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
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Abstract

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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chromosome Painting with Chromosome‐Specific BAC Clones of Arabidopsis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

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

  Materials
  • 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
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Figures

Videos

Literature Cited

Literature Cited
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