International Journal of Primatology

, Volume 34, Issue 2, pp 303–314 | Cite as

A New Method for Genome-wide Marker Development and Genotyping Holds Great Promise for Molecular Primatology

  • Christina M. Bergey
  • Luca Pozzi
  • Todd R. Disotell
  • Andrew S. Burrell


Over the last two decades primatologists have benefited from the use of numerous molecular markers to study various aspects of primate behavior and evolutionary history. However, most of the studies to date have been based on a single locus, usually mitochondrial DNA, or a few nuclear markers, e.g., microsatellites. Unfortunately, the use of such markers not only is unable to address successfully important questions in primate population genetics and phylogenetics (mainly because of the discordance between gene tree and species tree), but also their development is often a time-consuming and expensive task. The advent of next-generation sequencing allows researchers to generate large amounts of genomic data for nonmodel organisms. However, whole genome sequencing is still cost prohibitive for most primate species. We here introduce a second-generation sequencing technique for genotyping thousands of genome-wide markers for nonmodel organisms. Restriction site–associated DNA sequencing (RAD-seq) reduces the complexity of the genome and allows inexpensive and fast discovery of thousands of markers in many individuals. Here, we describe the principles of this technique and we demonstrate its application in five primates, Microcebus sp., Cebus sp., Theropithecus gelada, Pan troglodytes, and Homo sapiens, representing some of the major lineages within the order. Despite technical and bioinformatic challenges, RAD-seq is a promising method for multilocus phylogenetic and population genetic studies in primates, particularly in young clades in which a high number of orthologous regions are likely to be found across populations or species.


Genotyping Nonmodel organisms Phylogenetics Population genetics Restriction site–associated DNA sequencing Second-generation DNA sequencing Single-nucleotide polymorphisms 



The present study was supported by a Leakey Foundation General Grant and an NSF Graduate Research Fellowship. We thank the NYU Langone Medical Center’s Genome Technology Center for assistance with library preparation and sequencing, as well as two anonymous reviewers and the editors for their helpful comments.


  1. Anderson, E. C., & Garza, J. C. (2006). The power of single-nucleotide polymorphisms for large-scale parentage inference. Genetics, 172(4), 2567–2582.PubMedCrossRefGoogle Scholar
  2. Avise, J. C. (1994). Molecular markers, natural history, and evolution. New York: Chapman & Hall.CrossRefGoogle Scholar
  3. Baird, N. A., Etter, P. D., Atwood, T. S., Currey, M. C., Shiver, A. L., Lewis, Z. A., Selker, E. U., et al. (2008). Rapid SNP discovery and genetic mapping using sequenced RAD markers. PloS One, 3(10), e3376.PubMedCrossRefGoogle Scholar
  4. Barnett, D. W., Garrison, E. K., Quinlan, A. R., Strömberg, M. P., & Marth, G. T. (2011). BamTools: a C++ API and toolkit for analyzing and managing BAM files. Bioinformatics, 27(12), 1691–1692.PubMedCrossRefGoogle Scholar
  5. Burbano, H. A., Hodges, E., Green, R. E., Briggs, A. W., Krause, J., Meyer, M., et al. (2010). Targeted investigation of the Neanderthal genome by array-based sequence capture. Science, 328(5979), 723–725.PubMedCrossRefGoogle Scholar
  6. Catchen, J. M., Amores, A., Hohenlohe, P., Cresko, W., & Postlethwait, J. H. (2011). Stacks: Building and genotyping loci de novo from short-read sequences. G3, 1(3), 171–182.PubMedCrossRefGoogle Scholar
  7. Danecek, P., Auton, A., Abecasis, G., Albers, C. A., Banks, E., DePristo, M. A., Handsaker, R. E., et al. (2011). The variant call format and VCFtools. Bioinformatics, 27(15), 2156–2158.PubMedCrossRefGoogle Scholar
  8. Davey, J. W., Cezard, T., Fuentes-Utrilla, P., Eland, C., Gharbi, K., & Blaxter, M. L. (2012). Special features of RAD sequencing data: Implications for genotyping. Molecular Ecology. doi: 10.1111/mec.12084.
  9. Davey, J. W., Hohenlohe, P. A., Etter, P. D., Boone, J. Q., Catchen, J. M., & Blaxter, M. L. (2011). Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nature Reviews Genetics, 12(7), 499–510.PubMedCrossRefGoogle Scholar
  10. Degnan, J. H., & Rosenberg, N. A. (2009). Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends in Ecology and Evolution, 24(6), 332–340.PubMedCrossRefGoogle Scholar
  11. DePristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., Hartl, C., Philippakis, A. A., et al. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genetics, 43(5), 491–498.PubMedCrossRefGoogle Scholar
  12. Di Fiore, A. (2003). Molecular genetic approaches to the study of primate behavior, social organization, and reproduction. Yearbook of Physical Anthropology, 46, 62–99.CrossRefGoogle Scholar
  13. Edwards, S. V. (2009). Is a new and general theory of molecular systematics emerging? Evolution: International Journal of Organic Evolution, 63(1), 1–19.CrossRefGoogle Scholar
  14. Emerson, K., Merz, C., & Catchen, J. (2010). Resolving postglacial phylogeography using high-throughput sequencing. Proceedings of the National Academy of Sciences of the USA, 107(37), 16196–16200.PubMedCrossRefGoogle Scholar
  15. Enard, W., & Pääbo, S. (2004). Comparative primate genomics. Annual Review of Genomics and Human Genetics, 5, 351–378.PubMedCrossRefGoogle Scholar
  16. Etter, P. D., Bassham, S., Hohenlohe, P. A., Johnson, E., & Cresko, W. A. (2011). SNP discovery and genotyping for evolutionary genetics using RAD sequencing. In V. Orgogozo & M. V. Rockman (Eds.), Molecular methods for evolutionary genetics (pp. 157–178). New York: Humana Press.Google Scholar
  17. Goodman, M., Grossman, L. I., & Wildman, D. E. (2005). Moving primate genomics beyond the chimpanzee genome. Trends in Genetics, 21(9), 511–517.PubMedCrossRefGoogle Scholar
  18. Hauser, L., Baird, M., Hilborn, R., Seeb, L. W., & Seeb, J. E. (2011). An empirical comparison of SNPs and microsatellites for parentage and kinship assignment in a wild sockeye salmon (Oncorhynchus nerka) population. Molecular Ecology Resources, 11(Supplement 1), 150–161.PubMedCrossRefGoogle Scholar
  19. Hohenlohe, P. A., Amish, S. J., Catchen, J. M., Allendorf, F. W., & Luikart, G. (2011). Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout. Molecular Ecology Resources, 11(Supplement 1), 117–122.PubMedCrossRefGoogle Scholar
  20. Hohenlohe, P. A., Bassham, S., Currey, M., & Cresko, W. A. (2012). Extensive linkage disequilibrium and parallel adaptive divergence across threespine stickleback genomes. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 367(1587), 395–408.CrossRefGoogle Scholar
  21. Hohenlohe, P. A., Bassham, S., Etter, P. D., Stiffler, N., Johnson, E. A., & Cresko, W. A. (2010). Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags. PLoS Genetics, 6(2), e1000862.PubMedCrossRefGoogle Scholar
  22. Human Genome Sequencing Consortium (2001). Initial sequencing and analysis of the human genome. Nature, 409(6822), 860–921.Google Scholar
  23. Jones, A. G., Small, C. M., Paczolt, K. A., & Ratterman, N. L. (2010). A practical guide to methods of parentage analysis. Molecular Ecology Resources, 10(1), 6–30.PubMedCrossRefGoogle Scholar
  24. Keller, I., Wagner, C. E., Greuter, L., Mwaiko, S., Selz, O. M., Sivasundar, A., et al. (2012). Population genomic signatures of divergent adaptation, gene flow and hybrid speciation in the rapid radiation of Lake Victoria cichlid fishes. Molecular Ecology. doi: 10.1111/mec.12083.
  25. Krause, J., Fu, Q., Good, J. M., Viola, B., Shunkov, M. V., Derevianko, A. P., et al. (2010). The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature, 464(7290), 894–897.PubMedCrossRefGoogle Scholar
  26. Lemmon, A. R., Emme, S. A., & Lemmon, E. M. (2012). Anchored hybrid enrichment for massively high-throughput phylogenomics. Systematic Biology, 61(5), 727–744.PubMedCrossRefGoogle Scholar
  27. Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25(14), 1754–1760.PubMedCrossRefGoogle Scholar
  28. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., et al. (2009). The sequence alignment/map format and SAMtools. Bioinformatics, 25(16), 2078–2079.PubMedCrossRefGoogle Scholar
  29. Maddison, W. P. (1997). Gene trees in species trees. Systematic Biology, 46(3), 523.CrossRefGoogle Scholar
  30. Maddison, W. P., & Knowles, L. L. (2006). Inferring phylogeny despite incomplete lineage sorting. Systematic Biology, 55(1), 21–30.PubMedCrossRefGoogle Scholar
  31. Mason, V. C., Li, G., Helgen, K. M., & Murphy, W. J. (2011). Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens. Genome Research, 21(10), 1695–1704.PubMedCrossRefGoogle Scholar
  32. McCormack, J. E., Hird, S. M., Zellmer, A. J., Carstens, B. C., & Brumfield, R. T. (2012). Applications of next-generation sequencing to phylogeography and phylogenetics. Molecular Phylogenetics and Evolution. doi: 10.1016/j.ympev.2011.12.007.
  33. Meyer, L. R., Zweig, A. S., Hinrichs, A. S., Karolchik, D., Kuhn, R. M., Wong, M., Sloan, C. A., et al. (2013). The UCSC Genome Browser database: Extensions and updates 2013. Nucleic Acids Research, 41(D1), D46–D69.CrossRefGoogle Scholar
  34. Miller, M. R., Dunham, J. P., Amores, A., Cresko, W. A., & Johnson, E. A. (2007). Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. Genome Research, 17(2), 240–248.PubMedCrossRefGoogle Scholar
  35. Nadeau, N. J., Martin, S. H., Kozak, K. M., Salazar, C., Dasmahapatra, K. K., Davey, J. W., et al. (2012). Genome-wide patterns of divergence and gene flow across a butterfly radiation. Molecular Ecology. doi: 10.1111/j.1365-294X.2012.05730.x.
  36. Perelman, P., Johnson, W. E., Roos, C., Seuánez, H. N., Horvath, J. E., Moreira, M. A. M., et al. (2011). A molecular phylogeny of living primates. PLoS Genet, 7(3), e1001342.PubMedCrossRefGoogle Scholar
  37. Perry, G. H., Marioni, J. C., Melsted, P., & Gilad, Y. (2010). Genomic-scale capture and sequencing of endogenous DNA from feces. Molecular Ecology, 19(24), 5332–5344.PubMedCrossRefGoogle Scholar
  38. Perry, G. H., Reeves, D., Melsted, P., Ratan, A., Miller, W., Michelini, K., Louis, E. E., et al. (2012). A genome sequence resource for the aye-aye (Daubentonia madagascariensis), a nocturnal lemur from Madagascar. Genome Biology and Evolution, 4(2), 126–135.PubMedCrossRefGoogle Scholar
  39. Peterson, B. K., Weber, J. N., Kay, E. H., Fisher, H. S., & Hoekstra, H. E. (2012). Double digest RADseq: An inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PloS One, 7(5), e37135.PubMedCrossRefGoogle Scholar
  40. Quinlan, A. R., & Hall, I. M. (2010). BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics, 26(6), 841–842.PubMedCrossRefGoogle Scholar
  41. Rubin, B. E. R., Ree, R. H., & Moreau, C. S. (2012). Inferring phylogenies from RAD sequence data. PloS One, 7(4), e33394.PubMedCrossRefGoogle Scholar
  42. Steiper, M. E., & Seiffert, E. R. (2012). Evidence for a convergent slowdown in primate molecular rates and its implications for the timing of early primate evolution. Proceedings of the National Academy of Sciences of the USA, 109(16), 6006–6011.PubMedCrossRefGoogle Scholar
  43. Ting, N., & Sterner, K. N. (2012). Primate molecular phylogenetics in a genomic era. Molecular Phylogenetics and Evolution. doi: 10.1016/j.ympev.2012.08.021.
  44. Wagner, C. E., Keller, I., Wittwer, S., Selz, O. M., Mwaiko, S., Greuter, L., et al. (2012). Genome-wide RAD sequence data provide unprecedented resolution of species boundaries and relationships in the Lake Victoria cichlid adaptive radiation. Molecular Ecology. doi: 10.1111/mec.12023.
  45. Wilkinson, R. D., Steiper, M. E., Soligo, C., Martin, R. D., Yang, Z., & Tavaré, S. (2011). Dating primate divergences through an integrated analysis of palaeontological and molecular data. Systematic Biology, 60(1), 16–31.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Christina M. Bergey
    • 1
    • 2
  • Luca Pozzi
    • 1
    • 2
  • Todd R. Disotell
    • 1
    • 2
  • Andrew S. Burrell
    • 1
  1. 1.Department of AnthropologyNew York UniversityNew YorkUSA
  2. 2.New York Consortium in Evolutionary PrimatologyNew YorkUSA

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