Cytological Characterization of Arabidopsis arenosa Polyploids by SIM

  • Chris MorganEmail author
  • Eva Wegel
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)


Arabidopsis arenosa has recently become established as a model organism for investigating how meiosis has evolved to overcome the meiotic challenges faced by newly formed autotetraploids. Here, we describe a protocol for the preparation of spread, immunolabeled prophase I chromosomes from established A. arenosa autotetraploids for imaging with three-dimensional structured illumination microscopy (3D-SIM). This technique allows us to dissect the unique synaptic behavior in A. arenosa and identify synaptic partner switch sites that are unresolvable with conventional widefield microscopy.

Key words

Arabidopsis arenosa Meiosis Synaptonemal complex Synaptic partner switch Structured illumination microscopy 



We thank Dr. Kirsten Bomblies (John Innes Centre) for supporting the development of this protocol and Prof. Chris Franklin (University of Birmingham) for kindly supplying antibodies. This work was supported by a European Research Council Consolidator grant to K.B. (CoG EVO-MEIO 681946) and a UK Biological and Biotechnology Research Council (BBSRC) studentship to C.M. (DTP BB/MO1116 x/1 M1BTP). This work has also been supported by the BBSRC via grant BB/P013511/1 to the John Innes Centre.


  1. 1.
    Lloyd A, Bomblies K (2016) Meiosis in autopolyploid and allopolyploid Arabidopsis. Curr Opin Plant Biol 30:116–122CrossRefGoogle Scholar
  2. 2.
    Bomblies K, Higgins JD, Yant L (2015) Meiosis evolves: adaptation to external and internal environments. New Phytol 208:306–323CrossRefGoogle Scholar
  3. 3.
    Bomblies K, Jones G, Franklin C, Zickler D, Kleckner N (2016) The challenge of evolving stable polyploidy: could an increase in “crossover interference distance” play a central role? Chromosoma 125:287–300CrossRefGoogle Scholar
  4. 4.
    Yant L, Hollister JD, Wright KM, Arnold BJ, Higgins JD, Franklin FCH, Bomblies K (2013) Meiotic adaptation to genome duplication in Arabidopsis arenosa. Curr Biol 23:2151–2156CrossRefGoogle Scholar
  5. 5.
    Hollister JD, Arnold BJ, Svedin E, Xue KS, Dilkes BP, Bomblies K (2012) Genetic adaptation associated with genome-doubling in Autotetraploid Arabidopsis arenosa. PLoS Genet 8:e1003093CrossRefGoogle Scholar
  6. 6.
    Higgins JD, Wright KM, Bomblies K, Franklin FCH (2014) Cytological techniques to analyze meiosis in Arabidopsis arenosa for investigating adaptation to polyploidy. Front Plant Sci 4:546CrossRefGoogle Scholar
  7. 7.
    Pawlowski WP, Grelon M, Armstrong S (2013) Plant meiosis: methods and protocols. In: Methods in Molecular Biology, vol 990. Humana Press, New YorkGoogle Scholar
  8. 8.
    Grandont L, Cunado N, Coriton O, Huteau V, Eber F, Chevre AM, Grelon M, Chelysheva L, Jenczewski E (2014) Homoeologous chromosome sorting and progression of meiotic recombination in Brassica napus: Ploidy does matter. Plant Cell 26:1448–1463CrossRefGoogle Scholar
  9. 9.
    Gustafsson MGL, Shao L, Carlton PM, Wang CJR, Golubovskaya IN, Cande WZ, Agard DA, Sedat JW (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94:4957–4970CrossRefGoogle Scholar
  10. 10.
    Lloyd A, Morgan C, Franklin FCH, Bomblies K (2018) Plasticity of meiotic recombination rates in response to temperature in Arabidopsis. Genetics 208:1409–1420CrossRefGoogle Scholar
  11. 11.
    Ball G, Demmerle J, Kaufmann R, Davis I, Dobbie IM, Schermelleh L (2015) SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci Rep 5:15915CrossRefGoogle Scholar
  12. 12.
    Demmerle J, Innocent C, North AJ, Ball G, Muller M, Miron E, Matsuda A, Dobbie IM, Markaki Y, Schermelleh L (2017) Strategic and practical guidelines for successful structured illumination microscopy. Nat Protoc 12:988–1010CrossRefGoogle Scholar
  13. 13.
    Longair MH, Baker DA, Armstrong JD (2011) Simple Neurite tracer: open source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27:2453–2454CrossRefGoogle Scholar
  14. 14.
    Olivier N, Keller D, Rajan VS, Gonczy P, Manley S (2013) Simple buffers for 3D STORM microscopy. Biomed Opt Express 4:885–899CrossRefGoogle Scholar
  15. 15.
    Lam WS, Yang XH, Makaroff CA (2005) Characterization of Arabidopsis thaliana SMC1 and SMC3: evidence that AtSMC3 may function beyond chromosome cohesion. J Cell Sci 118:3037–3048CrossRefGoogle Scholar
  16. 16.
    Higgins JD, Sanchez-Moran E, Armstrong SJ, Jones GH, Franklin FCH (2005) The Arabidopsis synaptonemal complex protein ZYP1 is required for chromosome synapsis and normal fidelity of crossing over. Genes Dev 19:2488–2500CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.John Innes CentreNorwichUK

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