A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.     

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.     

Isolation and high-resolution mapping of new DNA markers from the pericentromeric region of chromosome 10.

The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including D10Z1, D10S94, D10S97, and D10S102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small subchromosomal fragments that include both D10S94 and D10Z1. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a lambda library made with pp11A DNA. We have completed regional mapping of 22 of those clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between D10S34 and RBP3. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A.     

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.     

Radiation hybrid maps of Medaka chromosomes LG 12, 17, and 22.

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was approximately 33 kb/cR. We estimate the potential resolution of the RH panel to be approximately 186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.     

A radiation hybrid map of chicken chromosome 15

We have constructed a radiation hybrid (RH) map of chicken chromosome (GGA) 15. This map can be used as a resource to efficiently map genes to this chromosome. The map has been developed using a 6000 rad chicken-hamster whole-genome radiation hybrid panel (ChickRH6). In total, six microsatellite loci, 18 sequence tagged sites (STSs) from BAC end sequences and 11 genes were typed on the panel. The initial framework map comprised eight markers, and an additional 23 markers were then added to generate the final map. The total map length was 334 centiRay6000 (cR6000). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR6000 and physical distance over GGA15 was estimated to be 0.063 Mb/cR6000. The present map increases the marker density and the marker resolution on GGA15 and enables fast mapping of new chicken genes homologous to genes from human chromosomes 12 and 22.     

Construction of a new porcine whole-genome framework map using a radiation hybrid panel

We have constructed a radiation hybrid (RH) map of the porcine genome using an RH panel generated by an irradiation dose of 5000-rad (Sus scrofa radiation hybrid map, SSRH map). Normal porcine aortic endothelial cells were irradiated and fused with a thymidine kinase-deficient mouse cell line, L-M (TK-). A total of 110 cell lines were selected and used for further analysis. Among 1091 microsatellite (MS) markers selected for mapping, 842 markers (77%) could be typed on the panel. The framework map comprised 342 MS markers and an additional 247 MS markers were then added to generate the whole-genome map. The average retention frequency for the data set was 30.6%. The total map length was 5596.2 centiRay (cR). Using an estimated physical length of 2718 Mbp, the average ratio between cR and physical distance over the porcine genome was estimated to be 0.49 Mb/cR.     

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. Received: 23 February 1999 / Accepted: 11 May 1999     

The functional significance of absence: the chromosomal segment harboring Tp53 is absent from the T55 rat radiation hybrid mapping panel

The T55 rat radiation hybrid (RH) mapping panel has been reported to retain the entire rat genome at retention frequencies between 22% and 37%. However, we found that a small segment of rat chromosome 10 harboring at least four different genes, including Tp53, was completely absent from the panel (retention frequency = 0%). Two other markers located in the vicinity exhibited much reduced retention (2-6%). RH clones are generated by transferring highly fragmented DNA into a recipient cell. There might be a strong selection against the transfer and retention of chromosome segments harboring an intact Tp53, as the action of this gene might prevent proliferation and establishment of the RH clone. Our finding further suggests that unexpected low retention or absence of chromosome segments in an RH panel may represent indications that the segments harbor genes with important functions in cell proliferation control.     

A first-generation porcine whole-genome radiation hybrid map

A whole-genome radiation hybrid (WG-RH) panel was used to generate a first-generation radiation map of the porcine (Sus scrofa) genome. Over 900 Type I and II markers were used to amplify the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) comprised of 118 hybrid clones. Average marker retention frequency of 29.3% was calculated with 757 scorable markers. The RHMAP program established 128 linkage groups covering each chromosome (n = 19) at a lod ≥ 4.8. Fewer than 10% of the markers (59) could not be placed within any linkage group at a lod score ≥4.8. Linkage group order for each chromosome was determined by incorporating linkage data from the swine genetic map as well as physical assignments. The current map has an estimated ratio of ∼70 kb/cR and a maximum theoretical resolution of 145 kb. This initial map forms a template for establishing accurate YAC and BAC contigs and eventual positional cloning of genes associated with complex traits. Received: 8 January 1999 / Accepted: 13 April 1999     

A high-resolution 15,000(rad) radiation hybrid panel for the domestic cat

The current genetic and recombination maps of the cat have fewer than 3,000 markers and a resolution limit greater than 1 Mb. To complement the first-generation domestic cat maps, support higher resolution mapping studies, and aid genome assembly in specific areas as well as in the whole genome, a 15,000(Rad) radiation hybrid (RH) panel for the domestic cat was generated. Fibroblasts from the female Abyssinian cat that was used to generate the cat genomic sequence were fused to a Chinese hamster cell line (A23), producing 150 hybrid lines. The clones were initially characterized using 39 short tandem repeats (STRs) and 1,536 SNP markers. The utility of whole-genome amplification in preserving and extending RH panel DNA was also tested using 10 STR markers; no significant difference in retention was observed. The resolution of the 15,000(Rad) RH panel was established by constructing framework maps across 10 different 1-Mb regions on different feline chromosomes. In these regions, 2-point analysis was used to estimate RH distances, which compared favorably with the estimation of physical distances. The study demonstrates that the 15,000(Rad) RH panel constitutes a powerful tool for constructing high-resolution maps, having an average resolution of 40.1 kb per marker across the ten 1-Mb regions. In addition, the RH panel will complement existing genomic resources for the domestic cat, aid in the accurate re-assemblies of the forthcoming cat genomic sequence, and support cross-species genomic comparisons.     

A Radiation Hybrid Map of 95 STSs Spanning Human Chromosome 13q

We have constructed a high-resolution physical map of the long arm of human chromosome 13 using a panel of 94 radiation hybrids. A comprehensive map of 95 chromosome 13-specific sequence tagged sites (STSs) spanning 13q from the presumed centromere at D13Z1 to the known telomere was obtained by multipoint maximum likelihood statistical methods. The 95 markers have an average retention frequency of 10%, with markers closer to the centromere having much greater retention frequencies (22-49%) than distal 13q markers (2-12%) The most likely radiation hybrid map localized the 95 STSs into 54 unique map positions, 34 with odds of 1000:1 or greater; the comprehensive map localized all but 17 STSs with odds exceeding 10:1. The total map length of 13q was 1302 cR₉₀₀₀ (range 6.4-94.4 cR₉₀₀₀) and a physical distance of 98 Mb, so that 1% breakage in the RH panel corresponds to 75 kb. A comparison of the comprehensive RH map to genetic maps of chromosome 13q shows identical locus orders for the common markers, with two exceptions over 1-cM distances. We discuss the possible relationships between the genetic and the radiation hybrid maps.     

A 12,000 rad whole genome radiation hybrid panel for high resolution mapping in cattle: characterization of the centromeric end of chromosome 1

We have constructed a 12,000 rad bovine whole-genome radiation hybrid panel (BovR12) to complement the 5000 rad panel (BovR5) currently available to the bovine genomics research community. Initial characterization with markers from chromosome 1 reveals a higher frequency of breakage between adjacent markers and subsequently a larger number of 'linkage' groups on this chromosome. For this set of markers, the retention frequency is also higher than in BovR5.     

Selected locus and multiple panel models for radiation hybrid mapping.

We develop two new types of models for whole-genome radiation hybrid mapping using the general multipoint framework. The first, selected locus models, are appropriate for mapping markers in the region of a selectable locus that was used in creation of the hybrids. The models allow for strong retention of the selectable locus, with retention rates decreasing with increasing distance from the selectable locus in both directions. We illustrate the application of these models with 10 chromosome 17 sequence-tagged site (STS) markers and the thymidine kinase (TK) locus typed on a whole-genome hybrid panel in which TK was used in the selection process. The second set of models are appropriate when loci typed on two or more independent panels are to be used to build maps. Maps can be built assuming interlocus distances are independent or proportional between the panels, and the hypothesis of proportional distances can be tested. We illustrate the application of these models by using 27 chromosome 21 STS markers typed on two hybrid panels created with radiation doses of approximately 10,000 and approximately 50,000 Rads.     

A radiation hybrid map for the bovine Y Chromosome

Screening a bovine Y Chromosome-specific DNA library resulted in 34 new microsatellites, six of which mapped to the pseudoautosomal region (PAR), and 28 localized to the Y-specific region. These microsatellites, together with 23 markers previously mapped to the bovine Y Chr, were scored on a 7000-rad cattle–hamster radiation hybrid (RH) panel. Retention frequency of individual markers ranged from 18.5% to 76.5% with an average of 48.4%. Markers with high retention frequency (>55%) were found to exist in multiple copies on the Y Chr. Thirteen markers were placed on the PAR RH map with the AmelY gene proximal to the pseudoautosomal boundary and 46 markers, including Sry and Tspy gene, on the Y-specific region of the RH map. The microsatellites developed and mapped in this work will be useful for comparative mapping of cattle, sheep, and goat, studying the origin, evolution, and migration of bovidae species and provide an initial platform to develop a high-resolution map of the Y Chr and positional cloning of Y-specific genes.     

A Radiation Hybrid Map of the BRCA1 Region

A locus on chromosome 17q, designated “BRCA1,” has been identified as a predisposition gene for breast cancer. A panel of chromosome 17–specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17. A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays. Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region. In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region.     

A radiation hybrid map of the proximal short arm of the human X chromosome spanning incontinentia pigmenti 1 (IP1) translocation breakpoints.

Radiation hybrid mapping was used in combination with physical mapping techniques to order and estimate distances between 14 loci in the proximal region of the short arm of the human X chromosome. A panel of radiation hybrids containing human X-chromosomal fragments was generated from a Chinese hamster-human cell hybrid containing an X chromosome as its only human DNA. Sixty-seven radiation hybrids were screened by Southern hybridization with sets of probes that mapped to the region Xp11.4-Xcen to generate a radiation hybrid map of the area. A physical map of 14 loci was constructed based on the segregation of the loci in the hybrid clones. Using pulsed-field gel electrophoresis (PFGE) analyses and a somatic cell hybrid mapping panel containing naturally occurring X; autosome translocations, the order of the 14 loci was verified and the loci nearest to the X-chromosomal translocation breakpoints associated with the disease incontinentia pigmenti 1 (IP1) were identified. The radiation hybrid panel will be useful as a mapping resource for determining the location, order, and distances between other genes and polymorphic loci in this region as well as for generating additional region-specific DNA markers.     

A comparative radiation hybrid map of sheep chromosome 10

Comparative radiation hybrid (RH) maps of individual ovine chromosomes are essential to identify genes governing traits of economic importance in sheep, a livestock species for which whole genome sequence data are not yet available. The USUoRH5000 radiation hybrid panel was used to generate a RH map of sheep chromosome 10 (OAR10) with 59 markers that span 1,422 cR over an estimated 92 Mb of the chromosome, thus providing markers every 2 Mb (equivalent to every 24 cR). The markers were derived from 46 BAC end sequences (BESs), a single EST, and 12 microsatellites. Comparative analysis showed that OAR10 shares remarkable conservation of gene order along the entire length of cattle chromosome 12 and that OAR10 contains four major homologous synteny blocks, each related to segments of the homologous human chromosome 13. Extending the comparison to the horse, dog, mouse, and chicken genome showed that these blocks share conserved synteny across species.     

Development of a gene-based radiation hybrid map of chicken Chromosome 7 and comparison to human and mouse

To validate the ChickRH6 whole-genome radiation hybrid (WGRH) panel, we constructed a map of chicken Chromosome 7 based on 19 microsatellite markers from the genetic map and 76 ESTs (expressed sequence tags), whose efficient targeted development was made possible by using the ICCARE software. This high-density radiation hybrid (RH) map of a chicken macrochromosome gives us indications on characteristics of ChickRH6. The potential resolution of the panel is 325 kb and the practical resolution of our framework map is 1.3 Mb. Based on these results, a complete framework map of the chicken genome would comprise 1000 markers. The marker order is in good agreement with the genetic map and comparison with the human and mouse sequence maps revealed a number of internal rearrangements.     

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1)

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.