologous region. The present map increases the marker density and the marker resolution on BTA13 and enables further insight into the evolutionary development of the chromosome as compared to man.

A comparative radiation hybrid map of bovine chromosome 18 and homologous chromosomes in human and mice.

A comprehensive radiation hybrid (RH) map and a high resolution comparative map of Bos taurus (BTA) chromosome 18 were constructed, composed of 103 markers and 76 markers, respectively, by using a cattle-hamster somatic hybrid cell panel and a 5,000 rad whole-genome radiation hybrid (WGRH) panel. These maps include 65 new assignments (56 genes, 3 expressed-sequence tags, 6 microsatellites) and integrate 38 markers from the first generation WGRH(5,000) map of BTA18. Fifty-nine assignments of coding sequences were supported by somatic hybrid cell mapping to markers on BTA18. The total length of the comprehensive map was 1666 cR(5,000). Break-point positions within the chromosome were refined and a new telomeric RH linkage group was established. Conserved synteny between cattle, human, and mouse was found for 76 genes of BTA18 and human chromosomes (HSA) 16 and 19 and for 34 cattle genes and mouse chromosomes (MMU) 7 and 8. The new RH map is potentially useful for the identification of candidate genes for economically important traits, contributes to the expansion of the existing BTA18 gene map, and provides new information about the chromosome evolution in cattle, humans, and mice.

Construction of a 5000rad 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.

Identification and chromosome assignment of 23 genes expressed in meat and dairy cattle.

Hepatic and intestinal expressed sequence tags (ESTs) of lactating German Holstein and Charolais cows were used to identify and map genes potentially involved in the regulation of metabolic processes in cattle. Seventeen ESTs were homologous to human gene sequences and six ESTs identified unknown coding sequences in human genomic DNA clones. A cattle-hamster somatic hybrid cell panel and cattle-hamster 5000 rad whole genome radiation hybrid (WGRH) panel were used to assign the ESTs in the bovine genome. Fifteen of the mapped genes and the six ESTs represent new assignments in the established cattle WGRH5000 map. Calculation of the radiation hybrid (RH) mapping data was performed with the software RHMAPPER. The new loci contribute to the completion of the bovine transcript maps and their comparative assignments increase our knowledge about genome evolution between cattle and human.

Generation and characterization of a 12,000-rad radiation hybrid panel for fine mapping in pig

We have constructed a 12,000-rad porcine whole-genome radiation hybrid panel to complement the first generation 7,000-rad panel (IMpRH) and allow higher resolution mapping studies both in specific areas of interest and on the whole genome. We analyzed 243 hybrid clones on the basis of their marker retention frequency to produce a final panel of 90 hybrid clones with an average retention frequency of 35.4%. The resolution of this 12,000-rad panel (IMNpRH2) was compared to the resolution of the 7,000-rad panel (IMpRH) by constructing framework maps in the 2.4-Mb region of porcine chromosome 15 containing the acid meat RN gene. In this region, two-point analysis was used to estimate RH distances and demonstrates their reliability with the estimation of physical distances. This study demonstrates that the 12,000-rad panel constitutes a powerful tool for constructing high-resolution maps. Indeed, the resolution of IMNpRH2 (12–14 kb/cR_12,000) is two to three times more than that of IMpRH (35–37 kb/cR_7,000). As expected, the increase in the radiation dose allows an increase of the mapping resolution in terms of kb/cR with the same suppleness of use for mapping experiments. In addition the RH map constructed in the region investigated proved to be more homogeneous on IMNpRH2 than on IMpRH.

A higher resolution radiation hybrid map of bovine chromosome 13.

In this paper, we present a radiation hybrid framework map of BTA13 composed of nine microsatellite loci, six genes and one EST. The map has been developed using a recently constructed 12'000 rad bovine-hamster whole-genome radiation hybrid panel. Moreover, we present a comprehensive map of BTA13 comprising 72 loci, of which 45 are microsatellites, 20 are genes and seven are ESTs. The map has an estimated length of 2694.7 cR12'000. The proposed order is in general agreement with published maps of BTA13. Our results only partially support previously published information of five blocks of conserved gene order between cattle and man. We found no evidence for the existence of an HSA20 homologous segment of coding DNA on BTA13 located centromeric of a confirmed HSA10 homologous region. The present map increases the marker density and the marker resolution on BTA13 and enables further insight into the evolutionary development of the chromosome as compared to man.