Painting of Arabidopsis Chromosomes with Chromosome‐Specific BAC Clones

Terezie Mandáková1, Martin A. Lysak1

1 Central European Institute of Technology (CEITEC), Masaryk University, Czech Republic
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20022
Online Posting Date:  June, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Chromosome painting (CP) refers to fluorescence in situ hybridization (FISH) of chromosome‐specific DNA probes to identify large chromosome regions, chromosome arms, and whole chromosomes. For CP and CCP (comparative chromosome painting) in plants, most often, contigs of chromosome‐specific bacterial artificial chromosomes (BAC) from the species of origin or a related species are used as painting probes. CP enables visualization and tracing of particular chromosome regions and/or chromosomes throughout all mitotic and meiotic stages as well as the corresponding interphase chromosome territories. CCP enables identification of large‐scale homeologous chromosome regions and chromosomes shared among two or more species. Here, a step‐by‐step protocol for carrying out CP in Arabidopsis thaliana (Arabidopsis) and CCP in other crucifer taxa based on the use of Arabidopsis chromosome‐specific BAC contigs is described. © 2016 by John Wiley & Sons, Inc.

Keywords: Arabidopsis; BAC FISH; Brassicaceae; chromosome painting; fluorescence in situ hybridization (FISH); nick translation; pachytene chromosomes

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Chromosome Painting with Chromosome‐Specific BAC Clones of Arabidopsis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Chromosome Painting with Chromosome‐Specific BAC Clones of Arabidopsis

  • Good‐quality chromosome preparations on slides (see Basic Protocol 1 in Mandáková and Lysak, , for preparing chromosome preparations from root tip meristems and generative organ tissues of Arabidopsis and other Brassicaceae species)
  • 2× saline sodium citrate (SSC, see recipe)
  • RNase solution: 100 μg/ml ribonuclease A (stock of 1 mg/ml in distilled water stored in aliquots at −20°C)
  • 0.1 mg/ml pepsin in 10 mM HCl (see recipe)
  • 70%, 80% and 96% ethanol
  • Vectashield antifade (Vector Laboratories) with 2 μg/ml DAPI (4′,6‐diamidino‐2‐phenylindole, Sigma), store at 4°C
  • 4% formaldehyde in 2× SSC, freshly prepared
  • BAC clones of Arabidopsis thaliana (Arabidopsis Biological Resource Center, Columbus, OH)
  • 10× NT buffer (see recipe)
  • Nucleotide mixture: 2 mM dATP, dCTP, dGTP, and 400 mM dTTP (Roche Applied Science)
  • 1 mM x‐dUTP (x = biotin, digoxigenin, Cy3, or other hapten/fluorochrome)
  • 0.1 M β‐mercaptoethanol
  • DNase I (Roche): 1:250 dilution of 1 mg/ml DNase I stock in 0.15 M NaCl in 50% glycerol
  • 10 U/μl DNA polymerase I (Fermentas, Glen Burnie)
  • 1% agarose gel
  • 100‐bp ladder DNA marker
  • 0.5 M EDTA, pH 8.0
  • 3 M sodium acetate, pH 5.2
  • 96% ice‐cold ethanol
  • Hybridization buffer (see recipe)
  • Rubber cement
  • 50% or 20% deionized formamide in 2× SSC, pH 7.0
  • 4T buffer (see recipe)
  • Blocking solution (see recipe)
  • Antibodies: avidin∼Texas Red (Vector Laboratories), biotinylated goat anti‐avidin (Vector Laboratories), mouse anti‐digoxigenin (Jackson ImmunoResearch Laboratories), goat anti‐mouse∼Alexa Fluor 488 (Invitrogen)
  • TNB buffer (see recipe)
  • TNT buffer (see recipe)
  • Coplin or Hellendahl jars (holding slides vertically)
  • Orbital shaker
  • Humid box (e.g., a slide storage box filled with wet paper towel)
  • 37°C incubator
  • Coverslips (24 × 24–mm, 24 × 32–mm, and 24 × 50–mm)
  • 37°C water bath
  • Plastic slide rack
  • Epifluorescence microscope equipped with optical filters for DAPI and other used fluorochromes, a digital CCD (charge‐coupled device) camera, and image acquisition software
  • 0.5‐ and 2‐ml microcentrifuge tubes
  • Vortex
  • Programmable temperature‐controlled heating block or thermal cycler at 15°C and 60°C
  • Electrophoresis system
  • Refrigerated centrifuge
  • Desiccator or vacuum centrifuge
  • 37°C thermomixer
  • 80°C heating block with exact temperature control
  • Forceps
  • Paper or plastic slide case
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant A. thaliana thaliana. Nature 408:796‐815. doi: 10.1038/35048692.
  Blennow, E. 2004. Reverse painting highlights the origin of chromosome aberrations. Chromosome Res. 12:25‐33. doi: 10.1023/B:CHRO.0000009277.42798.9a.
  Cremer, T. and Cremer, C. 2001. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet. 2:292‐301. doi: 10.1038/35066075.
  Ferguson‐Smith, M.A. and Trifonov, V. 2007. Mammalian karyotype evolution. Nat. Rev. Genet. 8:950‐962. doi: 10.1038/nrg2199.
  Heilig, J., Elbing, K.L., and Brent, R. 2001. Large‐scale preparation of plasmid DNA. Curr. Protoc. Mol. Biol. 41:1.7.1‐1.7.16. doi: 10.1002/0471142727.mb0107s41.
  Henegariu, O., Bray‐Ward, P., and Ward, D.C. 2000. Custom fluorescent nucleotide synthesis as an alternative method for nucleic acid labeling. Nat. Biotechnol. 18:345‐348. doi: 10.1038/73815.
  Langer, S., Kraus, J., Jentsch, I., and Speicher, M.R. 2004. Multicolor chromosome painting in diagnostic and research applications. Chromosome Res. 12:15‐23. doi: 10.1023/B:CHRO.0000009326.21752.88.
  Lysak, M.A. and Mandáková, T. 2013. Analysis of plant meiotic chromosomes by chromosome painting. Methods Mol. Biol. 990:13‐24. doi: 10.1007/978‐1‐62703‐333‐6_2.
  Lysak, M.A., Fransz, P.F., Ali, H.B.M., and Schubert, I. 2001. Chromosome painting in A. thaliana. Plant J. 28:689‐697. doi: 10.1046/j.1365‐313x.2001.01194.x.
  Mandáková, T. and Lysak, M.A. 2008. Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae). Plant Cell 20:2559‐2570. doi: 10.1105/tpc.108.062166.
  Mandáková, T. and Lysak, M.A. 2016. Chromosome preparation for cytogenetic analyses in Arabidopsis. Curr. Protoc. Plant Biol. 1:43‐51.
  Mandáková, T., de Jong, H., Schranz, M.E., and Lysak, M.A. 2015a. Karyotype evolution in apomictic Boechera: The origin of the aberrant chromosomes. Plant J. 82:785‐793. doi: 10.1111/tpj.12849.
  Mandáková, T., Singh, V., Krämer, U., and Lysak, M.A. 2015b. The genome of the heavy metal hyperaccumator Noccaea caerulescens and its stability on metalliferous and non‐metalliferous soils. Plant Phys. 169:674‐689. doi: 10.1104/pp.15.00619.
  Mandáková, T., Joly, S., Krzywinski, M., Mummenhoff, K., and Lysak, M.A. 2010. Fast diploidization in close mesopolyploid relatives of Arabidopsis. Plant Cell 22:2277‐2290. doi: 10.1105/tpc.110.074526.
  Meinkoth, J. and Wahl, G. 1984. Hybridization of nucleic acid immobilized on solid support. Anal. Biochem. 138:267‐284. doi: 10.1016/0003‐2697(84)90808‐X.
  Pecinka, A., Kato, N., Meister, A., Probst, A.V., Schubert, I., and Lam, E. 2005. Tandem repetitive transgenes and fluorescent chromatin tags alter the local interphase chromosome arrangement in Arabidopsis thaliana. J. Cell Sci. 118:3751‐3758. doi: 10.1242/jcs.02498.
  Pecinka, A., Schubert, V., Meister, A., Kreth, G., Klatte, M., Lysak, M.A., Fuchs, J., and Schubert, I. 2004. Chromosome territory arrangement and homologous pairing in nuclei of A. thaliana are predominantly random except for NOR‐bearing chromosomes. Chromosoma 113:258‐269. doi: 10.1007/s00412‐004‐0316‐2.
  Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  Schubert, I., Fransz, P.F., Fuchs, J., and de Jong, H. 2001. Chromosome painting in plants. Methods Cell Sci. 23:57‐69. doi: 10.1023/A:1013137415093.
  Sharma, A.K. and Sharma, A. 2001. Chromosome painting—Principles, strategies and scope. Methods Cell Sci. 23:1‐5. doi: 10.1023/A:1013108610550.
PDF or HTML at Wiley Online Library