Skip to main content
SpringerLink
Log in

Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation

  • Protocol
  • First Online:
Plant Cytogenetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1429))

Abstract

Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Keeney S, Giroux CN, Kleckner N (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88:375–384

    Article  CAS  PubMed  Google Scholar 

  2. Grelon M, Vezon D, Gendrot G, Pelletier G (2001) AtSPO11-1 is necessary for efficient meiotic recombination in plants. EMBO J 20:589–600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mezard C, Vignard J, Drouaud J, Mercier R (2007) The road to crossovers: plants have their say. Trends Genet 23:91–99

    Article  CAS  PubMed  Google Scholar 

  4. Franklin AE, McElver J, Sunjevaric I, Rothstein R, Bowen B, Cande WZ (1999) Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase. Plant Cell 11:809–824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pawlowski WP, Golubovskaya IN, Cande WZ (2003) Altered nuclear distribution of recombination protein RAD51 in maize mutants suggests the involvement of RAD51 in meiotic homology recognition. Plant Cell 15:1807–1816

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mezard C (2006) Meiotic recombination hotspots in plants. Biochem Soc Trans 34:531–534

    Article  CAS  PubMed  Google Scholar 

  7. Smagulova F, Gregoretti IV, Brick K, Khil P, Camerini-Otero RD, Petukhova GV (2011) Genome-wide analysis reveals novel molecular features of mouse recombination hotspots. Nature 472:375–378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Drouaud J, Camilleri C, Bourguignon PY, Canaguier A, Berard A, Vezon D et al (2006) Variation in crossing-over rates across chromosome 4 of Arabidopsis thaliana reveals the presence of meiotic recombination “hot spots”. Genome Res 16:106–114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Li X, Li L, Yan J (2015) Dissecting meiotic recombination based on tetrad analysis by single-microspore sequencing in maize. Nat Commun 6:6648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pratto F, Brick K, Khil P, Smagulova F, Petukhova GV, Camerini-Otero RD (2014) DNA recombination. Recombination initiation maps of individual human genomes. Science 346:1256442

    Article  PubMed  Google Scholar 

  11. Prieler S, Penkner A, Borde V, Klein F (2005) The control of Spo11’s interaction with meiotic recombination hotspots. Genes Dev 19:255–269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. He Y, Wang M, Dukowic-Schulze S, Bradbury P, Eichten S, Sidhu GK et al (2016) Genomic features shaping the landscape of meiotic double strand break hotspots in maize. Submitted

    Google Scholar 

  13. Bishop DK, Park D, Xu L, Kleckner N (1992) DMC1: A meiosis-specific yeast homolog of Escherichia coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69:439–456

    Article  CAS  PubMed  Google Scholar 

  14. Masson J-Y, West SC (2001) The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem Sci 26:131–136

    Article  CAS  PubMed  Google Scholar 

  15. Shibata T, Nishinaka T, Mikawa T, Aihara H, Kurumizaka H, Yokoyama S et al (2001) Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: a possible advantage of DNA over RNA as genomic material. Proc Natl Acad Sci U S A 98:8425–8432

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kurzbauer MT, Uanschou C, Chen D, Schlogelhofer P (2012) The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis. Plant Cell 24:2058–2070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. O’Neill LP, Turner BM (1996) Immunoprecipitation of chromatin. Methods Enzymol 274:189–197

    Article  PubMed  Google Scholar 

  18. Collas P (2010) The current state of chromatin immunoprecipitation. Mol Biotechnol 45:87–100

    Article  CAS  PubMed  Google Scholar 

  19. He Y, Sidhu GK, Pawlowski WP (2013) Chromatin immunoprecipitation for studying chromosomal localization of meiotic proteins in maize. Methods Mol Biol 990:191–201

    Article  CAS  PubMed  Google Scholar 

  20. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE et al (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9:R137

    Article  PubMed  PubMed Central  Google Scholar 

  22. Thorvaldsdottir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192

    Article  CAS  PubMed  Google Scholar 

  23. Francis KE, Lam SY, Harrison BD, Bey AL, Berchowitz LE, Copenhaver GP (2007) Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis. Proc Natl Acad Sci U S A 104:3913–3918

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Higgins JD, Perry RM, Barakate A, Ramsay L, Waugh R, Halpin C et al (2012) Spatiotemporal asymmetry of the meiotic program underlies the predominantly distal distribution of meiotic crossovers in barley. Plant Cell 24:4096–4109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sanchez-Moran E, Santos JL, Jones GH, Franklin FC (2007) ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis. Genes Dev 21:2220–2233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pawlowski WP, Golubovskaya IN, Timofejeva L, Meeley RB, Sheridan WF, Cande WZ (2004) Coordination of meiotic recombination, pairing, and synapsis by PHS1. Science 303:89–92

    Article  CAS  PubMed  Google Scholar 

  27. Pawlowski WP, Golubovskaya IN, Cande WZ (2003) Altered nuclear distribution of recombination protein RAD51 in maize mutants suggests involvement of RAD51 in the meiotic homology recognition. Plant Cell 8:1807–1816

    Article  Google Scholar 

Download references

Acknowledgment

Research to develop this protocol was supported by a grant from National Science Foundation (IOS-1025881) to WPP.

Author information

Authors and Affiliations

  1. National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, 100083, China

    Yan He

  2. Institute of Biotechnology, Biotechnology Resource Center and Section of Plant Biology in School of IntegrativePlant Science, Cornell University, Ithaca, NY, 14853, USA

    Minghui Wang

  3. Institute of Biotechnology, Biotechnology Resource Center, Cornell University, Ithaca, NY, 14853, USA

    Qi Sun

  4. Section of Plant Biology, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA

    Wojciech P. Pawlowski

Authors
  1. Yan He
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Minghui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wojciech P. Pawlowski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wojciech P. Pawlowski .

Editor information

Editors and Affiliations

  1. University of Minnesota, St. Paul, Minnesota, USA

    Shahryar F. Kianian

  2. University of Minnesota, St. Paul, Minnesota, USA

    Penny M. Avoles Kianian

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

He, Y., Wang, M., Sun, Q., Pawlowski, W.P. (2016). Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation. In: Kianian, S., Kianian, P. (eds) Plant Cytogenetics. Methods in Molecular Biology, vol 1429. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3622-9_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3622-9_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3620-5

  • Online ISBN: 978-1-4939-3622-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation