The Journal of Heredity 2001:92(6)
© 2001 The American Genetic Association 92:516-519
Brief Communication |
A Radiation Hybrid Mapping Panel for the Rhesus Macaque
From the Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702 (Murphy, Page, Smith, and O'Brien) and the New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772 (Desrosiers).
Address correspondence to William Murphy, Laboratory of Genomic Diversity, Bldg. 560, Rm. 11–10, National Cancer Institute–FCRCD, Frederick, MD 21702, or e-mail: murphywi@mail.ncifcrf.gov.
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Abstract |
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The genomes of nonhuman primates have recently become highly visible candidates for full genome analysis, as they provide powerful models of human disease and a better understanding of the evolution of the human genome. We describe the creation of a 5000 rad radiation hybrid (RH) mapping panel for the rhesus macaque. Duplicate genotypes of 84 microsatellite and coding gene sequence tagged sites from six macaque chromosomes produced an estimated whole genome retention frequency of 0.33. To test the mapping ability of the panel, we constructed RH maps for macaque chromosomes 7 and 9 and compared them to orthologous locus orders in existing human and baboon maps derived from different methodologies. Concordant marker order between all three species maps suggests that the current panel represents a powerful mapping resource for generating high-density comparative maps of the rhesus macaque and other species genomes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Radiation hybrid (RH) mapping is becoming a powerful tool for whole genome characterization in model organisms. RH maps provide a useful resource for high-resolution comparative mapping, comparative candidate positional cloning, and ultimately a guide to whole genome sequence assembly (Band et al. 2000; Kwitek et al. 2001; Murphy et al. 2000; O'Brien et al. 2001; van de Sluis et al. 2000). There is increasing interest in charting the genomes of nonhuman primates for both their close evolutionary kinship with mankind and the potential that nonhuman primates, especially the rhesus macaque, possess for effectively and accurately modeling human biological phenomena (Dawes 2001; McConkey and Varki 2000; VandeBerg et al. 2000). Here we describe the construction and initial characterization of a rhesus macaque RH panel as a resource for comparative evolutionary inference with other genomes and effective utilization of this species as an animal model. We demonstrate its potential resolving power by producing comparative chromosome maps for two macaque chromosomes and comparison of these to human and baboon maps of homologous chromosomes. These comparative mapping data are useful for elucidating the evolutionary history of human chromosome 14 and 15.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The 5000 rad RH panel was constructed by fusing approximately 2 x 107 irradiated cells from a male rhesus macaque fibroblast donor cell line with an equivalent number of cells from the A23 thymidine kinase (TK)-deficient hamster cell line. The origin of the donor fibroblasts was a male rhesus macaque (Mm123–87) housed at the New England Regional Primate Center. The radiation dosage was 5000 rads, similar to what has been used for other domestic species (Band et al. 2000; Murphy et al. 2000). Fusions were plated onto alpha-MEM+20% fetal bovine serum, penicillin, streptomycin, oubain, and HAT supplement, and grown at 37°C. Appropriate controls for the TK selection and irradiation procedures showed no growth 10 days after fusion. Seven to 14 days after fusion, colonies were isolated and grown separately in the wells of 24-well microtiter plates. Ninety-three clones were selected and expanded based on retention of macaque-specific DNA, determined using previously optimized markers (Rogers et al. 2000) and an Inter-Alu PCR assay. DNA harvests yielded an average of 1.9 mg of DNA per hybrid.
Genotyping was performed with 25 ng of hybrid DNA from the 93 cell lines as well as 3 control samples (macaque genomic DNA, hamster [A23] genomic DNA, and a water blank) in a 10 µl reaction volume using Perkin-Elmer dual 384-well 9700 thermal cyclers under the following conditions: 10 min denaturation at 95°C, followed by 35 cycles of 15 s at 95°C, 15 s at 50–60°C, and 30–45 s at 72°C, with a final 5-min extension at 72°C. PCR mixtures contained polymerase chain reaction (PCR) buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl), MgCl2 (1.5–3.0 mM), dNTPs (200 µM each), 40 nmol forward and reverse primers, and 0.5 units TaqGold DNA polymerase (ABI). Amplification products were resolved on 2% agarose gels, stained with Vista green, digitally recorded, and manually scored. RH genotypes were scored as either positive, negative, or ambiguous (clones which were discordant between duplicate typings). RH maps were constructed using RH2PT and RHMAXLIK, both from RHMAP version 3.0 (Boehnke et al. 1996).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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PCR-based duplicate genotyping of 84 loci distributed on 6 different macaque chromosomes (Table 1) revealed an average genome-wide retention frequency of 0.33 (range 0.15–0.62), suggesting the panel retains sufficient donor genome to construct moderate-resolution maps with full genome coverage. We noticed an increased retention frequency of markers adjacent to centromeres, as has been observed in RH panels from other species (e.g., Murphy et al. 2000; Stewart et al. 1997). Type I coding markers homologous to human chromosome 14 and 15 loci were initially amplified using touchdown PCR with conserved mammalian primers (Table 2) designed from multiple mammalian sequence alignments. PCR products were end sequenced for verification by BLAST search, from which macaque-specific primers were designed (GenBank accession nos. AF455790–AF455802). Human microsatellite loci, previously optimized for linkage mapping in the baboon genome (Rogers et al. 2000), were optimized for use in the RH panel using a touchdown PCR strategy. All markers were typed on the panel in duplicate, allowing us to assay two loci per 384-well plate.
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To test the mapping accuracy of the RH panel we analyzed RH scores from two chromosomes with a sufficient coverage of markers, Mma7 and Mma9. Two-point linkage analyses using RH2PT identified a single linkage group with an LOD of 8.0 for Mma7 and a single linkage group for Mma9 with an LOD of 6.0. The maximum likelihood order (assuming both equal retention and centromeric models) for the Mma7 loci was identical to the baboon linkage map and showed only a single discordant locus position with the human sequence-based map (Figure 1). It is noteworthy that this single discrepancy is between closely linked loci (D14S72 and APEX) for which the RH ordering confidence is less than 100:1, and probably represents a mapping artifact that is likely to be resolved by increasing marker density. Similarly concordant locus orders were obtained for the maximum likelihood locus order for 11 loci mapped to Mma9, in comparison to the human and baboon linkage maps (Figure 2). Comparison of these two preliminary macaque RH chromosome maps with the baboon linkage map predicts a 3 cR5000/cM ratio in the macaque, assuming recombination rates do not differ significantly between macaque and baboon, both of which are Cercopithecine monkeys and have identical G-banded karyotypes (Moore et al. 1999).
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Comparative RH maps can also reveal the evolutionary history of mammalian chromosomes (Murphy et al. 1999, 2001). Macaque chromosome 9 (Figure 1) represents a conserved synteny of human chromosomes 14 and 15, which likely reflects the ancestral condition for placental mammals (Chowdhary et al. 1998; Murphy et al. 2001; Wienberg et al. 2000). The interstitial centromere position within the human 15 homologous region is also inferred to be the ancestral condition for primates, and possibly placental mammals, given a similar centromere position in outgroup species such as cat and dolphin (Murphy et al. 2001; O'Brien et al. 1999). The conserved gene order between human, macaque, and baboon provides no evidence of an inversion moving the centromere to a position at the boundary of the chromosome 14 and 15 homologous segments, which then could have undergone a fission to produce the two chromosomes observed in humans and other apes. Instead these data imply a more complex evolutionary scenario, involving the loss of the interstitial centromere in the ancestral 14–15 chromosomes, followed by an evolutionary break at the 14–15 junction, and evolution of two new centromeres at the ends of both chromosomes.
Our initial characterization of a 5000 rad macaque RH panel suggests a mapping resolution similar to those panels described for other model organisms which have been utilized for construction of genome spanning maps (http://compgen.rutgers.edu/rhmap/). The high average retention frequency and concordance of locus orders with published baboon and human maps demonstrate the ability of the panel to construct accurate chromosome maps, while providing the means to dissect chromosome evolution within primates, and eventually across mammalian orders. The establishment of this mapping resource sets the stage for the generation of a high-density macaque RH map incorporating microsatellites and expressed sequence tags, to facilitate whole genome scans and provide an anchored comparative framework for whole genome sequencing.
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Acknowledgments |
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We thank Jennifer Tabler and Jason Morris for technical assistance and Roscoe Stanyon for helpful comments on the manuscript. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
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Footnotes |
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Corresponding Editor: Oliver A. Ryder
Received November 16, 2001
Accepted November 19, 2001
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Band MR, Larson JH, Rebeiz M, Green CA, Heyen DW, Donovan J, Windish R, Steining C, Mahyuddin P, Womack JE, and Lewin HA, 2000. An ordered comparative map of the cattle and human genomes. Genome Res 10:1359–1368.
Boehnke M, Lunetta K, Hauser E, Lange K, Uro J, et al., 1996. RHMAP: statistical package for multipoint radiation hybrid mapping (version 3.0).
Chowdhary BP, Raudsepp T, Fronicke L, and Scherthan H, 1998. Emerging patterns of comparative genome organization in some mammalian species as revealed by Zoo-FISH. Genome Res 8:577–589.
Dawes H, 2001. Humans seek clues from closest kin. Curr Biol 11:R760–R761.[Medline]
Kwitek AE, Tonellato PJ, Chen D, Gullings-Handley J, Cheng YS, Twigger S, Scheetz TE, Casavant TL, Stoll M, Nobrega MA, Shiozawa M, Soares MB, Sheffield VC, and Jacob HJ, 2001. Automated construction of high-density comparative maps between rat, human, and mouse. Genome Res 11:1935–1943
McConkey EH and Varki A, 2000. A primate genome project deserves high priority. Science 289:1295–1296.
Moore CM, Janish C, Eddy CA, Hubbard GB, Leland MM, and Rogers J, 1999. Cytogenetic and fertility studies of a rheboon, rhesus macaque, Macaca mulatta, x baboon, Papio hamadryas, cross: further support for a single karyotype nomenclature. Am J Phys Anthropol 110:119–127.[ISI][Medline]
Murphy WJ, Stanyon R, and O'Brien SJ, 2001. Evolution of comparative genome organization inferred from comparative gene mapping. Genome Biol 2:0005.1–0005.8.
Murphy WJ, Sun S, Chen Z, Pecon-Slattery J, and O'Brien SJ, 1999. Extensive conservation of sex chromosome organization between human and cat revealed by parallel radiation hybrid mapping. Genome Res. 9:1223–1230.
Murphy WJ, Sun S, Chen Z, Yuhki N, Hirschmann D, Menotti-Raymond M, and O'Brien SJ, 2000. A radiation hybrid map of the cat genome: implications for comparative mapping. Genome Res 10:691–702.
O'Brien SJ, Eisenberg JF, Miyamoto M, Hedges SB, Kumar S, Wilson DE, Menotti-Raymond M, Murphy WJ, Nash WG, Lyons LA, Menninger JC, Stanyon R, Wienberg J, Copeland NG, Jenkins NA, Gellin J, Yerle M, Andersson L, Womack J, Broad T, Postlethwait J, Serov O, Bailey E, James MR, Watanabe TK, Wakefield MJ, and Marshall Graves JA, 1999. Genome maps 10. Comparative genomics. Mammalian radiations. Wall Chart 286:463–478.
O'Brien SJ, Eizirik E, and Murphy WJ, 2001. On choosing mammalian genomes for sequencing. Science 292:2264–2266.
Rogers J, Mahaney MC, Witte SM, Nair S, Newman D, Wedel S, Rodriguez LA, Rice KS, Slifer SH, Perelygin A, Slifer M, Palladino-Negro P, Newman T, Chambers K, Joslyn G, Parry P, and Morin PA, 2000. A genetic linkage map of the baboon, Papio hamadryas, genome based on human microsatellite polymorphisms. Genomics 67:237–247.[ISI][Medline]
Stewart EA, McKusick KB, Aggarwal A, Bajorek E, Brady S, Chu A, Fang N, Hadley D, Harris M, Hussain S, Lee R, Maratukulam A, O'Connor K, Perkins S, Piercy M, Qin F, Reif T, Sanders C, She X, Sun WL, Tabar P, Voyticky S, Cowles S, Fan JB, Mader C, Quackenbush J, Myers RM, and Cox DR, 1997. An STS-based radiation hybrid map of the human genome. Genome Res 7:422–433.
van de Sluis B, Kole S, van Wolferen M, Holmes NG, Pearson PL, Rothuizen J, van Oost BA, and Wijmenga C, 2000. Refined genetic and comparative physical mapping of the canine copper toxicosis locus. Mammal Genome 11:455–460.[ISI][Medline]
VandeBerg JL, Williams-Blangero S, Dyke B, and Rogers J, 2000. Examining priorities for a primate genome project. Science 290:1504–1505.
Wienberg J, Froenicke L, and Stanyon R, 2000. Insights into mammalian comparative genome organization and evolution by molecular cytogenetics. In: Comparative genomics (Clark MS, ed.) Dordrecht: Kluwer Academic; 207–244.
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