Univariate Flow Karyotyping And Chromosome Sorting Of Plant Chromosomes

This protocol describes the analysis and sorting of plant chromosomes stained with DAPI. The flow cytometer must be equipped with a UV light source to excite this dye.

Materials

Chromosome suspension (see Basic Protocol 2) 0.1 mg/ml DAPI stock solution (see recipe) LB01 lysis buffer (see recipe) Collection liquid

Sheath fluid SF50 for flow cytometric analysis: 40 mM KCl/10 mM NaCl (sterilize by autoclaving)

Computer with spreadsheet or other software for theoretical flow karyotypes

(available from Dolezel) 20-^m-pore-size nylon mesh in 4 x 4-cm squares

Flow cytometer and sorter (e.g., Becton Dickinson FACSVantage) with a UV argon laser (e.g., Coherent Innova 305) and a 424 ± 44-nm band-pass filter Microscope slides

Fluorescence microscope with DAPI filter set

Additional reagents and equipment for aligning the flow cytometer and adjusting the sorting device (see Support Protocol 2), and for determining purity of sorted chromosomes (see Support Protocol 3)

Prepare theoretical flow karyotypes

1. Prepare theoretical flow karyotypes using either a spreadsheet or dedicated computer software (for details see Conia et al., 1989; Dolezel, 1991).

2. Predict the assignment of chromosomes to chromosome peaks on a flow karyotype.

3. Determine the resolution (coefficient of variation) of chromosome peaks needed to discriminate individual chromosome types.

Theoretical flow karyotypes can be modeled based on relative length or DNA content of individual chromosomes, and are very useful in planning experiments with chromosome analysis. The model predicts the complexity of the analysis and the limitations of univariate flow karyotyping. It may be used to predict positions of peaks representing specific chromosomes on a flow karyotype, and to study the effect of resolution (coefficient of variation) of chromosome peaks on discriminiation of individual chromosome types.

Perform flow cytometry

4. Stain a chromosome suspension (~1 ml/sample) by adding 0.1 mg/ml DAPI stock solution to a final concentration of 2 |g/ml.

Analysis can be performed immediately after addition of DAPI, without incubation. If necessary, the stained suspension can be kept on ice.

5. Filter the suspension through a 20-|m nylon mesh.

6. Make sure that the flow cytometer is properly aligned for univariate analysis (see Support Protocol 2) and that a 424 ± 44-nm band-pass filter is placed in front of the DAPI fluorescence detector.

7. Run a dummy sample (LB01 lysis buffer containing 2 |l/ml DAPI) to equilibrate the sample line.

This ensures stable peak positions during analysis and sorting.

8. Introduce the sample and let it stabilize at the appropriate flow rate (e.g., 200 particles/sec). If possible, do not change the flow rate during the analysis.

Significant changes in the flow rate during the analysis may result in peak shifts.

9. Set a gating region on a dot plot of forward scatter (FS) and DAPI peak/pulse height to exclude debris, nuclei, and large clumps.

10. Adjust photomultiplier voltage and amplification gains so that chromosome peaks are evenly distributed on a histogram of DAPI signal pulse area/integral.

11. Collect 20,000 to 50,000 chromosomes and save the results on a computer disk.

Sort chromosomes

12. Make sure that the sorting device is properly adjusted (see Support Protocol 2).

Specimen Handling, Storage, and Preparation

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