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Plant Meiosis pp 197-206 | Cite as

Quantification of Synapsis Using Immunolocalization in Embedded Nuclei of Lolium

  • Dylan W. PhillipsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)

Abstract

The establishment, formation and disassembly of the synaptonemal complex (SC) is intimately associated with other essential processes that occur during prophase I of meiosis, including recombination. Labeling the SC using primary antibodies raised against key proteins, detected using secondary antibodies conjugated to fluorescent dyes, differentiate between synapsed and unsynapsed regions, revealing the dynamics of the process. Embedding meiotic nuclei in acrylamide pads preserves the three-dimensional (3D) organization of the chromosomes, which can be optically sectioned using confocal laser scanning microscopy to produce a faithful representation of the SC at the point of fixation. Deconvolution, and processing using Imaris allows the axes to be isolated from the nucleus and their features measured. Here, I describe a robust protocol to quantify the SC using immunofluorescence in Lolium perenne and L. temulentum.

Key words

Meiosis Lolium Synapsis Synaptonemal complex Immunolocalization 

Notes

Acknowledgments

Financial support is provided through a UK Biotechnology and Biological Sciences Research Council (BBSRC) Strategic Programme Grant (BB/CSP1730/1).

References

  1. 1.
    Loidl J, Bonini N (2016) Conservation and variability of meiosis across the eukaryotes. Annu Rev Genet 50:293–316CrossRefGoogle Scholar
  2. 2.
    Jenkins G (1985) Synaptonemal complex formation in hybrids of Lolium temulentum x Lolium perenne (L) – 1. High chiasma frequency diploid. Chromosoma 92:81–88CrossRefGoogle Scholar
  3. 3.
    Urata Y, Parmelee SJ, Agard DA, Sedat JW (1995) A three-dimensional structural dissection of Drosophila polytene chromosomes. J Cell Biol 131:2279–2295CrossRefGoogle Scholar
  4. 4.
    Bass H, Marshall W, Sedat J, Agard D, Cande W (1997) Telomeres cluster de novo before the initiation of synapsis: a three-dimensional spatial analysis of telomere positions before and during meiotic prophase. J Cell Biol 137:5–18CrossRefGoogle Scholar
  5. 5.
    Wang C, Carlton P, Golubovskaya I, Cande W (2009) Interlock formation and coiling of meiotic chromosome axes during synapsis. Genetics 183:905–915CrossRefGoogle Scholar
  6. 6.
    Phillips D, Mikhailova E, Timofejeva L, Mitchell J, Osina O, Sosnikhina S, Jones RN, Jenkins G (2008) Dissecting meiosis of rye using translational proteomics. Ann Bot 101:873–880CrossRefGoogle Scholar
  7. 7.
    Phillips D, Nibau C, Wnetrzak J, Jenkins G (2012) High resolution analysis of meiotic chromosome structure and behaviour in barley (Hordeum vulgare L.). PLoS One 7(6):e39539CrossRefGoogle Scholar
  8. 8.
    Hurel A, Phillips D, Vrielynck N, Mezard C, Grelon M, Christophorou N (2018) A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions. Plant J 95:385–396CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Institute of Biological, Environmental and Rural SciencesAberystwyth UniversityAberystwythUK

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