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.     

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.     

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.     

A radiation hybrid map of chicken Chromosome 4

The mapping resolution of the physical map for chicken Chromosome 4 (GGA4) was improved by a combination of radiation hybrid (RH) mapping and bacterial artificial chromosome (BAC) mapping. The ChickRH6 hybrid panel was used to construct an RH map of GGA4. Eleven microsatellites known to be located on GGA4 were included as anchors to the genetic linkage map for this chromosome. Based on the known conserved synteny between GGA4 and human Chromosomes 4 and X, sequences were identified for the orthologous chicken genes from these human chromosomes by BLAST analysis. These sequences were subsequently used for the development of STS markers to be typed on the RH panel. Using a logarithm of the odds (LOD) threshold of 5.0, nine linkage groups could be constructed which were aligned with the genetic linkage map of this chromosome. The resulting RH map consisted of the 11 microsatellite markers and 50 genes. To further increase the number of genes on the map and to provide additional anchor points for the physical BAC map of this chromosome, BAC clones were identified for 22 microsatellites and 99 genes. The combined RH and BAC mapping approach resulted in the mapping of 61 genes on GGA4 increasing the resolution of the chicken–human comparative map for this chromosome. This enhanced comparative mapping resolution enabled the identification of multiple rearrangements between GGA4 and human Chromosomes 4q and Xp.     

Mapping and Expression Analysis of Chicken NDRG1 and NDRG3 Genes

N-myc downstream-regulated genes 1 and 3 (NDRG1 and NDRG3) are members of the alpha/beta hydrolase superfamily. Phylogenetic analysis of the family demonstrated that human NDRG1 and 3 belong to a subfamily. The mapping and gene expression patterns of these genes represent one step toward further investigation into their possible roles in the chicken (Gallus gallus). To map these genes in the chicken chromosome, a 6000 rads chicken-hamster radiation hybrid panel (ChickRH6) was used. Primers were designed according to the published human sequences for amplification of those two genes. We compared the corresponding human mRNA sequences with the predicted coding sequences of the chicken NDRG1 and 3 genes and found that the assembled contigs shared a high percentage of similarity with the human genes. PCR of samples from ChickRH6 revealed that the locations of NDRG1 and 3 are linked to the markers MYC (58 cRs away, LOD score 4.52) and SEQ0265 (10 cRs away, LOD score 17.81), respectively. This result adds two new markers to the chicken RH map, and it reinforces that the RH technique is indeed a powerful tool for mapping genes due to its rapidity, precision, convenience, and reproducibility. In addition, we detected the gene expression and distribution of chicken NDRG1 and 3 in seven tissues, including heart, liver, spleen, lung, muscle, brain, and thymus, by RT-PCR, and found that NDRG1 is relatively ubiquitously expressed in all the tested tissues and highly expressed in heart and liver, whereas NDRG3 is high in heart, muscle, and brain.     

The chicken RH map: current state of progress and microchromosome mapping

The ChickRH6 radiation hybrid panel has been used to construct consensus chromosome radiation hybrid (RH) maps of the chicken genome. Markers genotyped were either from throughout the genome or targeted to specific chromosomes and a large proportion (one third) of data was the result of collaborative efforts. Altogether, 2,531 markers were genotyped, allowing the construction of RH reference maps for 20 chromosomes and linkage groups for four other chromosomes. Amongst the markers, 581 belong to the framework maps, while 1,721 are on the comprehensive maps. Around 800 markers still have to be assigned to linkage groups. Our attempt to assign the supercontigs from the chrun (virtual chromosome containing all the genome sequence that could not be attributed to a chromosome) as well as EST (Expressed Sequence Tag) contigs that do not have a BLAST hit in the genome assembly led to the construction of new maps for microchromosomes either absent or for which very little data is present in the genome assembly. RH data is presented through our ChickRH webserver (http://chickrh.toulouse.inra.fr/), which is a mapping tool as well as the official repository RH database for genotypes. It also displays the RH reference maps and comparison charts with the sequence thus highlighting the possible discrepancies. Future improvements of the RH maps include complete coverage of the sequence assigned to chromosomes, further mapping of the chrun and mapping of EST contigs absent from the assembly. This will help finish the mapping of the smallest gene-rich microchromosomes.     

Construction of bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping

High-density whole-genome maps are essential for ordering genes or markers and aid in the assembly of genome sequence. To increase the density of markers on the bovine radiation hybrid map, and hence contribute to the assembly of the bovine genome sequence, an Illumina BeadStation was used to simultaneously type large numbers of markers on the Roslin-Cambridge 3000 rad bovine-hamster whole-genome radiation hybrid panel (WGRH3000). In five multiplex reactions, 6738 sequence tagged site (STS) markers were successfully typed on the WGRH3000 panel DNA. These STSs harboured SNPs that were developed as a result of the bovine genome sequencing initiative. Typically, the most time consuming and expensive part of creating high-density radiation hybrid (RH) maps is genotyping the markers on the RH panel with conventional approaches. Using the method described in this article, we have developed a high-density whole-genome RH map with 4690 loci and a linkage map with 2701 loci, with direct comparison to the bovine whole-genome sequence assembly (Btau_2.0) in a fraction of the time it would have taken with conventional typing and genotyping methods.     

Cross-referencing radiation hybrid data to the recombination map: lessons from mouse chromosome 18

We are building a framework map of known-order anchor markers between the mouse T31 radiation hybrid (RH) panel and the recombination map based on The Jackson Laboratory (TJL) interspecific backcross panels using the established genetic order to evaluate and strengthen the RH results. In making this map comparison, we have elucidated several problems inherent in RH mapping and minimized these by careful attention to data gathering and interpretation methods. We describe lessons and pitfalls of developing radiation hybrid maps, using the example of mouse Chromosome 18, for which we have built a framework map of microsatellite anchor loci spanning the entire chromosome at significant LOD with no gaps. Sixty-five D18Mit- simple sequence length polymorphism (SSLP) markers form a continuous linkage along the T31 RH Chromosome 18 (RH map length 1598 cR, genetic length 41 cM) with all LODs greater than 6. These markers are also placed on TJL interspecific backcrosses, and the order of the markers in the two systems is in complete agreement. We are continuing to cross-reference the RH data to TJL backcross data for the other mouse chromosomes to improve further the power of RH mapping and to integrate more precisely the extensive existing recombination mapping data for the mouse with the incoming radiation hybrid map data.     

用放射杂交板定位鸡的MC4R基因以及其在鸡和人染色体上同源区的比较分析

黑素皮质素受体(melanocortin-4 receptor,MC4R)基因的突变与猪、鼠和人等的食欲、肥胖和生长有关联性,然而对鸡的MC4R基因的功能却知之甚少。为了确定鸡的MC4R基因在染色体上的位置,使用鸡-仓鼠杂交板(ChickRH6)做了该基因的定位工作。通过扩增ChickRH6杂交板上的93个样品,然后经整合分析将mC4R基因定位在2号染色体上的标记MCW0062、BCL2和OVY附近,即2q12。这个连锁图上的5个标记基于两点分析与MC4R的LOD值都大于5。同时,以MC4R基因为标记做了鸡和人的染色体比较分析。结果显示鸡的2号染色体(GGA2)和人的18号染色体(HSA18)存在同源区,且基因BCL2和肥胖基因(obesity)位于MC4R基因附近。推测鸡的MC4R基因与人的MC4R基因可能具有相似的功能。该研究揭示了鸡和人MC4R基因的染色体分布,并用杂交放射板将鸡的MC4R基因定位在2号染色体的12区带。     

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 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.     

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 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 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.     

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.     

Physical mapping of CSF2RA, ANT3 and STS on the pseudoautosomal region of bovine chromosome X

The pseudoautosomal region (PAR) of bovine chromosome X (BTA X) has a particularly low representation of genes and markers, making comparative gene mapping in this region difficult. We describe the localization of three genes, colony-stimulating factor 2 receptor alpha (CSF2RA), ADP/ATP translocase 3 (ANT3) and steroid sulphatase (STS) on PAR of BTA X using a 5000 rad whole-genome radiation hybrid panel. The relationship of these genes to a number of previously mapped simple sequence repeat (microsatellite) markers is determined by physical and radiation hybrid mapping methods. The resulting radiation hybrid map resolves a discrepancy between the two major bovine linkage maps in the PAR of BTA 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.     

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

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