Preparing Maize Synaptonemal Complex Spreads and Sequential Immunofluorescence and Fluorescence In Situ Hybridization

  • Stephen M. StackEmail author
  • Lindsay A. Shearer
  • Leslie D. Lohmiller
  • Lorinda K. Anderson
Part of the Methods in Molecular Biology book series (MIMB, volume 2061)


Immunofluorescence and fluorescence in situ hybridization (FISH) can be used to locate specific proteins and DNA sequences, respectively, in chromosomes by light microscopy. Here we describe sequential use of these techniques on spreads of maize synaptonemal complexes (SCs) to determine whether crossing over can occur in knob heterochromatin. We used antibodies to AFD1, an SC protein, and MLH1, a class I (interference-sensitive) crossover protein found in most recombination nodules (RNs) to identify crossovers (COs) along SCs. Next, we used FISH to localize a 180 bp knob-specific tandem repeat. Combining immunofluorescence and FISH images of the same SC spreads showed that heterochromatic knobs do not prohibit class I COs. This technique is broadly applicable to investigations of plant prophase I chromosomes where meiotic recombination takes place.

Key words

AFD1 Crossing over Fluorescence in situ hybridization Heterochromatin Immunofluorescence Knobs Maize MLH1 Recombination nodules Synaptonemal complex 



We thank the National Science Foundation for supporting our work and Associate Professor Song-Bin Chang in the Department of Life Sciences at National Cheng Kung University, Taiwan for selecting the PCR priming sequences for knob DNA amplification.


  1. 1.
    Stack S, Shearer LA, Lohmiller L, Anderson LK (2017) Meiotic crossing over in maize knob heterochromatin. Genetics 205:1101–1112CrossRefGoogle Scholar
  2. 2.
    Carpenter ATC (1975) Electron microscopy of meiosis in Drosophila melanogaster females: II: the recombination nodule—a recombination-associated structure at pachytene? Proc Natl Acad Sci U S A 72:3186–3189CrossRefGoogle Scholar
  3. 3.
    Anderson LK, Stack SM (2005) Recombination nodules in plants. Cytogenet Genome Res 109:198–204CrossRefGoogle Scholar
  4. 4.
    Lhuissier FGP, Offenberg HH, Wittich PE, Vischer NOE, Heyting C (2007) The mismatch repair protein MLH1 marks a subset of strongly interfering crossovers in tomato. Plant Cell 19:862–876CrossRefGoogle Scholar
  5. 5.
    Moses M (1968) Synaptinemal complex. Annu Rev Genet 2:363–412CrossRefGoogle Scholar
  6. 6.
    Golubovskaya IN, Hamant O, Timofejeva L, Wang CJR, Braun D, Meeley R, Cande WZ (2006) Alleles of afd1 dissect Rec8 functions during meiotic prophase I. J Cell Sci 119(3306):3315Google Scholar
  7. 7.
    Mercier R, Mezard C, Jenczewski E, Macaisne N, Grelon M (2015) The molecular biology of meiosis in plants. Annu Rev Plant Biol 66:5.1–5.31CrossRefGoogle Scholar
  8. 8.
    Charlesworth B, Langley CH, Stephan W (1986) The evolution of restricted recombination and the accumulation of repeated sequences. Genetics 112(947):962Google Scholar
  9. 9.
    Longley AE (1939) Knob positions on corn chromosomes. J Agric Res 59:475–490Google Scholar
  10. 10.
    Ananiev EV, Phillips RL, Rines HW (1998) Complex structure of the knob DNA on maize chromosome 9: Retrotransposon invasion into heterochromatin. Genetics 149:2025–2037PubMedPubMedCentralGoogle Scholar
  11. 11.
    Harlow E, Lane D (1988) Antibodies a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  12. 12.
    Chang MT, Neuffer MG (1994) Chromosomal behavior during microsporogenesis. In: Freeling M, Walbot V (eds) The Maize Handbook. Springer-Verlag, New York, pp 460–475CrossRefGoogle Scholar
  13. 13.
    Stack SM, Anderson LK (2009) Electron microscopic immunogold localization of recombination-related proteins in spreads of synaptonemal complexes from tomato microsporocytes. In: Keeney S (ed) Meiosis Volume 2. Cytological Methods, Methods in Molecular Biology. Humana Press, Inc., Totowa, NJ, pp 147–169Google Scholar
  14. 14.
    Herickhoff L, Stack S, Robertson J (1992) Staining plant cells with silver. III. The mechanism. Biotech Histochem 67:171–182CrossRefGoogle Scholar
  15. 15.
    Tarkowska J (1965) Experimental analysis of the mechanism of cytomixis. I. Cytomixis in vegetative tissues. Acta Soc Bot Pol 34:27–44CrossRefGoogle Scholar
  16. 16.
    Tarkowska J (1966) Experimental analysis of the mechanism of cytomixis. II. Cytomixis in the pollen mother cells of lily: Lilium candidum L. Acta Soc Bot Pol 35:25–40CrossRefGoogle Scholar
  17. 17.
    Schwarzacher T, Heslop-Harrison P (2000) Practical in situ Hybridization. BIOS Scientific Publishers Ltd., New York, pp 1–203Google Scholar

Copyright information

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

Authors and Affiliations

  • Stephen M. Stack
    • 1
    Email author
  • Lindsay A. Shearer
    • 1
  • Leslie D. Lohmiller
    • 1
  • Lorinda K. Anderson
    • 1
  1. 1.Department of BiologyColorado State UniversityFort CollinsUSA

Personalised recommendations