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Radiation hybrid (RH) mapping is based on radiation-induced chromosome breakage rather than meiotic recombination, as a means to induce marker segregation for mapping. To date, the implementation of this mapping approach in hexaploid (Triticum aestivum L.; 2n = 6x = 42; AABBDD) and tetraploid (T. turgidum L.; 2n = 4x = 28; AABB) wheat has concentrated on the production of mapping panels for individual chromosomes. In order to extend the usefulness of this approach, we have devised a method to produce panels for the simultaneous mapping of all chromosomes of the D subgenome of hexaploid wheat. In this approach, seeds of hexaploid wheat (AABBDD) are irradiated and the surviving plants are crossed to tetraploid wheat (AABB) to produce a mapping panel based on quasi-pentaploids (AABBD). Chromosome lesions in the A and B genomes are largely masked in the quasi-pentaploids due to the presence of A- and B-genome chromosomes from the tetraploid parent. On the other hand, the chromosomes from the D-genome are present in one copy (hemizygous) and allow radiation hybrid mapping of all D-genome chromosomes simultaneously. Our analyses showed that transmission of D-genome chromosomes was apparently normal and that radiation-induced chromosome breakage along D-genome chromosomes was homogeneous. Chromosome breakage levels between D-genome chromosomes were comparable except for chromosome 6D which suffered greater chromosome breakage. These results demonstrate the feasibility of constructing D-genome radiation hybrids (DGRHs) in wheat.

A method of estimating distances between pairs of genetic markers is described that directly uses their observed joint frequency distribution in a panel of radiation hybrids (RHs). The distance measure is based on the strength of association between marker pairs, which is high for close markers and decays with distance. These distances are then submitted to a previous method that generates linear coordinates for the markers directly from the intermarker distance matrix. This method of map building from RH data is simpler than others, because it uses only the observed joint frequency distributions of markers in the panel, and does not attempt to model unobserved quantities such as the retention of different sized fragments that contain the markers. It also incorporates directly the observed variation in retention of different markers, without needing a model for differential fragment retention dependent on chromosomal location, which is generally not known. Only small, precise distances are used in map construction, thereby reducing any effects of different fragment retention frequencies and local variations in X-ray sensitivity. The method is tested by simulation, and known marker distances and locations are successfully recovered from RH raw data. The method is also applied to publicly available data sets related to the recent transcript map of the human genome.

We have constructed a physical map of the human genome by using a panel of 83 whole genome radiation hybrids (the Stanford G3 panel) in conjunction with 10,478 sequence-tagged sites (STSs) derived from random genomic DNA sequences, previously mapped genetic markers, and expressed sequences. Of these STSs, 5049 are framework markers that fall into 1766 high-confidence bins. An additional 945 STSs are indistinguishable in their map location from one or more of the framework markers. These 5994 mapped STSs have an average spacing of 500 kb. An additional 4484 STSs are positioned with respect to the framework markers. Comparison of the orders of markers on this map with orders derived from independent meiotic and YAC STS-content maps indicates that the error rate in defining high-confidence bins is

Members of the ICE/CED-3 protease family appear to play an essential role in programmed cell death process. In this paper the chromosomal localization of the human genes CPP32, Mch2, Mch3 and Ich-1 is reported, obtained by Radiation Hybrid Mapping. CPP32 was assigned to chromosome 4q33-q35.1, Mch2 to chromosome 4q25-q26, Mch3 to chromosome 10q25.1-q25.2 and Ich-1 to chromosome 7q35. Ich-1 was found to map very close to the marker WI-9353. The possible overlapping of the two independent locus assignments is considered. The genomic distribution of these genes is discussed, with particular reference to the co-location with some human genetic diseases all characterized by autosomal dominant inheritance and by similar malformative features.

Radiation hybrids have become a widely used tool for physical mapping. A drawback of the technique is that large numbers of hybrids are required to construct robust, high-resolution maps. The information contained within a panel of radiation hybrids is limited by the frequency of retention of chromosomal fragments from the donor cell line. In almost all experiments to date, the retention frequency has been below the optimal level; therefore, many hybrids are needed to produce high-quality maps. Because of the labor-intensive nature of large-scale mapping projects, it is important to make panels as small as possible. One method that has been adopted is to produce initially a large number of hybrids that are all typed with a few loci. Those hybrids showing satisfactorily high retention are admitted to the final panel and the rest are discarded. In this way, a panel of radiation hybrids with higher than expected retention can be created. Methods for conducting such a selection regime are discussed. To investigate the potential advantages of selecting hybrids based on their retention frequency, simulations were run under a variety of conditions. As expected panels with high retention (40%) provided better mapping resources than panels with lower (20%) retention. Beginning with an initial panel of 200 hybrids, comparisons of a random sample of 100 hybrids and the set of those 100 hybrids showing the highest marker retention demonstrated that selection may not be always the best strategy despite the increase in mean retention it yields. The selection of hybrids containing large numbers of fragments leads to an overestimation of the frequency of radiation-induced breaks. When breaks occur with high frequency (for example, when high radiation doses are used), the selection of hybrids leads to a loss of linkage and hence an inability to order the markers. As such, the merits of screening hybrids depends on both the radiation dose and the desired map resolution.

Summary PRKAG1, PRKAG2 and PRKAG3 encode three isoforms of AMP-activated protein kinase γ chain. A major effect on meat quality and a medium effect on back fat thickness of the RN− mutation in the PRKAG3 gene has previously been reported. We have now mapped PRKAG1 and PRKAG2 at expected locations on SSC5 and SSC18 by analysis of radiation hybrids (IMpRH panel). PRKAG2 has been mapped in a region where no quantitative trait loci (QTL) has been reported. PRKAG1 has been mapped close to (but probably outside) a region containing a QTL influencing fatness traits. We have determined the full coding sequence of PRKAG1. No missense mutation was identified when comparing the coding sequence of one Meishan and one Large White boars. Further work is, however, required to determine if a polymorphism in PRKAG1 could be responsible for a part of the variability observed on fatness traits.

For the positional cloning of genes and other novel types of genetic experiments, in humans and other organisms, there is a crucial need for techniques with which genome-wide high-resolution ordered clone maps can be rapidly constructed. Current best methods, such as sequence-tagged site (STS) content mapping, entail a large number of experiments and, in practice, require large low-resolution yeast artificial chromosome (YAC) clones and very many STSs. In this paper, we introduce a new approach, inner product mapping (IPM), that overcomes these limitations. IPM uses radiation hybrids (RHs) to provide localizing signatures for YACs. Two independent data tables that compare YACs against RHs and RHs against STSs are obtained; these tables are combined to produce a computed map of the YACs against ordered STSs. IPM maps each YAC independently, requires relatively few RH comparisons to map a YAC, and can work with small (or large) YACs and few (or many) STSs. This paper describes IPM and presents computer simulations supporting the efficiency of IPM over that of competing methods.

Artemis Pharmaceuticals have just announced a second saturation mutagenesis screen of the zebrafish, Danio rerio. In this interview with Stefan Schulte-Merker, who is Head of Fish Genetics at Artemis, we find out more about the purpose of the second screen and contrast it with the first screen which was carried out in Christaine Nusslein-Volhard laboratory and which is now called ‘Tubingen One’. Stefan Schulte-Merker has been working with zebrafish for over ten years and was involved in a number of genetic screens. He was also part of a team that produced a first-generation map of the ‘Goodfellow panel’ of radiation hybrids, which was intended to help in the cloning of the mutated genes. He is currently Head of Fish Genetics at Artemis Pharmaceuticals, in Tubingen, who are in the process of a second-round genetic screen, called ‘Tubingen 2000’.

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

Radiation hybrid mapping is a method that combines aspects of both genetic and physical mapping and is extensively used in the construction of whole-genome maps. A panel of radiation hybrid cell lines, each retaining a different portion of the donor genome in the background of a recipient cell, is used to score the presence or absence of sequence tagged sites. Keywords: radiation hybrids; panels; mapping; map; genome

Oat–maize radiation hybrids are oat (Avena sativa L.) plants carrying radiation-induced subchromosome fragments of a given maize (Zea mays L.) chromosome. Since first-generation radiation hybrids contain various maize chromosome rearrangements in a hemizygous condition, variation might be expected in the transmission of these rearrangements to subsequent generations. The transmission and integrity of maize chromosome 9 rearrangements were evaluated in progenies of 30 oat–maize radiation hybrids by using a series of DNA-based markers and by genomic in situ hybridization. Maize chromosome 9 rearrangements were reisolated by self-fertilization in 24 of the 30 radiation hybrid lineages. Normal and deleted versions of maize chromosome 9 were transmitted at similar frequencies of 9.1% and 7.6%, respectively, while intergenomic translocations were transmitted at a significantly higher frequency of 47.6%. Most lines (93%) that inherited a rearrangement had it in the hemizygous condition. Lines with a rearrangement in the homozygous state (7%) were only identified in lineages with intergenomic translocations. Homozygous lines are more desirable from the perspective of stock maintenance, since they may stably transmit a given rearrangement to a subsequent generation. However, their isolation is not strictly required, since hemizygous lines can also be used for genome mapping studies.Key words: Avena sativa, Zea mays, addition lines, chromosome rearrangements.

International audience

Mouse A9 cells containing human chromosome 7 tagged with pSV2neo were irradiated with X-rays and fused to A9 cells to isolate G418-resistant clones. From these clones, we selected radiation hybrids that contained 10-40 Mb of human DNA apparently at a single site of their genome by FISH analysis using human repetitive sequences as a probe. Then we made a panel of hybrids that contained various fragments of the 7q31-q32 region and cover its entire region altogether by PCR with STS markers of human chromosome 7. This panel is useful in chromosome transfer experiments since the dominant selective marker neo gene is attached to human DNA.

Background Bovine chromosome (BTA) 15 contains a quantitative trait loci (QTL) for meat tenderness, as well as several breaks in synteny with human chromosome (HSA) 11. Both linkage and radiation hybrid (RH) maps of BTA 15 are available, but the linkage map lacks gene-specific markers needed to identify genes underlying the QTL, and the gene-rich RH map lacks associations with marker genotypes needed to define the QTL. Integrating the maps will provide information to further explore the QTL as well as refine the comparative map between BTA 15 and HSA 11. A recently developed approach to integrating linkage and RH maps uses both linkage and RH data to resolve a consensus marker order, rather than aligning independently constructed maps. Automated map construction procedures employing this maximum-likelihood approach were developed to integrate BTA RH and linkage data, and establish comparative positions of BTA 15 markers with HSA 11 homologs. Results The integrated BTA 15 map represents 145 markers; 42 shared by both data sets, 36 unique to the linkage data and 67 unique to RH data. Sequence alignment yielded comparative positions for 77 bovine markers with homologs on HSA 11. The map covers approximately 32% of HSA 11 sequence in five segments of conserved synteny, another 15% of HSA 11 is shared with BTA 29. Bovine and human order are consistent in portions of the syntenic segments, but some rearrangement is apparent. Comparative positions of gene markers near the meat tenderness QTL indicate the region includes separate segments of HSA 11. The two microsatellite markers flanking the QTL peak are between defined syntenic segments. Conclusions Combining data to construct an integrated map not only consolidates information from different sources onto a single map, but information contributed from each data set increases the accuracy of the map. Comparison of bovine maps with well annotated human sequence can provide useful information about genes near mapped bovine markers, but bovine gene order may be different than human. Procedures to connect genetic and physical mapping data, build integrated maps for livestock species, and connect those maps to more fully annotated sequence can be automated, facilitating the maintenance of up-to-date maps, and providing a valuable tool to further explore genetic variation in livestock.

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.

The 26S proteasome is a multicatalytic enzyme complex that plays a central role in all regulatory pathways such as cell cycle regulation, differentiation, and apoptosis. The 26S proteasome has three components, 20S proteasome, PA28 (Proteasome Activator 28) and PA700 (Proteasome Activator 700) subunits. The combination of PA28 and 20S proteasomes greatly increases the proteasome-mediated proteolysis activity, which is important in the processing and presentation of some antigens. The three genes related to PA28 and seven genes related to PA700 have been mapped by our group (Wang et al., 2003, 2004; Yu et al., 2004). Here we report our mapping results on the PSMA5, PSMB3 and PSMB6 genes, which were thought to be part of the 20S proteasome, the core of the 26S proteasome. Materials and methods

International audience

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 centiRay(6000) (cR(6000)). The estimated retention frequency for the data set was 18%. Using an estimated physical length of 21 Mb, the ratio between cR(6000) and physical distance over GGA15 was estimated to be 0.063 Mb/cR(6000). 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.

International audience

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience

National audience

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.

In the 1970s Goss and Harris [12] developed a new method for mapping human chromosomes. This method was based on irradiating human cells, rescuing some of the irradiated cells by hybridization to rodent cells, and analyzing the hybrid cells for surviving fragments of a particular human chromosome. For various technical reasons, radiation hybrid mapping languished for nearly a decade and a half until revived by Cox et al. [10]. The current, more sophisticated and successful versions raise many fascinating statistical problems. We will first discuss the mathematically simpler case of haploid radiation hybrids. Once this case is thoroughly digested, we will turn to the mathematically subtler case of polyploid radiation hybrids.

International audience

To resolve a candidate chromosome region on the middle part of bovine chromosome 6 (BTA6) containing several different quantitative trait locus (QTL) intervals, we constructed a high-resolution, integrated, comprehensive, and comparative map using a 12,000-rad, wholegenome, cattle–hamster radiation hybrid (RH) panel. The RH map includes a total of 71 loci either selected from bovine and comparative maps or targeted directly from a microdissection library specific for the BTA6 region. All loci typed were placed in one linkage group at a lod score threshold of 4.0. The length of the comprehensive RH map, which is the first highresolution RH map in cattle, spans 2568.8 cR12,000. The order of markers obtained principally agrees with the order on published bovine genetic maps. Our RH map integrates markers as well as genes and ESTs available from several physical and genetic maps of BTA6 and the orthologous ovine chromosome 6, human chromosome 4, and mouse chromosomes 5/3. Comparative analysis confirms and refines current knowledge about conservation and rearrangements in corresponding chromosomal regions on BTA6. We identified and localized two new breakpoints for intrachromosomal rearrangements between human chromosome 4 and BTA6. This RH map is a powerful tool in all aspects of genetic, physical, transcript, and comparative mapping. Due to its links to the gene-dense maps of human and mouse, it can serve as a prerequisite to identify possible candidate genes for quantitative trait loci localized in the targeted BTA6 region.

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Acces; International audience

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.

This unit describes the irradiation and fusion of cells to produce radiation hybrids and the harvesting and expansion of RH colonies. A support protocol describes amplification of human chromosome fragments by PCR and analysis of those fragments by agarose gel electrophoresis.

Chantier qualité spécifique "Auteurs Externes" département de Génétique animale : uniquement liaison auteur au référentiel HR-Access; International audience

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.

International audience

Biotinidase deficiency is an autosomal recessive defect in the recycling of biotin that can lead to a variety of neurologic and cutaneous symptoms. The disease can be prevented or effectively treated with exogenous biotin. The biotinidase locus (BTD) has been maped to 3p25 by in situ hybridization. The gene has been cloned, the coding region sequenced, the genomic organization determined, and a spectrum of mutations has been characterized in more than 90 individuals with profound or partial biotinidase deficiency. We have conducted haplotype analysis of 10 consanguineous and 39 nonconsanguineous probands from the United States and 8 consanguineous probands from Turkey to localize BTD with respect to polymorphic markers on 3p and to investigate the origins of five common mutations. The inbred probands were homozygous for overlapping regions of 3p ranging in size from 1.1 to 80 cM which were flanked most narrowly by D3S1259 and D3S1293. Radiation hybrids and haplotype analysis of markers within this region suggest that BTD is located within a 0.1-cM region flanked by D3S3510 and D3S1286. The radiation hybrid data suggest that the BTD gene is oriented 5′ to 3′ between the centromere and the 3p telomere. Association studies indicate that the gene is closer to a third locus D3S3613 than D3S3510, two markers which cannot be resolved by existing linkage data. The BTD locus and D3S3613 must therefore lie between D3S3510 and D3S1286. Comparison of haplotypes reveals evidence for possible founder effects for four of the five common mutations.

Twenty nine hybrids retaining fragments of human chromosome 2 were characterized by reverse-FISH and by a panel of 106 STSs. Most of the hybrids are radiation hybrids retaining fragments of chromosome 2 as the only human contribution. The hybrid panel dissected chromosome 2 in 69 distinct physical regions, allowing a fine mapping of the sequences. These hybrids are particularly useful as starting points for generation, via Alu-PCR, of specific partial chromosome paints (PCP). We also report the mapping by FISH of 60 YACs located on chromosome 2. These resources can be advantageously used in cytogenetic investigations, with particular reference to cancer cytogenetics, as illustrated with the renal carcinoma cell line KRC/Y.

In this paper the chromosomal localization of the human skeletal muscle genes Troponin-I slow-twitch (TNNI1), Troponin-I fast-twitch (TNNI2), and Troponin-C fast (TNNC2) and the refinement of the position for alpha-Tropomyosin (TPM1) and beta-Tropomyosin (TPM2) are reported. By radiation hybrid mapping, TPM1 was assigned to chromosome 15q22.1, TPM2 to chromosome 9p13.2-p13.1, TNNI1 to chromosome 1q31.3, TNNI2 to chromosome 11p15.5, and TNNC2 to chromosome 20q12-q13.11. The genomic distribution of these genes is discussed, with particular emphasis on the cluster organization of the Troponin genes.

Genome maps are crucial tools in human genetic research, providing known landmarks for locating disease genes and frameworks for large-scale sequencing. Radiation hybrid mapping is one technique for building genome maps. In this paper, we describe the methods used to build radiation hybrid maps of the entire human genome. We present the hidden Markov model that we employ to estimate the likelihood of a map despite uncertainty about the data, and we discuss the problem of searching for maximum-likelihood maps. We describe the graph algorithms used to find sparse but reliable initial maps and our methods of extending them. Finally, we show results validating our software on simulated data, and we describe our genome-wide human radiation hybrid maps and the evidence supporting them.