Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells.

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

Mapping of leptin and its syntenic genes to chicken chromosome 1p.

BACKGROUND: Misidentification of the chicken leptin gene has hampered research of leptin signaling in this species for almost two decades. Recently, the genuine leptin gene with a GC-rich (~70%) repetitive-sequence content was identified in the chicken genome but without indicating its genomic position. This suggests that such GC-rich sequences are difficult to sequence and therefore substantial regions are missing from the current chicken genome assembly. RESULTS: A radiation hybrid panel of chicken-hamster Wg3hCl2 cells was used to map the genome location of the chicken leptin gene. Contrary to our expectations, based on comparative genome mapping and sequence characteristics, the chicken leptin was not located on a microchromosome, which are known to contain GC-rich and repetitive regions, but at the distal tip of the largest chromosome (1p). Following conserved synteny with other vertebrates, we also mapped five additional genes to this genomic region (ARF5, SND1, LRRC4, RBM28, and FLNC), bridging the genomic gap in the current Galgal5 build for this chromosome region. All of the short scaffolds containing these genes were found to consist of GC-rich (54 to 65%) sequences comparing to the average GC-content of 40% on chromosome 1. In this syntenic group, the RNA-binding protein 28 (RBM28) was in closest proximity to leptin. We deduced the full-length of the RBM28 cDNA sequence and profiled its expression patterns detecting a negative correlation (R = - 0.7) between the expression of leptin and of RBM28 across tissues that expressed at least one of the genes above the average level. This observation suggested a local regulatory interaction between these genes. In adipose tissues, we observed a significant increase in RBM28 mRNA expression in breeds with lean phenotypes. CONCLUSION: Mapping chicken leptin together with a cluster of five syntenic genes provided the final proof for its identification as the true chicken ortholog. The high GC-content observed for the chicken leptin syntenic group suggests that other similar clusters of genes in GC-rich genomic regions are missing from the current genome assembly (Galgal5), which should be resolved in future assemblies of the chicken genome.

Dissecting Plant Chromosomes by the Use of Ionizing Radiation.

Radiation treatment of genomes is used to generate chromosome breaks for numerous applications. This protocol describes the preparation of seeds and the determination of the optimal level of irradiation dosage for the creation of a radiation hybrid (RH) population. These RH lines can be used to generate high-resolution physical maps for the assembly of sequenced genomes as well as the fine mapping of genes. This procedure can also be used for mutation breeding and forward/reverse genetics.

Fast-forward genetics by radiation hybrids to saturate the locus regulating nuclear-cytoplasmic compatibility in Triticum.

The nuclear-encoded species cytoplasm specific (scs) genes control nuclear-cytoplasmic compatibility in wheat (genus Triticum). Alloplasmic cells, which have nucleus and cytoplasm derived from different species, produce vigorous and vital organisms only when the correct version of scs is present in their nucleus. In this study, bulks of in vivo radiation hybrids segregating for the scs phenotype have been genotyped by sequencing with over 1.9 million markers. The high marker saturation obtained for a critical region of chromosome 1D allowed identification of 3318 reads that mapped in close proximity of the scs. A novel in silico approach was deployed to extend these short reads to sequences of up to 70 Kb in length and identify candidate open reading frames (ORFs). Markers were developed to anchor the short contigs containing ORFs to a radiation hybrid map of 650 individuals with resolution of 288 Kb. The region containing the scs locus was narrowed to a single Bacterial Artificial Chromosome (BAC) contig of Aegilops tauschii. Its sequencing and assembly by nano-mapping allowed rapid identification of a rhomboid gene as the only ORF existing within the refined scs locus. Resequencing of this gene from multiple germplasm sources identified a single nucleotide mutation, which gives rise to a functional amino acid change. Gene expression characterization revealed that an active copy of this rhomboid exists on all homoeologous chromosomes of wheat, and depending on the specific cytoplasm each copy is preferentially expressed. Therefore, a new methodology was applied to unique genetic stocks to rapidly identify a strong candidate gene for the control of nuclear-cytoplasmic compatibility in wheat.

Radiation hybrid maps of the D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes.

BACKGROUND: The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay. METHODS: In this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat 'Chinese Spring', and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii. RESULTS: A total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average deletion size of 42.0 Mb. A total of 520 markers were anchored to 216 Ae. tauschii sequence scaffolds, 116 of which were not anchored earlier to the D-genome. CONCLUSION: This study reports the development of first high resolution RH maps for the D-genome of Ae. tauschii accession AL8/78, which were then used for the anchoring of unassigned sequence scaffolds. This study demonstrates how RH mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes.

Second generation physical and linkage maps of yellowtail (Seriola quinqueradiata) and comparison of synteny with four model fish.

BACKGROUND: Physical and linkage maps are important aids for the assembly of genome sequences, comparative analyses of synteny, and to search for candidate genes by quantitative trait locus analysis. Yellowtail, Seriola quinqueradiata, is an economically important species in Japanese aquaculture, and genetic information will be useful for DNA-assisted breeding. We report the construction of a second generation radiation hybrid map, its synteny analysis, and a second generation linkage map containing SNPs (single nucleotide polymorphisms) in yellowtail. RESULTS: Approximately 1.4 million reads were obtained from transcriptome sequence analysis derived from 11 tissues of one individual. To identify SNPs, cDNA libraries were generated from a pool of 500 whole juveniles, and the gills and kidneys of 100 adults. 9,356 putative SNPs were detected in 6,025 contigs, with a minor allele frequency ≥ 25%. The linkage and radiation hybrid maps were constructed based on these contig sequences. 2,081 markers, including 601 SNPs markers, were mapped onto the linkage map, and 1,532 markers were mapped in the radiation hybrid map. CONCLUSIONS: The second generation linkage and physical maps were constructed using 6,025 contigs having SNP markers. These maps will aid the de novo assembly of sequencing reads, linkage studies and the identification of candidate genes related to important traits. The comparison of marker contigs in the radiation hybrid map indicated that yellowtail is evolutionarily closer to medaka than to green-spotted pufferfish, three-spined stickleback or zebrafish. The synteny analysis may aid studies of chromosomal evolution in yellowtail compared with model fish.

An update of the goat genome assembly using dense radiation hybrid maps allows detailed analysis of evolutionary rearrangements in Bovidae.

BACKGROUND: The domestic goat (Capra hircus), an important livestock species, belongs to a clade of Ruminantia, Bovidae, together with cattle, buffalo and sheep. The history of genome evolution and chromosomal rearrangements on a small scale in ruminants remain speculative. Recently completed goat genome sequence was released but is still in a draft stage. The draft sequence used a variety of assembly packages, as well as a radiation hybrid (RH) map of chromosome 1 as part of its validation. RESULTS: Using an improved RH mapping pipeline, whole-genome dense maps of 45,953 SNP markers were constructed with statistical confidence measures and the saturated maps provided a fine map resolution of approximate 65 kb. Linking RH maps to the goat sequences showed that the assemblies of scaffolds/super-scaffolds were globally accurate. However, we observed certain flaws linked to the process of anchoring chromosome using conserved synteny with cattle. Chromosome assignments, long-range order, and orientation of the scaffolds were reassessed in an updated genome sequence version. We also present new results exploiting the updated goat genome sequence to understand genomic rearrangements and chromosome evolution between mammals during species radiations. The sequence architecture of rearrangement sites between the goat and cattle genomes presented abundant segmental duplication on regions of goat chromosome 9 and 14, as well as new insertions in homologous cattle genome regions. This complex interplay between duplicated sequences and Robertsonian translocations highlights the rearrangement mechanism of centromeric nonallelic homologous recombination (NAHR) in mammals. We observed that species-specific shifts in ANKRD26 gene duplication are coincident with breakpoint reuse in divergent lineages and this gene family may play a role in chromosome stabilization in chromosome evolution. CONCLUSIONS: We generated dense maps of the complete whole goat genome. The chromosomal maps allowed us to anchor and orientate assembled genome scaffolds along the chromosomes, annotate chromosome rearrangements and thereby get a better understanding of the genome evolution of ruminants and other mammals.

Reliable Radiation Hybrid Maps: An Efficient Scalable Clustering-Based Approach.

The process of mapping markers from radiation hybrid mapping (RHM) experiments is equivalent to the traveling salesman problem and, thereby, has combinatorial complexity. As an additional problem, experiments typically result in some unreliable markers that reduce the overall quality of the map. We propose a clustering approach for addressing both problems efficiently by eliminating unreliable markers without the need for mapping the complete set of markers. Traditional approaches for eliminating markers use resampling of the full data set, which has an even higher computational complexity than the original mapping problem. In contrast, the proposed approach uses a divide-and-conquer strategy to construct framework maps based on clusters that exclude unreliable markers. Clusters are ordered using parallel processing and are then combined to form the complete map. We present three algorithms that explore the trade-off between the number of markers included in the map and placement accuracy. Using an RHM data set of the human genome, we compare the framework maps from our proposed approaches with published physical maps and with the results of using the Carthagene tool. Overall, our approaches have a very low computational complexity and produce solid framework maps with good chromosome coverage and high agreement with the physical map marker order.

Physical mapping resources for large plant genomes: radiation hybrids for wheat D-genome progenitor Aegilops tauschii.

BACKGROUND: Development of a high quality reference sequence is a daunting task in crops like wheat with large (~17Gb), highly repetitive (>80%) and polyploid genome. To achieve complete sequence assembly of such genomes, development of a high quality physical map is a necessary first step. However, due to the lack of recombination in certain regions of the chromosomes, genetic mapping, which uses recombination frequency to map marker loci, alone is not sufficient to develop high quality marker scaffolds for a sequence ready physical map. Radiation hybrid (RH) mapping, which uses radiation induced chromosomal breaks, has proven to be a successful approach for developing marker scaffolds for sequence assembly in animal systems. Here, the development and characterization of a RH panel for the mapping of D-genome of wheat progenitor Aegilops tauschii is reported. RESULTS: Radiation dosages of 350 and 450 Gy were optimized for seed irradiation of a synthetic hexaploid (AABBDD) wheat with the D-genome of Ae. tauschii accession AL8/78. The surviving plants after irradiation were crossed to durum wheat (AABB), to produce pentaploid RH1s (AABBD), which allows the simultaneous mapping of the whole D-genome. A panel of 1,510 RH1 plants was obtained, of which 592 plants were generated from the mature RH1 seeds, and 918 plants were rescued through embryo culture due to poor germination (<3%) of mature RH1 seeds. This panel showed a homogenous marker loss (2.1%) after screening with SSR markers uniformly covering all the D-genome chromosomes. Different marker systems mostly detected different lines with deletions. Using markers covering known distances, the mapping resolution of this RH panel was estimated to be <140kb. Analysis of only 16 RH lines carrying deletions on chromosome 2D resulted in a physical map with cM/cR ratio of 1:5.2 and 15 distinct bins. Additionally, with this small set of lines, almost all the tested ESTs could be mapped. A set of 399 most informative RH lines with an average deletion frequency of ~10% were identified for developing high density marker scaffolds of the D-genome. CONCLUSIONS: The RH panel reported here is the first developed for any wild ancestor of a major cultivated plant species. The results provided insight into various aspects of RH mapping in plants, including the genetically effective cell number for wheat (for the first time) and the potential implementation of this technique in other plant species. This RH panel will be an invaluable resource for mapping gene based markers, developing a complete marker scaffold for the whole genome sequence assembly, fine mapping of markers and functional characterization of genes and gene networks present on the D-genome.

A duck RH panel and its potential for assisting NGS genome assembly.

BACKGROUND: Owing to the low cost of the high throughput Next Generation Sequencing (NGS) technology, more and more species have been and will be sequenced. However, de novo assemblies of large eukaryotic genomes thus produced are composed of a large number of contigs and scaffolds of medium to small size, having no chromosomal assignment. Radiation hybrid (RH) mapping is a powerful tool for building whole genome maps and has been used for several animal species, to help assign sequence scaffolds to chromosomes and determining their order. RESULTS: We report here a duck whole genome RH panel obtained by fusing female duck embryonic fibroblasts irradiated at a dose of 6,000 rads, with HPRT-deficient Wg3hCl2 hamster cells. The ninety best hybrids, having an average retention of 23.6% of the duck genome, were selected for the final panel. To allow the genotyping of large numbers of markers, as required for whole genome mapping, without having to cultivate the hybrid clones on a large scale, three different methods involving Whole Genome Amplification (WGA) and/or scaling down PCR volumes by using the Fluidigm BioMark(TM) Integrated Fluidic Circuits (IFC) Dynamic Array(TM) for genotyping were tested. RH maps of APL12 and APL22 were built, allowing the detection of intrachromosomal rearrangements when compared to chicken. Finally, the panel proved useful for checking the assembly of sequence scaffolds and for mapping EST located on one of the smallest microchromosomes. CONCLUSION: The Fluidigm BioMark(TM) Integrated Fluidic Circuits (IFC) Dynamic Array(TM) genotyping by quantitative PCR provides a rapid and cost-effective method for building RH linkage groups. Although the vast majority of genotyped markers exhibited a picture coherent with their associated scaffolds, a few of them were discordant, pinpointing potential assembly errors. Comparative mapping with chicken chromosomes GGA21 and GGA11 allowed the detection of the first chromosome rearrangements on microchromosomes between duck and chicken. As in chicken, the smallest duck microchromosomes appear missing in the assembly and more EST data will be needed for mapping them. Altogether, this underlines the added value of RH mapping to improve genome assemblies.

Mammalian genes preferentially co-retained in radiation hybrid panels tend to avoid coexpression.

Coexpression has been frequently used to explore modules of functionally related genes in eukaryotic genomes. However, we found that genetically interacting mammalian genes identified through radiation hybrid (RH) genotypes tend not to be coexpressed across tissues. This pattern remained unchanged after controlling for potential confounding factors, including chromosomal linkage, chromosomal distance, and gene duplication. Because >99.9% of the genetically interacting genes were identified according to the higher co-retention frequencies, our observation implies that coexpression is not necessarily an indication of the need for the co-presence of two genes in the genome, which is a prerequisite for cofunctionality of their coding proteins in the cell. Therefore, coexpression information must be applied cautiously to the exploration of the functional relatedness of genes in a genome.

Isolation, mapping, SNP detection and association with backfat traits of the porcine CTNNBL1 and DGAT2 genes.

Both the CTNNBL1 (catenin, ß-like1) and DGAT2 (diacylglycerol acyltransferase2) genes play important roles in adipose metabolism. In this study, we cloned these two genes in pigs. Semi-quantitative RT-PCR results showed that both genes were extensively expressed, and CTNNBL1 was at a high level in the heart and spleen, while DGAT2 was most abundant in the liver. In CTNNBL1, one synonymous mutation c.555C>T was identified in the coding region, and association analysis showed that different genotypes of CTNNBL1 were significantly associated with backfat at the shoulder and backfat at the rump (P < 0.05). In 3'-UTR of DGAT2, an A/G variation was detected by the Bcn I PCR-RFLP method, and different genotypes were significantly associated with backfat between the 6th and 7th ribs (P < 0.05). The allele frequency was tested among 188 unrelated pigs from six breeds. The results showed that for CTNNBL1, the Chinese indigenous breeds had higher frequencies of the C allele whereas the western breed had higher frequency of the T allele; and for DGAT2, allele A or G were distributed with no obvious difference in allele frequency. IMpRH was employed to localize these two genes, and CTNNBL1 was assigned to SSC17q21-23 and DGAT2 was assigned to SSC9p23-p24. The results suggest that the porcine CTNNBL1 and DGAT2 genes affect porcine fat deposition and further investigation will be necessary to illustrate the underlying mechanisms.

Statistical confidence measures for genome maps: application to the validation of genome assemblies.

MOTIVATION: Genome maps are imperative to address the genetic basis of the biology of an organism. While a growing number of genomes are being sequenced providing the ultimate genome maps-this being done at an even faster pace now using new generation sequencers-the process of constructing intermediate maps to build and validate a genome assembly remains an important component for producing complete genome sequences. However, current mapping approach lack statistical confidence measures necessary to identify precisely relevant inconsistencies between a genome map and an assembly. RESULTS: We propose new methods to derive statistical measures of confidence on genome maps using a comparative model for radiation hybrid data. We describe algorithms allowing to (i) sample from a distribution of maps and (ii) exploit this distribution to construct robust maps. We provide an example of application of these methods on a dog dataset that demonstrates the interest of our approach. AVAILABILITY: Methods are implemented in two freely available softwares: Carthagene (http://www.inra.fr/mia/T/CarthaGene/) and a companion software (metamap, available at: http://snp.toulouse.inra.fr/~servin/index.cgi/Metamap).

A radiation hybrid map of the European sea bass (Dicentrarchus labrax) based on 1581 markers: Synteny analysis with model fish genomes.

The selective breeding of fish for aquaculture purposes requires the understanding of the genetic basis of traits such as growth, behaviour, resistance to pathogens and sex determinism. Access to well-developed genomic resources is a prerequisite to improve the knowledge of these traits. Having this aim in mind, a radiation hybrid (RH) panel of European sea bass (Dicentrarchus labrax) was constructed from splenocytes irradiated at 3000 rad, allowing the construction of a 1581 marker RH map. A total of 1440 gene markers providing ~4400 anchors with the genomes of three-spined stickleback, medaka, pufferfish and zebrafish, helped establish synteny relationships with these model species. The identification of Conserved Segments Ordered (CSO) between sea bass and model species allows the anticipation of the position of any sea bass gene from its location in model genomes. Synteny relationships between sea bass and gilthead seabream were addressed by mapping 37 orthologous markers. The sea bass genetic linkage map was integrated in the RH map through the mapping of 141 microsatellites. We are thus able to present the first complete gene map of sea bass. It will facilitate linkage studies and the identification of candidate genes and Quantitative Trait Loci (QTL). The RH map further positions sea bass as a genetic and evolutionary model of Perciformes and supports their ongoing aquaculture expansion.

A method to produce radiation hybrids for the D-genome chromosomes of wheat (Triticum aestivum L.).

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 high-resolution radiation hybrid map of sheep chromosome X and comparison with human and cattle.

A radiation hybrid (RH) map of sheep X chromosome (Ovisaries; OARX) containing 146 physically anchored loci was generated in this study, providing information for comparative X chromosome analysis between the maps of sheep, human, and cattle. Primers typed on the USUoRH5000 ovine whole-genome radiation hybrid panel were designed from sequences predicted to be on the ovine X chromosome, based on comparative mapping within the virtual sheep genome browser (v1.2). The resulting RH map for the ovine X chromosome consists of 4 linkage groups composed of 76 BAC end sequences (BES), 28 gene loci that were confirmed within ovine BAC clones in the CHORI-243 ovine BAC library, 28 additional gene loci from the ovine comparative map and 14 polymorphic sequence tagged sites (STS) from the OARX linkage map. This first-generation RH map of OARX contributes to the expansion of a comprehensive ovine genome map for sheep and provides evidence of rearrangements in loci order compared to the human and cattle orders.

Cytogenetic anchoring of radiation hybrid and virtual maps of sheep chromosome X and comparison of X chromosomes in sheep, cattle, and human.

A comprehensive physical map was generated for Ovis aries chromosome X (OARX) based on a cytogenomics approach. DNA probes were prepared from bacterial artificial chromosome (BAC) clones from the CHORI-243 sheep library and were assigned to G-banded metaphase spreads via fluorescence in-situ hybridization (FISH). A total of 22 BACs gave a single hybridization signal to the X chromosome and were assigned out of 32 tested. The positioned BACs contained 16 genes and a microsatellite marker which represent new cytogenetically mapped loci in the sheep genome. The gene and microsatellite loci serve to anchor between the existing radiation hybrid (RH) and virtual sheep genome (VSG) maps to the cytogenetic OARX map, whilst the BACs themselves also serve as anchors between the VSG and the cytogenetic maps. An additional 17 links between the RH and cytogenetic maps are provided by BAC end sequence (BES) derived markers that have also been positioned on the RH map. Comparison of the map orders for the cytogenetic, RH, and virtual maps reveals that the orders for the cytogenetic and RH maps are most similar, with only one locus, represented by BAC CH243-330E18, mapping to relatively different positions. Several discrepancies, including an inverted segment are found when comparing both the cytogenetic and RH maps with the virtual map. These discrepancies highlight the value of using physical mapping methods to inform the process of future in silico map construction. A detailed comparative analysis of sheep, human, and cattle mapping data allowed the construction of a comparative map that confirms and expands the knowledge about evolutionary conservation and break points between the X chromosomes of the three mammalian species.

An integrated RH map of porcine chromosome 10.

BACKGROUND: Whole genome radiation hybrid (WG-RH) maps serve as "scaffolds" to significantly improve the orientation of small bacterial artificial chromosome (BAC) contigs, order genes within the contigs and assist assembly of a sequence-ready map for virtually any species. Here, we report the construction of a porcine: human comparative map for pig (Sus scrofa) chromosome 10 (SSC10) using the IMNpRH2(12,000-rad) porcine WG-RH panel, integrated with the IMpRH(7000-rad) WG-RH, genetic and BAC fingerprinted contig (FPC) maps. RESULTS: Map vectors from the IMNpRH2(12,000-rad) and IMpRH(7,000-rad) panels were merged to construct parallel framework (FW) maps, within which FW markers common to both panels have an identical order. This strategy reduced map discrepancies between the two panels and significantly improved map accuracy. A total of 216 markers, including 50 microsatellites (MSs), 97 genes and ESTs, and 69 BAC end sequences (BESs), were ordered within two linkage groups at two point (2 pt) LOD score of 8. One linkage group covers SSC10p with accumulated map distances of 738.2 cR(7,000) and 1814.5 cR(12,000), respectively. The second group covers SSC10q at map distances of 1336.9 cR(7,000) and 3353.6 cR(12,000), yielding an overall average map resolution of 16.4 kb/cR(12,000) or 393.5 kb per marker on SSC10. This represents an approximately 2.5-fold increase in map resolution over the IMpRH(7,000-rad) panel. Based on 127 porcine markers that have homologous sequences in the human genome, a detailed comparative map between SSC10 and human (Homo sapiens) chromosome (HSA) 1, 9 and 10 was built. CONCLUSION: This initial comparative RH map of SSC10 refines the syntenic regions between SSC10 and HSA1, 9 and 10. It integrates the IMNpRH2(12,000-rad) and IMpRH(7,000-rad), genetic and BAC FPC maps and provides a scaffold to close potential gaps between contigs prior to genome sequencing and assembly. This map is also useful in fine mapping of QTLs on SSC10.

A high resolution RH map of the bovine major histocompatibility complex.

BACKGROUND: The cattle MHC is termed the bovine leukocyte antigen (BoLA) and, along with the MHCs of other ruminants, is unique in its genomic organization. Consequently, correct and reliable gene maps and sequence information are critical to the study of the BoLA region. The bovine genome sequencing project has produced two assemblies (Btau_3.1 and 4.0) that differ substantially from each other and from conventional gene maps in the BoLA region. To independently compare the accuracies of the different sequence assemblies, we have generated a high resolution map of BoLA using a 12,000rad radiation hybrid panel. Seventy-seven unique sequence tagged site (STS) markers chosen at approximately 50 kb intervals from the Btau 2.0 assembly and spanning the IIa-III-I and IIb regions of the bovine MHC were mapped on a 12,000rad bovine radiation hybrid (RH) panel to evaluate the different assemblies of the bovine genome sequence. RESULTS: Analysis of the data generated a high resolution RH map of BoLA that was significantly different from the Btau_3.1 assembly of the bovine genome but in good agreement with the Btau_4.0 assembly. Of the few discordancies between the RH map and Btau_4.0, most could be attributed to closely spaced markers that could not be precisely ordered in the RH panel. One probable incorrectly-assembled sequence and three missing sequences were noted in the Btau_4.0 assembly. The RH map of BoLA is also highly concordant with the sequence-based map of HLA (NCBI build 36) when reordered to account for the ancestral inversion in the ruminant MHC. CONCLUSION: These results strongly suggest that studies using Btau_3.1 for analyses of the BoLA region should be reevaluated in light of the Btau_4.0 assembly and indicate that additional research is needed to produce a complete assembly of the BoLA genomic sequences.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC) genes but is located on a chromosomal region distinct from the LRC and FcR gene clusters.

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common gamma-chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20.