Flow Analysis and Sorting of Plant Chromosomes

Jan Vrána1, Petr Cápal1, Hana Šimková1, Miroslava Karafiátová1, Jana Čížková1, Jaroslav Doležel1

1 Institute of Experimental Botany, Center of the Region Haná for Biotechnological and Agricultural Research, Olomouc
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 5.3
DOI:  10.1002/cpcy.9
Online Posting Date:  October, 2016
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Abstract

Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.

Keywords: cell cycle synchronization; chromosome isolation; chromosome sorting; chromosome‐specific DNA libraries; chromosome genomics; flow cytometry; high‐molecular‐weight DNA; genome sequencing; plant chromosomes; physical genome mapping

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Accumulation of Root‐Tip Cells in Mitotic Metaphase
  • Alternate Protocol 1: Accumulation of Root‐Tip Cells in Mitotic Metaphase in Large Seeded Legumes
  • Alternate Protocol 2: Accumulation of Root‐Tip Cells at Mitotic Metaphase by Nitrous Oxide Treatment
  • Alternate Protocol 3: Accumulation of Root‐Tip Cells in Mitotic Metaphase in Plant Species Where Seeds are Unavailable
  • Support Protocol 1: Analysis of the Degree of Metaphase Synchrony
  • Basic Protocol 2: Preparation of Suspensions of Intact Plant Chromosomes
  • Basic Protocol 3: Univariate Flow Karyotyping of Plant Chromosomes
  • Alternate Protocol 4: Bivariate Flow Karyotyping of Plant Chromosomes After FISHIS
  • Support Protocol 2: Preparation of Flow Sorter for Chromosome Sorting
  • Basic Protocol 4: Chromosome Sorting for DNA Amplification
  • Alternate Protocol 5: Single Chromosome Sorting and Amplification
  • Alternate Protocol 6: Chromosome Sorting for Preparation of High‐Molecular Weight DNA (HMW DNA)
  • Alternate Protocol 7: Chromosome Sorting for Proteomic Analyses
  • Support Protocol 3: Estimation of Purity in Sorted Fractions Using FISH
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Accumulation of Root‐Tip Cells in Mitotic Metaphase

  Materials
  • Deionized water (dH 2O)
  • Seeds
  • Hydroxyurea treatment solution (see recipe)
  • 1× or 0.1× Hoagland's nutrient solution (see reciperecipes and Critical Parameters)
  • Amiprophos‐methyl treatment solution (see recipe)
  • Oryzalin treatment solution (see recipe)
  • 18‐cm‐diameter glass petri dish with a lid
  • Paper towels cut to 18‐cm diameter
  • Filter paper cut to 18‐cm diameter
  • Biological incubator (heating/cooling) with internal temperature adjusted to 25° ± 0.5°C
  • 750‐ml plastic tray (e.g., 14 cm long × 8 cm wide × 10 cm high) including an open‐mesh basket to hold germinated seeds
  • Aquarium bubbler with tubing and aeration stones
  • Iced water bath

Alternate Protocol 1: Accumulation of Root‐Tip Cells in Mitotic Metaphase in Large Seeded Legumes

  Additional Materials (also see protocol 1)
  • Inert substrate for seed germination (e.g., perlite)
  • 4‐liter plastic tray (e.g., 25 cm long × 15 cm wide × 11 cm high)
  • Aluminum foil

Alternate Protocol 2: Accumulation of Root‐Tip Cells at Mitotic Metaphase by Nitrous Oxide Treatment

  Additional Materials (also see protocol 1)
  • Pressure chamber and nitrous oxide (N 2O) cylinder

Alternate Protocol 3: Accumulation of Root‐Tip Cells in Mitotic Metaphase in Plant Species Where Seeds are Unavailable

  Additional Materials (also see protocol 1)
  • Wire netting covers with holes suitable for holding crocus bulbs

Support Protocol 1: Analysis of the Degree of Metaphase Synchrony

  Materials
  • Root tips synchronized in metaphase (see protocol 1, Alternate Protocols protocol 21, protocol 32, or protocol 43)
  • Deionized water (dH 2O)
  • 3:1 (v/v) ethanol/glacial acetic acid, freshly prepared
  • 70% and 96% (v/v) ethanol
  • 5 N HCl
  • Schiff's reagent (see recipe)
  • 45% (v/v) acetic acid
  • Fructose syrup (see recipe)
  • DePeX mounting medium (Serva)
  •  
  • 4°C incubator
  • Microscope slides and coverslips
  • 18 × 18–mm coverslips
  • Coplin staining jars
  • Microscope

Basic Protocol 2: Preparation of Suspensions of Intact Plant Chromosomes

  Materials
  • Root tips synchronized in metaphase (see protocol 1 and Alternate Protocols protocol 21, protocol 32, or protocol 43)
  • Deionized water (dH 2O)
  • Formaldehyde fixative (see recipe)
  • Tris buffer (see recipe)
  • LB01 lysis buffer (see recipe)
  • LB01‐P lysis buffer (see recipe)
  • Ice
  • Isolation buffer, 1.5× (1.5× IB, see recipe)
  • 0.1 mg/ml DAPI stock solution (see recipe)
  • Cooled water bath (5°C)
  • 5‐ml polystyrene tubes (e.g., Falcon 352008; Corning)
  • Mechanical homogenizer (e.g., Polytron PT1300D with a PT‐DA 1205/5 probe; Kinematica AG)
  • 50‐μm (pore size) nylon mesh in 4 × 4‐cm squares
  • 0.5‐ml tubes for polymerase chain reaction (PCR)
  • Microscope slides
  • Fluorescence microscope with 10× to 20× objective, UV light source, and DAPI filter set

Basic Protocol 3: Univariate Flow Karyotyping of Plant Chromosomes

  Materials
  • Chromosome suspension (see protocol 6)
  • 0.1 mg/ml DAPI stock solution (see recipe)
  • LB01 buffer (see recipe)
  • Collection liquid
  • Sheath fluid for flow cytometric analysis (SF50): 50 mM NaCl in dH 2O (sterilize by autoclaving)
  • 20‐μm‐pore‐size nylon mesh in 4 × 4–cm squares
  • Flow cytometer and sorter (e.g., FACSAria, BD Biosciences) with a UV laser (e.g., Coherent Genesis) and appropriate optical band‐pass filter (e.g., 450 ± 25–nm)
  • Additional reagents and equipment for aligning the flow cytometer and adjusting the sorting device (see protocol 9), and for determining purity of sorted chromosomes (see protocol 14)

Alternate Protocol 4: Bivariate Flow Karyotyping of Plant Chromosomes After FISHIS

  Materials
  • Chromosome suspension (see protocol 6)
  • Ice
  • 10 M NaOH (see recipe)
  • 1 M Tris·Cl (see recipe)
  • Oligonucleotide microsatellite probe [e.g., (GAA) 7 labeled with FITC (Sigma‐Aldrich)]
  • DAPI stock solution
  • 20‐μm‐pore‐size nylon mesh in 4 × 4‐cm squares
  • pH meter
  • Flow cytometer and sorter (e.g., FACSAria, BD Biosciences) with UV laser and optical path for DAPI excitation and detection (see protocol 7) and blue laser (488 nm; e.g., Coherent Sapphire) for FITC excitation, and appropriate optical band‐pass filter (e.g., 530 ± 15 nm) for FITC detection

Support Protocol 2: Preparation of Flow Sorter for Chromosome Sorting

  Additional Materials (also see protocol 7)
  • Calibration beads for checking cytometer performance: e.g., BD FACSDiva CS&T beads (BD Biosciences)
  • Calibration beads for sorting setup: e.g., Accudrop beads (BD Biosciences)
  • (Optional) Commercial fluorescent calibration beads e.g., AlignFlow from Molecular Probes or Rainbow from Spherotec

Basic Protocol 4: Chromosome Sorting for DNA Amplification

  Materials
  • Deionized water (dH 2O)
  • Chromosome suspension (see protocol 6)
  • Proteinase K buffer 40× (see recipe)
  • Proteinase K stock solution (10 mg/ml; see recipe)
  • Illustra GenomiPhi V2 DNA Amplification kit (GE Healthcare)
  • 0.5‐ml PCR tubes (sterilized by autoclaving)
  • Microcentrifuge
  • PCR cycler with heated lid
  • Vivacon 500 columns, MWCO 100,000 Dalton (Sartorius)
  • 1.5‐ml PCR tubes
  • Fluorimeter

Alternate Protocol 5: Single Chromosome Sorting and Amplification

  Materials
  • Illustra GenomiPhi V2 DNA Amplification kit (GE Healthcare)
  • Proteinase K (10 mg/ml; Sigma‐Aldrich, cat. no. P4850)
  • Lysis buffer (see recipe)
  • Neutralization buffer (see recipe)
  • Ice
  • 0.2‐ml PCR tubes (sterilized by autoclaving)
  • Microcentrifuge (e.g., MiniStar silverline, VWR)
  • PCR cycler with heated lid
  • Sterile filter pipet tips (e.g., Axygen, Corning)

Alternate Protocol 6: Chromosome Sorting for Preparation of High‐Molecular Weight DNA (HMW DNA)

  Materials
  • 1.5× isolation buffer (1.5× IB; see recipe)
  • 2% low‐gelling agarose (InCert Agarose, Lonza) in 1× IB without 2‐mercaptoethanol
  • Lysis buffer B (see recipe)
  • Lysis buffer C (see recipe)
  • ET buffer (see recipe)
  • Proteinase K
  • 1.5‐ml polystyrene tubes with conical bottom
  • Centrifuge with swinging‐bucket rotor
  • Vortex mixer
  • 50°C water bath
  • Plug mold (Bio‐Rad)

Alternate Protocol 7: Chromosome Sorting for Proteomic Analyses

  Materials
  • Phenylmethanesulfonylfluoride (PMSF) stock solution (see recipe)
  • LB01‐P buffer (see recipe)
  • 15‐ml tubes with conical bottom
  • Centrifuge

Support Protocol 3: Estimation of Purity in Sorted Fractions Using FISH

  Materials
  • Deionized water (dH 2O)
  • P5 buffer (see recipe)
  • Chromosome suspension (see protocol 6)
  • Hybridization mix (see recipe)
  • Rubber cement
  • 2× SSC washing buffer (see recipe)
  • 0.1× SSC stringent washing buffer (see recipe)
  • 4× SSC washing buffer (see recipe)
  • Blocking buffer (Amersham Biosciences)
  • Fluorescently or hapten‐labeled DNA probes
  • Vectashield DAPI antifade solution (Vector Laboratories)
  • Antidigoxigen‐FITC (Roche)
  • Streptavidin‐Cy3 (Invitrogen)
  • Microscopic slides and coverslips
  • PCR cycler with heated lid and in situ hybridization adapter
  • Humidity chamber (37°C)
  • Tweezers
  • Parafilm
  • Coplin staining jars
  • Heated water bath with controlled temperature
  • Fluorescence microscope with epi‐illumination and filter sets for selected fluorochromes
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Literature Cited

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Internet Resources
  http://olomouc.ueb.cas.cz/
  Web site that contains useful information and protocols on plant flow cytogenetics.
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