Genome Radiation Hybrid Panel Research Articles

Construction of a radiation hybrid panel and the first yellowtail (Seriola quinqueradiata) radiation hybrid map using a nanofluidic dynamic array

BackgroundYellowtail (Seriola quinqueradiata) are an economically important species in Japan. However, there are currently no methods for captive breeding and early rearing for yellowtail. Thus, the commercial cultivation of this species is reliant upon the capture of wild immature fish. Given this, there is a need to develop captive breeding techniques to reduce pressure on wild stocks and facilitate the sustainable development of yellowtail aquaculture. We constructed a whole genome radiation hybrid (RH) panel for yellowtail gene mapping and developed a framework physical map using a nanofluidic dynamic array to use SNPs (single nucleotide polymorphisms) in ESTs (expressed sequence tags) for the DNA-assisted breeding of yellowtail.ResultsClonal RH cell lines were obtained after ionizing radiation; specifically, 78, 64, 129, 55, 42, and 53 clones were isolated after treatment with 3,000, 4,000, 5,000, 6,000, 8,000, or 10,000 rads, respectively. A total of 421 hybrid cell lines were obtained by fusion with mouse B78 cells. Ninety-four microsatellite markers used in the genetic linkage map were genotyped using the 421 hybrid cell lines. Based upon marker retention and genome coverage, we selected 93 hybrid cell lines to form an RH panel. Importantly, we performed the first genotyping of yellowtail markers in an RH panel using a nanofluidic dynamic array (Fluidigm, CA, USA). Then, 580 markers containing ESTs and SNPs were mapped in the first yellowtail RH map.ConclusionsWe successfully developed a yellowtail RH panel to facilitate the localization of markers. Using this, a framework RH map was constructed with 580 markers. This high-density physical map will serve as a useful tool for the identification of genes related to important breeding traits using genetic structural information, such as conserved synteny. Moreover, in a comparison of 30 sequences in the RH group 1 (SQ1), yellowtail appeared to be evolutionarily closer to medaka and the green-spotted pufferfish than to zebrafish. We suggest that synteny analysis may be potentially useful as a tool to investigate chromosomal evolution by comparison with model fish.

Cloning and expression of activation induced cytidine deaminase from Bos taurus

Activation induced cytidine deaminase is an enzyme crucial to somatic hypermutation and gene conversion, processes that are essential for the diversification of Ig V genes. The bovine Ig repertoire appears to be diversified by mechanisms that are significantly different to those that operate in humans and mice. This study set out to test the hypothesis that differences in the organization, coding sequence, expression or genomic location of the bovine AICDA gene enables the encoded enzyme to catalyse the unusual Ig diversification mechanism seen in cattle as well as conventional antigen-driven mutation. Characterization of bovine AICDA excluded the first two possibilities. AICDA expression was detected in lymphoid tissues from neonatal and older cattle, but AICDA cDNA could not be detected in muscle tissue. The pattern of gene expression did not therefore differ from that in other vertebrates. The AICDA cDNA was cloned and expressed successfully in Escherichia coli generating a phenotype consistent with the mutating action of this deaminase. Using a whole genome radiation hybrid panel, bovine AICDA was mapped to a region of bovine chromosome 5 syntenic with the location of human AICDA on chromosome 12. We conclude that the unusual nature of Ig diversification in cattle is unlikely to be attributable to the structure, sequence, activity or genomic location of bovine AICDA.

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 cR₅₀₀₀. The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH₅₀₀₀) 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.

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) not only is a useful source of milk, it also provides meat and works as a natural source of labor and biogas. To establish a project for buffalo genome mapping a 5,000-rad whole genome radiation hybrid panel was constructed for river buffalo and used to build preliminary RH maps from two chromosomes (BBU 3 and BBU10). The preliminary maps contain 66 markers, including coding genes, cattle ESTs and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci, in particular, genes and expressed sequence tags that will allow detailed comparative maps between buffalo, cattle and other species to be constructed. A large quantity of DNA has been prepared from the cell lines forming the RH panel reported here and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.

Assignment of 204 genes localized on HSA17 to a porcine RH (IMpRH) map to generate a dense comparative map between pig and human/mouse

Bi- and uni-directional chromosome painting (ZOO-FISH) and gene mapping have revealed correspondences between human chromosome (HSA) 17 and porcine chromosome (SSC) 12 harboring economically important quantitative trait loci. In the present study, we have assigned 204 genes localized on HSA17 to SSC12 to generate a comprehensive comparative map between HSA17 and SSC12. Two hundred fifty-five primer pairs were designed using porcine sequences orthologous with human genes. Of the 255 primer pairs, 208 (81.6%) were used to assign the corresponding genes to porcine chromosomes using the INRA-Minnesota 7000-rad porcine × Chinese hamster whole genome radiation hybrid (IMpRH) panel. Two hundred three genes were integrated into the SSC12 IMpRH linkage maps; and one gene, PPARBP, was found to link to THRA1 located in SSC12 but not incorporated into the linkage maps. Three genes (GIT1, SLC25A11, and HT008) were suggested to link to SSC12 markers, and the remaining gene (RPL26) did not link to any genes/expressed sequence tags/markers registered, including those in the present study. A comparison of the gene orders among SSC12, HSA17, and mouse chromosome 11 indicates that intra-chromosomal rearrangements occurred frequently in this ancestral mammalian chromosome during speciation.

Construction of a radiation hybrid map of chicken chromosome 2 and alignment to the chicken draft sequence

BackgroundThe 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.ResultsEighty 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.ConclusionWe 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.

An integrated 4249 marker FISH/RH map of the canine genome.

BackgroundThe 156 breeds of dog recognized by the American Kennel Club offer a unique opportunity to map genes important in genetic variation. Each breed features a defining constellation of morphological and behavioral traits, often generated by deliberate crossing of closely related individuals, leading to a high rate of genetic disease in many breeds. Understanding the genetic basis of both phenotypic variation and disease susceptibility in the dog provides new ways in which to dissect the genetics of human health and biology.ResultsTo facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH) map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs) localized to 1423 unique positions, 851 of which have also been mapped by fluorescence in situ hybridization (FISH). The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS) between the dog and human genomes, dramatically extending the length of most previously described CS.ConclusionsThese results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps.

Adiponectin을 암호화하는 돼지 APM1 유전자의 염색체상 위치파악과 돌연변이 탐색

APM1(adipose most abundant gene transcript 1)은 지방세포에서 분비된는 단백질로서 Adiponectin을 암호화하며 GBP-28, AdipoQ, Acrp30으로 불리워졌다. 이 유전자는 쥐의 16번 염색체 및 인간의 3번 염색체 q27 부위에 존재하며 지방의 대사 및 호르몬의 여러 과정에 중요하게 작용하는 것으로 알려져 왔다. 돼지에서 APM1과 양적형질과의 연관성을 알아보기 위하여 somatic cell hybrid panel과 radiation hybrid panel을 이용하여 염색체상의 위치를 밝혀냈다. 분석결과 이 유전자는 돼지 13번 염색체의 q41이나 q46-49에 존재하는 것으로 밝혀졌다. RH pnael의 분석 결과, 이 APM1과 가장 연관이 된 marker는 SIAT1(LOD score 20.29)로 밝혀졌으며 이미 보고된 지방과 관련된 양적형질 유전자 좌위와 일치하였다. 8개의 다른 품종을 이용한 SSCP 분석으로, 한국재래돼지와 한국야생돼지에서 두 개의 특이한 SSCP 타입이 발견되었으며 sequence 분석결과 두 개의 염기가 치환(T672C and C705G)되어 있음을 알 수 있었다. 이 유전자는 사람, 마우스, 소의 염기서열과 약 79-87%의 상동성을 가지고 있으며 다른 포유동물에서 밝혀진 signal sequence, hypervariable region, collagenous region, globular domain과 유사한 구조로 되어 있음을 알 수 있었다. Adiponectin is adipocyte complement-related protein which is highly specialized to play important roles in metabolic and honnonal processes. This protein, called GBP-28, AdipoQ, and Acrp30, is encoded by the adipose most abundant gene transcript 1 (APM1) which locates on human chromosome 3q27 and mouse chromosome 16. In order to determine chromosomal localization of the porcine APM1, we carried out PCR analysis using somatic cell hybrid panel as well as porcine whole genome radiation hybrid (RH) panel. The result showed that the porcine APM1 located on chromosome 13q41 or 13q46-49. These locations were further investigated with the two point analysis of RH panel, revealed the most significant linked marker (LOD score 20.29) being SIAT1 (8 cRs away), where the fat-related QTL located. From the SSCP analysis of APM1 using 8 pig breeds, two distinct SSCP types were detected from K~ native and Korean wild pigs. The determined sequences in Korean native and Korean wild pigs showed that two nucleotide positions (T672C and C705G) were substituted. The primary sequence of the porcine APM1 has 79 to 87% identity with those of human, mouse, and bovine APM1. The domain structures of the porcine APM1 such as signal sequence, hypervariable region, collagenous region. and globular domain are also similar to those of mammalian genes.

High-resolution comparative mapping between human chromosomes 4 and 8 and bovine chromosome 27 provides genes and segments serving as positional candidates for udder health in cattle

To get more information about the order of genes located in Bos taurus (BTA) chromosome 27 segments, supposed to harbor loci influencing clinical mastitis and somatic cell count, and to identify genes that serve as positional candidates for the mentioned traits, we constructed a high-resolution, comparative, and comprehensive gene map for BTA27. The map includes 57 loci in a 5000-rad cattle–hamster whole genome radiation hybrid panel supported by 50 syntenic assignments in a cattle–murine somatic hybrid cell panel. Thirty-eight new loci (36 genes, 2 microsatellites) together with repeated mappings of 5 genes and 7 microsatellites and integration of existing data from 7 microsatellites were used to generate a comprehensive RH 5000 map. The RH map, constructed at lod score criterion 8 using the software RHMAP v.3.0, consisted of three linkage groups 23, 22, and 590 cR 5000 in length. Gene assignments on BTA27 and the localization of 8 more genes on BTA8 and BTA14 previously predicted on BTA8/BTA27 and BTA14/BTA27 narrowed down significantly the chromosome break points between the three cattle chromosomes and segments on Homo sapiens chromosomes HSA4 and HSA8. Defined evolutionary break points increase the accuracy of comparative in silico mapping of further human genes in conserved chromosome segments of BTA27.

A 1.4-Mb interval RH map of horse chromosome 17 provides detailed comparison with human and mouse homologues

Comparative genomics has served as a backbone for the rapid development of gene maps in domesticated animals. The integration of this approach with radiation hybrid (RH) analysis provides one of the most direct ways to obtain physically ordered comparative maps across evolutionarily diverged species. We herein report the development of a detailed RH and comparative map for horse chromosome 17 (ECA17). With markers distributed at an average interval of every 1.4 Mb, the map is currently the most informative among the equine chromosomes. It comprises 75 markers (56 genes and 19 microsatellites), of which 50 gene specific and 5 microsatellite markers were generated in this study and typed to our 5000-rad horse × hamster whole genome RH panel. The markers are dispersed over six RH linkage groups and span 825 cR 5000. The map is among the most comprehensive whole chromosome comparative maps currently available for domesticated animals. It finely aligns ECA17 to human and mouse homologues (HSA13 and MMU1, 3, 5, 8, and 14, respectively) and homologues in other domesticated animals. Comparisons provide insight into their relative organization and help to identify evolutionarily conserved segments. The new ECA17 map will serve as a template for the development of clusters of BAC contigs in regions containing genes of interest. Sequencing of these regions will help to initiate studies aimed at understanding the molecular mechanisms for various diseases and inherited disorders in horse as well as human.

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

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

Application of disease-associated differentially expressed genes--mining for functional candidate genes for mastitis resistance in cattle.

In this study the mRNA differential display method was applied to identify mastitis-associated expressed DNA sequences based on different expression patterns in mammary gland samples of non-infected and infected udder quarters of a cow. In total, 704 different cDNA bands were displayed in both udder samples. Five hundred-and-thirty two bands, (75.6%) were differentially displayed. Ninety prominent cDNA bands were isolated, re-amplified, cloned and sequenced resulting in 87 different sequences. Amongst the 19 expressed sequence tags showing a similarity with previously described genes, the majority of these sequences exhibited homology to protein kinase encoding genes (26.3%), to genes involved in the regulation of gene expression (26.3%), to growth and differentiation factor encoding genes (21.0%) and to immune response or inflammation marker encoding genes (21.0%). These sequences were shown to have mastitis-associated expression in the udder samples of animals with and without clinical mastitis by quantitative RT-PCR. They were mapped physically using a bovine-hamster somatic cell hybrid panel and a 5000 rad bovine whole genome radiation hybrid panel. According to their localization in QTL regions based on an established integrated marker/gene-map and their disease-associated expression, four genes (AHCY, PRKDC, HNRPU, OSTF1) were suggested as potentially involved in mastitis defense.

Application of disease-associated differentially expressed genes – Mining for functional candidate genes for mastitis resistance in cattle

In this study the mRNA differential display method was applied to identify mastitis-associated expressed DNA sequences based on different expression patterns in mammary gland samples of non-infected and infected udder quarters of a cow. In total, 704 different cDNA bands were displayed in both udder samples. Five hundred-and-thirty two bands, (75.6%) were differentially displayed. Ninety prominent cDNA bands were isolated, re-amplified, cloned and sequenced resulting in 87 different sequences. Amongst the 19 expressed sequence tags showing a similarity with previously described genes, the majority of these sequences exhibited homology to protein kinase encoding genes (26.3%), to genes involved in the regulation of gene expression (26.3%), to growth and differentiation factor encoding genes (21.0%) and to immune response or inflammation marker encoding genes (21.0%). These sequences were shown to have mastitis-associated expression in the udder samples of animals with and without clinical mastitis by quantitative RT-PCR. They were mapped physically using a bovine-hamster somatic cell hybrid panel and a 5000 rad bovine whole genome radiation hybrid panel. According to their localization in QTL regions based on an established integrated marker/gene-map and their disease-associated expression, four genes (AHCY, PRKDC, HNRPU, OSTF1) were suggested as potentially involved in mastitis defense.

Clarifications on breakpoints in HSAX and BTAX by comparative mapping of F9, HPRT, and XIST in cattle

The coagulation factor IX gene (F9), the hypoxanthine phosphoribosyl transferase 1 gene (HPRT1), and the X-inactive specific transcript gene (XIST) were physically assigned in cattle to analyze chromosomal breakpoints on BTAX recently identified by radiation hybrid (RH) mapping experiments. Whereas the FISH assignment of XIST indicates a similar location on the q-arm of the human and cattle X chromosomes, the locus of HPRT1 supported the assumption of a chromosome rearrangement between the distal half of the q-arm of HSAX and the p-arm of BTAX identified by RH mapping. F9 previously located on the q-arm of BTAX was assigned to the p-arm of BTAX using RH mapping and FISH. The suggested new position of F9 close to HPRT1 supports the homology between HSAXq and BTAXp. The F9 locus corresponds with the gene order found in the homologous human chromosome segment. XIST was assigned on BTAXq23, HPRT1 and F9 were mapped to BTAXp22, and the verification of the location of F9 in a 5000 rad cattle-hamster whole genome radiation hybrid panel linked the gene to markers URB10 and HPRT1.

Localization of the mutation responsible for osteopetrosis in the op rat to a 1.5-cM genetic interval on rat chromosome 10: identification of positional candidate genes by radiation hybrid mapping.

Osteopetrosis is caused by a heterogenous group of bone diseases that result in an increase in skeletal mass because of inadequate osteoclastic bone resorption. In the op osteopetrotic rat, the disease has been linked to a single genetic locus located at the proximal end of rat chromosome 10. In this study, we identified a 1.5-cM genetic interval that contains the mutation. We then generated an improved radiation hybrid (RH) map of this region to identify potential functional and positional candidates for the op gene. Using the rat genome radiation hybrid panel, we mapped 57 markers including 24 genes (14 that have not yet been mapped in the rat) and 10 expressed sequence tag markers. Included in the mapped genes are several candidate genes that might significantly influence the biochemical pathways involved in osteopetrosis. These include genes involved in osteoclast differentiation, apoptosis, and the functional capabilities of mature osteoclasts to resorb bone. Further analysis of the genes and expressed transcripts mapped to this region may yield important insights into the multifactorial control of osteoclast function and the mechanisms of failed bone homeostasis in diseases such as osteopetrosis, osteoporosis, and rheumatoid arthritis in which failed bone homeostasis is an instigating or exacerbating circumstance of the disease process.

Cloning and Localization of the Bovine and Ovine Lysophosphatidic Acid Acyltransferase (LPAAT) Genes that Codes for an Enzyme Involved in Triglyceride Biosynthesis

Lysophosphatidic acid acyltransferase (LPAAT) catalyzes the addition of fatty acyl moieties to the sn-2 position of the glycerol backbone of lysophosphatidic acid in triglyceride biosynthesis. In this study, we have cloned, sequenced, and characterized the bovine and ovine LPAAT cDNA. Both encode proteins of 287 amino acids with molecular masses of 32 and 31.9 kDa, respectively, differing only by a single amino acid residue. The bovine and ovine LPAAT are predicted to be transmembrane enzymes localized to the endoplasmic reticulum. We also characterized the sequence and genomic organization of the bovine LPAAT gene. The gene consists of seven exons and six introns, spanning a 7.5-kb distance. With the use of a whole genome radiation hybrid panel, we localized the bovine LPAAT to the central region of chromosome 23.

A radiation hybrid map of the X-chromosome of the dog (Canis familiaris)

The dog serves as an animal model for several human diseases including X-chromosome diseases. Although the canine X-chromosome is one of the largest chromosomes in the dog, only a few markers have been mapped to it to date. Using a commercially available canine whole genome radiation hybrid (RH) panel we have localized 14 microsatellite markers, 18 genes and 13 STSs on the canine X-chromosome, extending the total number of mapped markers to 45 covering an estimated 830 cR. Out of these 45 markers, seven distinct groups of markers could be established with an average spacing of 18.8 cR₃₀₀₀ and ten markers remained unlinked. Using FISH analysis, six markers could be mapped physically to the p- or q-arm of the X-chromosome. Combined with the FISH mapping, three RH groups could be assigned to the p-arm and two RH groups to the q-arm. Comparison with the human X-chromosome map revealed conserved synteny up to 234 cR (TIMP1–ALAS2–AR–IL2RG–XIST). We show here that the similarity of the canine and human X-chromosomes is the largest for any mammalian species beyond the primates.

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

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

Comparative mapping of five coding DNA sequences on cattle chromosomes 7 and 25

Comparative mapping of four genes and one unknown coding DNA sequence in breakpoint positions of bovine chromosomes (BTA) 7 and 25 are presented. Performing a genome data base search five bovine expressed sequence tags from the MARC library matched with human genes coding for the general transcription factor IIIC polypeptide 1 (GTF3C1), the hypothetical protein KIAA0556, the interleukin 4 receptor (IL4R), the regulatory factor X-associated ankyrin-containing protein (RFXANK), and with an unknown human coding sequence partially homologous to the genomic cosmid clone R30923. Loci for these sequences were COMPASS predicted on BTA7 or BTA18 and to BTA18 or BTA25. Mapping was performed in a cattle-hamster somatic hybrid cell panel and a cattle-hamster 5000 rad whole genome radiation hybrid panel. GTF3C1, KIAA0556 and IL4R were assigned to the centromere region of BTA25 and RFXANK and R30923 close to the centromere of BTA7. The assignments contribute to the identification of evolutionary chromosome break points between human chromosomes 16 and 19 and BTA7, BTA18, and BTA25.

Use of radiation hybrid panels to map genetic loci.

The mapping of genetic loci within organisms has been accelerated by the advent of Radiation Hybrid (RH) panels. These panels are available for humans and non-humans including mice, baboon, rat, and canine. This article contains a general protocol for the use of the Genebridge 4 whole genome RH panel to map a human locus. This protocol may also be adjusted to suit the other RH panels currently available.