Fluorescence In Situ Hybridization to Maize (Zea mays) Chromosomes

Morgan McCaw1, Nathaniel Graham1, Jon Cody1, Nathan Swyers1, Changzeng Zhao1, James Birchler1

1 University of Missouri, Division of Biological Sciences, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20033
Online Posting Date:  September, 2016
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Fluorescence In Situ Hybridization (FISH) is the annealing of fluorescent DNA probes to their complementary sequences on prepared chromosomes and subsequent visualization with a fluorescent microscope. In maize, FISH is useful for distinguishing each of the ten chromosomes in different accessions (karyotyping), roughly mapping single genes, transposable elements, transgene insertions, and identifying various chromosomal alterations. FISH can also be used to distinguish chromosomes between different Zea species in interspecific hybrids by use of retroelement painting. © 2016 by John Wiley & Sons, Inc.

Keywords: FISH; karyotyping; fluorescence in situ hybridization; maize (Zea mays)

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Table of Contents

  • Introduction
  • Basic Protocol 1: Target Selection and DNA Preparation
  • Basic Protocol 2: Labeling Probe DNA by Nick Translation
  • Basic Protocol 3: Fluorescence in Situ Hybridization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Target Selection and DNA Preparation

  • Forward and reverse primers
  • Target plasmids: many plasmids contain common primer binding sites, which can be used to amplify inserted DNA; if a unique primer is desired, it is suggested that a program such as Primer3 (http://bioinfo.ut.ee/primer3‐0.4.0/) be used to ensure proper primer design
  • JumpStart REDTaq ReadyMix Reaction Mix (Sigma‐Aldrich, cat. no. P0982)
  • Deionized, distilled (dd) H 2O
  • 3 M sodium acetate (pH 5.2)
  • 100% ethanol
  • 70% ethanol
  • 1 × TE or nuclease‐free water
  • 1 × TAE electrophoresis buffer
  • Agarose (Sigma‐Aldrich, cat. no. A9539)
  • DNA Gel Loading Dye (6×) (Life Technologies, cat. no. R0611)
  • GeneRuler 1‐kb DNA Ladder (Life Technologies, cat. no. SM0311)
  • Ethidium bromide (Sigma‐Aldrich, cat. no. E7637)
  • Target DNA cloned into a plasmid
  • 0.6‐μl PCR tubes
  • Pipets
  • Microcentrifuge
  • Thermal cycler
  • 1.7‐ml microcentrifuge tubes
  • Vortex mixer
  • Nanodrop spectrophotometer
  • Gel electrophoresis equipment
  • UV transilluminator

Basic Protocol 2: Labeling Probe DNA by Nick Translation

  • Ice
  • Template DNA (PCR product or plasmid)
  • 10× nick translation buffer (see recipe)
  • Labeled dNTP mixture (1 nM)
  • Nonlabeled dNTPs (2 mM each/mixed)
  • DNA polymerase I
  • DNase I (100 mU/μl)
  • 1× TE‐saturated Bio‐Gel P‐60 (see recipe)
  • 1× TE (see recipe)
  • Autoclaved salmon sperm (10 mg/ml; see recipe)
  • 3 M sodium acetate (pH = 5.2; see recipe)
  • 100% ethanol
  • 70% ethanol
  • 2× SSC
  • Thermal cycler or a lidded Styrofoam container filled with cold water
  • Silane‐treated glass wool
  • Forceps
  • Pasteur pipets
  • Inoculating loops
  • 1.6‐ml collection tubes
  • Vortex mixer
  • Microcentrifuge (>16,000 × g max)
  • Lint‐free tissues
  • Heat block or water bath set to 65°C

Basic Protocol 3: Fluorescence in Situ Hybridization

  • Probe
  • Prepared cells (see McCaw et al., )
  • 2× SSC: diluted from 20× SSC
  • Vectashield (with or without DAPI)
  • 22‐mm × 22‐mm plastic coverslips
  • Square wooden dowel rods
  • Metal tray
  • Lint‐free tissues
  • Boiling water bath
  • Airtight box
  • Paper towels
  • 55°C incubator
  • Coplin jar
  • 24‐mm × 50‐mm glass coverslips
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Literature Cited

  Albert, P.S., Gao, Z., Danilova, T.V., and Birchler, J.A. 2010. Diversity of chromosomal karyotypes in maize and its relatives. Cytogenet. Genome Res. 129:6‐16. doi: 10.1159/000314342
  Bishop, R. 2010. Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance. Bioscience Horizons 3:85‐95. doi: 10.1093/biohorizons/hzq009
  Gaeta, R.T., Danilova, T.V., Zhao, C., Masonbrink, R.E., McCaw, M.E., and Birchler, J.A. 2011. Recovery of a telomere‐truncated chromosome via a compensating translocation in maize. Genome 54:184‐195. doi: 10.1139/G10‐108.
  Kato, A., Lamb, J.C., and Birchler, J.A. 2004. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proc. Natl. Acad Sci. U.S.A. 101:13554‐13559. doi: 10.1073/pnas.0403659101.
  Lamb, J. and Birchler, J. 2006. Retroelement genome painting: Cytological visualization of retroelement expansions in the genera Zea and Tripsacum. Genetics 173:1007‐1021. doi: 10.1534/genetics.105.053165.
  Lamb, J., Meyer, J., Corcoran, B., Kato, A., Han, F., and Birchler, J. 2007. Distinct chromosomal distributions of highly repetitive sequences in maize. Chromosome Res. 15:33‐49. doi: 10.1007/s10577‐006‐1102‐1.
  Lamb, J.C., Danilova, T., Bauer, M.J., Meyer, J.M., Holland, J.J., Jensen, M.D., and Birchler, J.A. 2007. Single‐gene detection and karyotyping using small‐target fluorescence in situ hybridization on maize somatic chromosomes. Genetics 175:1047‐1058. doi: 10.1534/genetics.106.065573.
  McCaw, M., Swyers, N., Graham, N., Cody, J., Zhao, C., and Birchler, J. 2016. Preparation of chromosomes from Zea mays. Curr. Protoc. Plant Biol. 1:501‐508. doi: 10.1002/cppb.20032.
  Yu, W., Lamb, J.C., Han, F., and Birchler, J.A. 2006. Telomere‐mediated chromosomal truncation in maize. Proc. Natl. Acad Sci. U.S.A. 103:17331‐17336. doi: 10.1073/pnas.0605750103.
  Yu, W., Lamb, J.C., Han, F., and Birchler, J.A. 2007. Cytological visualization of DNA transposons and their transposition pattern in somatic cells of maize. Genetics 175:31‐39. doi: 10.1534/genetics.106.064238.
Key References
  The FISH manual upon which this protocol is based is available online at the above Web site.
Internet Resources
  This site provides karyotypes of many inbred lines and other resources related to karyotyping and FISH.
  This video demonstrates the FISH procedure step by step with many helpful hints for producing high‐quality FISH images.
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