Flow Cytometric Analysis and Sorting of Plant Chromosomes

A general outline of the procedure for flow cytometric analysis and sorting of plant chromosomes consists of the following steps: 

  1. accumulation of cells in metaphase 
  2. preparation of chromosome suspensions 
  3. flow analysis and sorting 
  4. processing of flow-sorted chromosomes 

Although plant chromosome suspensions have also been prepared from cultured cells and mesophyll protoplasts, the most frequently used procedure, developed by Dolezel et al. (1992), involves the use of root tips.

Protocols that describe inpidual steps of chromosome analysis and sorting are given here. Support protocols are also included describing the estimation of cell-cycle synchrony in root tips, instrument alignment, and estimation of purity of flow-sorted chromosome fractions.

Key Methodological Papers

Recent Review Papers

Papers Reporting Chromosome Analysis and Sorting in Cereals

Papers Reporting Chromosome Analysis and Sorting in Legumes

Cell Cycle Synchronisation

These protocols are used to induce high degree of metaphase synchrony in meristem root-tip cells. The procedures use a combination of hydroxyurea, a DNA synthesis inhibitor, and the anti-microtubular drug amiprophos-methyl or oryzalin. The original version of the procedure has been published by Dolezel et al. (1992).

Cell Cycle Synchronisation in Barley

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing 2 mM hydroxyurea in 0.1x Hoagland\s nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 6.5 h.
  10. Transfer the basket with seedlings to a tray filled with 2.5 ľM amiprophos-methyl in 0.1x Hoagland\s nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. Place the container in a refrigerator and leave overnight

Notes

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Research 7: 431 - 444 (1999)

Cell Cycle Synchronisation in Chickpea

  1. Imbibe the seeds for 24 h in deionized H2O with aeration.
  2. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  3. Moisten the paper layers with deionized H2O.
  4. Spread the seeds on the filter paper surface.
  5. Cover the petri dish and leave the seeds to germinate for 1 - 2 days to achieve optimal root length (2 - 3 cm).
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. Transfer the basket with seedlings to a second plastic tray containing 1.25 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18 h.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4 h.
  11. Transfer the basket with seedlings to a tray filled with 5 ľM oryzalin in 1x Hoagland's nutrient solution and incubate for 2h.
  12. Place the container in a refrigerator and leave overnight.

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). 
Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Vlacilova K, Ohri D, Vrana J, Cihalikova J, Kubalakova M, Kahl G, Dolezel J. Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.). Chromosome Research 10: 695 - 706 (2002) Abstract

Cell Cycle Synchronisation in Durum Wheat

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing the 1.25 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 5 h.
  10. Transfer the basket with seedlings to a tray filled with 2.5 ľM amiprophos-methyl in 0.1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. Place the container in a refrigerator and leave overnight.

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF

Cell Cycle Synchronisation in Field Bean

  1. Imbibe the seeds for 24 h in deionized H2O with aeration.
  2. Wet inert substrate (e.g., perlite) with 1x Hoagland's nutrient solution and put it into a plastic tray.
  3. Wash the seeds in deionized H2O, spread them over the surface of the substrate, cover them with 1-cm layer of wet substrate.
  4. Cover the tray with aluminium foil and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (approx. 4 cm).
  5. Remove the seedlings from the substrate and wash them in deionized H2O.
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. Transfer the basket with seedlings to a second plastic tray containing 1.25 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18.5 h.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4.5h.
  11. Transfer the basket with seedlings to a tray filled with 2.5 ľM amiprophos-methyl in 1x Hoagland's nutrient solution and incubate for 2h.

Notes

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). 
Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992)

Cell Cycle Synchronisation in Garden Pea

  1. Imbibe the seeds for 24 h in deionized H2O with aeration.
  2. Wet inert substrate (e.g., perlite) with 1x Hoagland's nutrient solution and put it into a plastic tray.
  3. Wash the seeds in deionized H2O, spread them over the surface of the substrate, cover them with 1-cm layer of wet substrate.
  4. Cover the tray with aluminium foil and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (approx. 4 cm).
  5. Remove the seedlings from the substrate and wash them in deionized H2O.
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. Transfer the basket with seedlings to a second plastic tray containing 1.25 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18 h.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 4.5 h.
  11. Transfer the basket with seedlings to a tray filled with 10 ľM amiprophos-methyl in 1x Hoagland's nutrient solution and incubate for 2h.
  12. Place the container in a refrigerator and leave overnight.

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). 
Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Neumann P, Pozarkova D, Vrana J, Dolezel J, Macas J. Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.). Chromosome Research 10: 63 - 71 (2002) Abstract

Cell Cycle Synchronisation in Oat

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing 2 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 4.5 h.
  10. Transfer the basket with seedlings to a tray filled with 10 ľM oryzalin in 0.1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. Place the container in a refrigerator and leave overnight

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

Cell Cycle Synchronisation in Rye

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing 2.5 mM hydroxyurea in 1x Hoaglands nutrient solution and incubate for 18 h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 1x Hoaglands nutrient solution and incubate for 6.5 h.
  10. Transfer the basket with seedlings to a tray filled with 10 ľM oryzalin in 1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. Place the container in a refrigerator and leave overnight.

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF

Cell Cycle Synchronisation in Vicia sativa

  1. Imbibe the seeds for 8 h in deionized H2O with aeration.
  2. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  3. Moisten the paper layers with deionized H2O.
  4. Spread the seeds on the filter paper surface.
  5. Cover the petri dish and leave the seeds to germinate for 1 day to achieve optimal root length (2 - 3 cm).
  6. Select seedlings with similar length of their primary roots.
  7. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  8. Transfer the basket with seedlings to a second plastic tray containing 2.5 mM hydroxyurea in 1x Hoagland's nutrient solution and incubate for 18.5 h.
  9. Wash the roots vigorously in several changes of deionized H2O.
  10. Incubate in hydroxyurea-free 1x Hoagland's nutrient solution for 3.5 h.
  11. Transfer the basket with seedlings to a tray filled with 5 ľM oryzalin in 1x Hoagland's nutrient solution and incubate for 2h.

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). 
Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Kovarova et al. (in press)

Cell Cycle Synchronisation in Wheat

  1. Place several layers of paper towels into a glass petri dish (18-cm diameter); top them with a single sheet of filter paper.
  2. Moisten the paper layers with deionized H2O.
  3. Spread the seeds on the filter paper surface.
  4. Cover the petri dish and leave the seeds to germinate for 2 - 3 days to achieve optimal root length (2 - 3 cm).
  5. Select seedlings with similar length of their primary roots.
  6. Thread seedling roots through the holes of the open-mesh basket positioned on a plastic tray filled with deionized H2O.
  7. Transfer the basket with seedlings to a second plastic tray containing the 2 mM hydroxyurea in 0.1x Hoaglands nutrient solution and incubate for 18h.
  8. Wash the roots vigorously in several changes of deionized H2O.
  9. Transfer the basket with seedlings to a plastic tray containing hydroxyurea-free 0.1x Hoaglands nutrient solution and incubate for 4.5 h.
  10. Transfer the basket with seedlings to a tray filled with 2.5 µM amiprophos-methyl in 0.1x Hoaglands nutrient solution and incubate for 2h.
  11. Transfer the basket with seedlings to a plastic tray filled with a mixture of ice cubes and deionized H2O (1 - 2°C).
  12. Place the container in a refrigerator and leave overnight.

Notes 

Approximately 30 seedlings are needed to prepare one sample (1 ml of chromosome suspension). Germinate the seeds at 25 ą 0.5°C in a biological incubator in the dark. 
Adjust the temperature of all solutions to 25 ą 0.5°C prior their use. Perform all incubations in the dark in a biological incubator at 25 ą 0.5°C. 
Aerate all solutions. The aeration stones and tubing must be kept clean to avoid extensive contamination by bacteria and fungi. 
The degree of metaphase synchrony may be checked microscopically or after flow cytometric analysis of nuclear DNA content.

References 

Vrana J, Kubalakova M, Simkova H, Cihalikova J, Lysak MA, Dolezel J. Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033 - 2041 (2000) Abstract PDF 
Kubalakova M, Vrana J, Cihalikova J, Simkova H, Dolezel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics 104: 1362 - 1372 (2002) Abstract

Analysis of Cell Cycle Synchrony

Microscopic Analysis of Cell Cycle Synchrony

This protocol is used to estimate the extent of mitotic synchrony and the frequency of cells in metaphase in synchronized root tips.

Harvest the root tips (1 cm) in deionized H2O.

  1. Fix them in 3:1 fixative overnight at 4°C.
  2. Remove fixative with several washes in 70% ethanol.
  3. If needed, store the tips in 70% ethanol at 4°C.
  4. Wash the tips in several changes of deionized H2O.
  5. Hydrolyse tips in 5N HCl at room temperature for 25 min.
  6. Wash in deionized H2O and incubate in Schiff's reagent for 1 hour.
  7. Wash tips in deionized H2O and macerate for about 1 min in 45% (v/v) acetic acid.
  8. Cut off the darkly stained meristem tip and squash it in a drop of fructose syrup under an 18 x 18-mm coverslip.
  9. Prepare five different slides. On each slide, analyse at least 1000 cells and determine the proportion of cells in metaphase.

Notes

Squash preparations prepared in fructose syrup can be maintained for few days in a refrigerator but they are not permanent. To make permanent slides, perform the following procedure:

  1. Squash meristem tip in a drop of 45% (v/v) acetic acid.
  2. Immediately after that, place the slide on a block of dry ice.
  3. After freezing, peel off the coverslip.
  4. Dehydrate the slide in two changes of 96% ethanol in Coplin jars.
  5. Leave to air-dry overnight.
  6. Dip the slide in xylene and mount it in a drop of DePeX (Serva).

Flow Cytometric Analysis of Cell Cycle Synchrony

This procedure is used to estimate of cell cycle synchrony induced by hydroxyurea and APM treatments. The protocol is based on the analysis of DNA content of isolated nuclei after staining with 4-,6-diamidino-2-phenylindole (DAPI). This dye is excited at UV and thus the flow cytometer must be equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

  1. Harvest the root tips (1 cm) in deionized H2O.
  2. Immediately transfer the root tips into 25 ml of formaldehyde fixative and fix them at 5°C for 30 min (legumes) or 20 min (cereals).
  3. Wash the roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them in 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer.
  5. Isolate nuclei by homogenising at 9500 rpm for 15 sec.
  6. Stain the nuclei in suspension by adding DAPI stock solution to final concentration of 2 µg/ml.
  7. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  8. Make sure that the flow cytometer is aligned well for univariate analysis and that the band-pass filter 424/44 is placed in front of the fluorescence 1 (FL1) detector.
  9. Introduce the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s).
  10. Set a gating region on a dot plot of forward scatter height (FSC-H) and FL1 pulse height (FL1-H) to exclude small debris and large clumps.
  11. Adjust photomultiplier voltage and amplification gains so that the peaks corresponding to G1 and G2 nuclei are seen on a histogram of FL1 pulse area (FL1-A).
  12. Analyse 10 - 20 thousand nuclei and save the result on a disc.

Notes

Although the nuclei suspension can be stored overnight, it is recommended to perform the analysis on the same day of isolation.

Preparation of Chromosome Suspensions

In this procedure, chromosomes are released from synchronized root tips mechanically after a mild fixation with formaldehyde. Chromosomes are released into a polyamine lysis buffer (LB01) which stabilises their structure. The method has been originally developed for chromosome isolation in field bean (Dolezel et al. 1992). Chromosome suspensions prepared according to this procedure are suitable for flow cytometric analysis and sorting.

Chromosome Isolation in Barley

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 20 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 15000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

References 

Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Research 7: 431 - 444 (1999)

Chromosome Isolation in Chickpea

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 10000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 15 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

References 

Vlacilova K, Ohri D, Vrana J, Cihalikova J, Kubalakova M, Kahl G, Dolezel J. Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.). Chromosome Research 10: 695 – 706 (2002) Abstract

Chromosome Isolation in Durum Wheat

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 20 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 20000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

References 

Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF

Chromosome Isolation in Field Bean

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 4 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 7.5).
  5. Isolate chromosomes by homogenising at 15000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes 

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 
Root tips may be homogenised also using a razor blade. This method is more laborious. However, it results in higher yield of longer chromosomes in field bean:

  1. Add 1.25 ml of LB01 lysis buffer into a glass petri dish (6-cm diameter).
  2. Transfer fixed root tips into the petri dish.
  3. Chop meristem root-tips individually using a sharp razor blade, avoid dispersion or drying.
  4. Filter the suspension through a 50-µm nylon mesh into a polystyrene tube.
  5. Syringe the suspension once through a 22-gauge needle to disperse intact metaphases.
  6. Store the suspension on ice.

References

Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992)

Chromosome Isolation in Garden Pea

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 3 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 7.5).
  5. Isolate chromosomes by homogenising at 13000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 25 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

References

Neumann P, Pozarkova D, Vrana J, Dolezel J, Macas J. Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L.). Chromosome Research 10: 63 - 71 (2002) Abstract

Chromosome Isolation in Oat

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 25 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 25000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 25 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

Chromosome Isolation in Rye

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 15000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

References 

Kubalakova M, Kovarova P, Suchankova P, Cihalikova J, Bartos J, Lucretti S, Watanabe N, Kianian SF, Dolezel J. Chromosome sorting in tetraploid wheat and its potential for genome analysis. Genetics 170: 823 - 829 (2005) Abstract PDF

Chromosome Isolation in Vicia sativa

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 30 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 10000 rpm for 15 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 15 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation.

References 

Kovarova et al. (in press)

Chromosome Isolation in Wheat

  1. Harvest root tips (1 cm) and transfer them into deionized H2O.
  2. Immediately transfer root tips into 25 ml of 2 % formaldehyde fixative and fix at 5°C for 20 min.
  3. Wash roots in 25 ml of Tris buffer three times for 5 min at 5°C.
  4. Excise root meristems and transfer them into 5-ml polystyrene tube containing 1 ml of LB01 lysis buffer (pH 9.0).
  5. Isolate chromosomes by homogenising at 20000 rpm for 10 sec.
  6. Filter the suspension through a 50-µm nylon mesh into 5-ml polystyrene tube.
  7. Store the suspension on ice.
  8. Transfer 50 µl of chromosome suspension into a 0.5-ml PCR tube.
  9. Add 1 µl of DAPI stock solution.
  10. Place a small drop (~ 10 µl) of DAPI-stained suspension on a microscope slide.
  11. Observe under a fluorescence microscope with low magnification (10 - 20x lens). Do not cover by a coverslip.

Notes

Approximately 30 root tips are used to prepare one sample (1 ml of chromosome suspension). 
The suspension should contain intact nuclei and chromosomes. The concentration of chromosomes in the sample should be 5 x 105 / ml or higher. If the chromosomes are damaged (broken and/or appear as long extended fibres) than the formaldehyde fixation was too weak and should be prolonged. If the chromosomes are aggregated and/or the cells remain intact, then the fixation was too strong and should be shortened. 
Although the chromosome suspension can be stored overnight, it is recommended to analyse the chromosomes on the same day of isolation. 

References 

Vrana J, Kubalakova M, Simkova H, Cihalikova J, Lysak MA, Dolezel J. Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156: 2033 - 2041 (2000) Abstract PDF 
Kubalakova M, Vrana J, Cihalikova J, Simkova H, Dolezel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.). Theoretical and Applied Genetics 104: 1362 - 1372 (2002) Abstract

Flow Karyotyping and Chromosome Sorting

Univariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes stained with 4-,6-diamidino-2-phenylindole (DAPI). This method was originally developed by Lucretti et al. (1993) for field bean and requires a flow cytometer equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

Flow Karyotyping

  1. Stain a chromosome suspension (approx. 1ml) by adding DAPI stock solution to final concentration of 2 µg/ml.
  2. Filter the suspension through a 20-µm nylon mesh.
  3. Make sure that the flow cytometer is properly aligned for univariate analysis. Make sure that the band-pass filter 424/44 is placed in front of the fluorescence 1 (FL1) detector.
  4. Run a dummy sample (LB01 buffer containing 2 µg/ml DAPI) to equilibrate the sample line.
  5. Introduce the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s). If possible, do not change the flow rate during the analysis!
  6. Set a gating region on a dot plot of forward scatter height (FSC-H) and FL1 pulse height (FL1-H) to exclude debris, nuclei and large clumps.
  7. Adjust photomultiplier voltage and amplification gains so that chromosome peaks are evenly distributed on a histogram of FL1 pulse area (FL1-A).
  8. Analyse 20 - 50 thousand chromosomes and save the result on computer disc.

Chromosome Sorting

  1. Make sure that the sorting device is properly adjusted.
  2. Run the sample and display the signals on a dot plot of FL1 pulse width (FL1-W) versus FL1-A.
  3. Adjust the FL1-W amplifier gain and width offset as needed to achieve optimal resolution of the width signal.
  4. Check for stability of the break-off point and of the side streams.
  5. Define sorting region for the largest chromosome on the FL1-W versus FL1-A dot-plot.
  6. Select 1 drop sort envelope (number of deflected drops) and - counter - sort mode giving the highest purity and count precision.
  7. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
  8. Check the number of chromosomes using a fluorescence microscope (do not cover the drop with a coverslip!).
  9. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
  10. Define a sorting region for the chromosome to be sorted on the dot-plot of FL1-W versus FL1-A.
  11. Select sort mode and sort envelope according to required purity, number of chromosomes to be sorted, and desired volume for the sorted fraction.
  12. Sort the required number of chromosomes into a polystyrene tube containing the appropriate amount of the collection liquid. The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use. For PCR, use a small amount (20 - 60 µl) of sterile deionized H2O in 0.5-ml PCR tube).
  13. Briefly spin the tube at room temperature.
  14. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

References

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993)

Bivariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes after dual staining with 4’,6-diamidino-2-phenylindole (DAPI) that binds preferentially to AT-rich regions of DNA, and with mithramycin that binds preferentially to GC-rich regions of DNA. This method was originally developed by Lucretti and Dolezel (1997) for broad bean and requires a flow cytometer equipped with two lasers (one of them UV). We use BD FACSVantage equipped with Coherent Innova 305 and Coherent Innova 70C argon-ion lasers.

Bivariate Flow Karyotyping

  1. Add MgSO4   stock solution to chromosome suspension (approx. 1ml) to a final concentration of 10 mM.
  2. Stain the chromosomes in suspension by adding DAPI stock solution to a final concentration of 1.5 µg/ml and mithramycin stock solution to a final concentration of 20 µg/ml. Leave to equilibrate for 30 min on ice.
  3. Make sure that the flow cytometer is properly aligned for bivariate analysis. Use a half mirror to split the fluorescence of DAPI to FL1 detector through the 424/44 band-pass filter and mithramycin fluorescence to fluorescence 4 (FL4) detector through a 575/26 band-pass filter.
  4. Run a dummy sample (LB01 buffer containing 1.5 µg/ml DAPI and 20 µg/ml mithramycin) to equilibrate the sample line.
  5. Filter the sample through a 20-µm nylon mesh.
  6. Run the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s). If possible, do not change the flow rate during the analysis.
  7. Set a gating region on a dot plot of FSC-H versus FL1-H.
  8. Adjust photomultiplier voltages and amplification gains so that chromosome peaks are evenly distributed on histograms of FL1-A and FL4 pulse area (FL4-A).
  9. Display the data on a dot-plot of FL1-A versus FL4-A.
  10. Analyse 20 - 50 thousand chromosomes and save the result on a disc.

Chromosome Sorting

  1. Make sure that the sorting device is properly adjusted.
  2. Run the sample and display the signals on a dot plot of FL1-A versus FL4-A.
  3. Check for stability of the break-off point and of the side streams.
  4. Define sorting region for the largest chromosome on the FL1-A versus FL4-A dot-plot.
  5. Select 1 drop sort envelope (number of deflected drops) and counter sort mode giving the highest purity and count precision.
  6. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
  7. Check the number of chromosomes using a fluorescence microscope (do not cover the drop with a coverslip!).
  8. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
  9. Define a sorting region for the chromosome to be sorted on a dot plot of FL1-A versus FL4-A.
  10. Select sort mode and sort envelope according to required purity, number of chromosomes to be sorted and desired volume for the sorted fraction.
  11. Sort the required number of chromosomes into a polystyrene tube containing the appropriate amount of the collection liquid. The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use. For PCR, use a small amount (20 - 60 µl) of sterile deionized H2O in 0.5-ml PCR tube.).
  12. Briefly spin the tube at room temperature.
  13. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

References

Lucretti S, Dolezel J. Bivariate flow karyotyping in broad bean (Vicia faba). Cytometry 28: 236 - 242 (1997)

Two-Step Sorting

This protocol is used to sort chromosomes when their frequency in the original suspension is too low. This is frequently the case for large chromosomes, which break more easily than smaller chromosomes during chromosome isolation (Lucretti et al. 1993). During the first sort, the sample is enriched for the required chromosome. During the second sort, the chromosomes are sorted with a high purity. We use this protocol with BD FACSVantage dual laser flow cytometer and sorter.

First Sort

  1. Make sure that the sorting device is properly adjusted.
  2. Run the sample and display the signals on a suitable distribution.
  3. Check for stability of the break-off point and of the side streams.
  4. Define sorting region for the largest chromosome on a suitable distribution.
  5. Select 1 drop sort envelope (number of deflected drops) and counter sort mode giving the highest purity and count precision.
  6. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
  7. Check the number of chromosomes using a fluorescence microscope (do not cover with a coverslip!).
  8. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
  9. Select the enrich mode and 3 deflected drops sort envelope that allows for the highest recovery.
  10. On a suitable distribution, define a sorting region for the chromosome(s) to be sorted.
  11. Sort approximately 105 chromosomes into 400 µl of LB01T into a 1.5 ml sample polystyrene cup (Deltalab).

Second Sort

  1. Add fluorescent dye(s) to pre-sorted chromosome suspension to reach recommended final concentration(s).
  2. Run the sample and define sorting region for the chromosome to be sorted.
  3. Select sort mode and sort envelope. (Sort mode and sort envelope are selected according to the required purity, number of chromosomes to be sorted and desired volume for the sorted fraction.).
  4. Sort the required number of chromosomes into a polystyrene tube containing corresponding amount of the collection liquid. (The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use). For PCR, use a small amount (20 - 60 µl) of sterile deionized H2O in 0.5-ml PCR tube).
  5. Briefly spin the tube at room temperature
  6. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

Notes  
The actual number of chromosomes that should be sorted depends on the number of chromosomes that will be sorted during the second sort. It is recommended to sort at least five times more chromosomes than the final number required.  
In some cases, it may be practical to enrich the sample for more than one chromosome. Individual chromosomes are sorted during the second sort.

References

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993)  

Estimation of Purity of Flow-Sorted Chromosomes

Estimation of Purity of Sorted Chromosomes Using FISH

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using hybridization of specific probes labelled with biotin or digoxigenin and detected with avidin, streptavidin or anti-digoxigenin antibody conjugated with fluorochromes. The chromosomes are evaluated using a fluorescence microscope.

I. Sort Chromosomes
1.Pipet 10 µl of P5 buffer containing 5% sucrose (w/v) onto a clean microscope slide.
2.Immediately sort 1000 chromosomes into the drop.
II. Perform FISH Reaction
1.Pipette 10µl P5 Buffer onto a clean microscopic slide and immediately sort 1000 chromosomes into the drop
2.Air-dry the slide and store at room temperature
3.Add 25µl Hybridization Mix to the area containing flow-sorted chromosomes, cover with a glass coverslip and seal up with rubber cement.
4.Denature for 45 sec at 80°C by placing the slide on a temperature controlled hot plate.
5.Transfer the slide to a wet chamber and incubate at 37°C overnight.
6.Remove carefully the coverslip and wash in 2 × SSC for 10 min at 42°C
7.Perform a stringent wash in 0.1 × SSC for 5 min at 42°C.
8.Wash again 10 min in 2 × SSC and next 10 min in 2 × SSC decrease gradually temperature to room temperature.
9.Wash in 4 × SSC for 10 min at room temperature.
10.Apply 60µl 1% Blocking Buffer onto the slide, cover with parafilm and incubate for 10 min in at room temperature. Repeat this step two times.
11.Apply fluorescently labeled antibody and/or fluorescently labeled avidin in 1% Blocking Buffer for 1 hour at 37°C
12.Wash the slide three times in 4 × SSC for 5 min at 40°C.
13.Mount the slide with Vectashield containing DAPI.
14.Use fluoresce microscope to identify sorted chromosomes and determine the presence of contaminating chromosomes by evaluating at least 100 chromosomes on three different slides.

References 

Kubaláková, M., Valárik, M., Bartoš, J., Vrána, J., Číhalíková, J., Molnár-Láng, M., Doležel, J.: Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. - Genome 46: 893-905, 2003. 
Doležel, J., Kubaláková, M., Bartoš, J., Macas, J.: Flow cytogenetics and plant genome mapping. - Chrom. Res. 12: 77-91, 2004.
Šafář, J., Bartoš, J., Janda, J., Bellec, A., Kubaláková, M., Valárik, M., Pateyron, S., Weiserová, J., Tušková, R., Číhalíková, J., Vrána, J., Šimková, H., Faivre-Rampant, P., Sourdille, P., Caboche, M., Bernard, M., Doležel, J., Chalhoub, B.: Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat. – Plant J. 39: 960-968, 2004.
Kubaláková, M., Kovářová, P., Suchánková, P., Číhalíková, J., Bartoš, J., Lucretti, S., Watanabe, N., Kianian, S.F., Doležel, J.: Chromosome sorting in tetraploid wheat and its potential for genome analysis. – Genetics, 170: 823-829, 2005.

Estimation of Purity of Sorted Chrmosomes Using PRINS

This protocol describes a procedure that allows precise estimation of the purity of a sorted fraction (Kubalakova et al. 2000). The procedure is based on specific fluorescent labelling of repetitive DNA sequences that show characteristic pattern of distribution among the chromosomes. The sequences are labelled using a primed in situ labelling (PRINS) and the chromosomes are evaluated using a fluorescence microscope.

I. Sort Chromosomes

  1. Pipet 15 µl of LB01 buffer (for barley) or PRINS buffer (for legumes and wheat) containing 5% sucrose (w/v) onto a clean microscope slide.
  2. Immediately sort 1000 chromosomes into the drop.
  3. Air-dry in an aseptic box for about 1h.

II. Perform PRINS Reaction

  1. Stick 'Frame-Seal' incubation chamber to the slide over the specimen area.
  2. Pipet 25-µl of PRINS reaction mix into the frame and place a polyester coverslip over the frame.
  3. Run the PRINS reaction using the following PCR cycles:
    1 cycle: 5 min 94°C (denaturation)
    5 min 55°C (annealing)
    10 min 72°C (extension)
    8 cycles: 1 min 91°C (denaturation)
    1 min 55°C (annealing)
    3 min 72°C (extension)
    1 cycle: 1 min 91°C (denaturation)
    5 min 55°C (annealing)
    10 min 72°C (extension)
  4. Remove the cover, add 100 µl of the stop buffer, and incubate for 2 min at 70°C.
  5. Remove the stop buffer and transfer the slide to a 10-cm petri dish.
  6. Add 70 µl wash buffer and incubate at room temperature for 5 min. Repeat the washing step twice.

III. Examine Slides

  1. Add 70 µl of wash buffer containing DAPI (0.2 µg/ml) to counterstain the chromosomes.
  2. Drain excess fluid, but not dry.
  3. Add 8 µl of Vectashield antifade solution and cover with a glass coverslip.
  4. Gently squeeze out excess solution and seal with rubber cement.
  5. Examine the slide with fluorescence microscope.

Notes

The drops are dried within 1 h. However, the PRINS reaction should be performed next day after overnight incubation at room temperature. 
The actual conditions for the PRINS reaction (i.e., denaturation and annealing temperature) must be optimised by for given species and primer pair. 
During slide observation, use a DAPI filter first to localise sorted chromosomes. Avoid prolonged exposure to excitation light that fades rapidly both DAPI and fluorescein.

References 

Kubalakova M, Lysak MA, Vrana J, Simkova H, Cihalikova J, Dolezel J. Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS. Cytometry 41: 102 - 108 (2000) Abstract PDF

Estimation of Purity of Sorted Chrmosomes Using PCR with Chromosome-Specific Primers

I. Sort Chromosomes

  1. Prepare PCR tubes containing 19 µl of sterile deionized H2O (final volume after sorting will be approximately 20 µl).
  2. Sort 500 chromosomes into each tube.
  3. Freeze the tube and store it at -20°C.

II. Perform PCR Reaction

  1. Thaw a chromosome fraction.
  2. Add 30 µl of PCR premix, vortex and spin briefly.
  3. Perform PCR amplification using the following cycles:
    1 cycle: 5 min denaturation at 94°C
    35-40 cycles: 30 sec denaturation at 94°C
    1 min annealing at the optimised temperature
    2-3 min extension at 72°C
    1 cycle: 10 min extension at 72°C
  4. Hold the samples at 4°C.

III. Analyze PCR Products

  1. Take equal amounts of PCR products (5 - 10 µl) from each tube and add 1 - 2 µl of loading buffer.
  2. Load the samples onto the agarose gel bathed in TAE buffer.
  3. Load DNA molecular weight markers.
  4. Run electrophoresis; stop when bromophenol blue reaches a point 3 cm from the edge of the gel.
  5. Stain the gel with ethidium bromide working solution (0.5 mg/ml).
  6. Photograph the gel and analyse the presence of products in individual lanes.

Notes

This protocol was optimised for RFLP-derived markers in barley. For other types of markers and plant species, slight modification might be necessary. 
It is important to freeze the tubes even if the reaction is to be performed on the same day of sorting. 
Annealing temperature must be optimised by for given primer pair and the template. 
The electrophoresis should be run at constant voltage of 4 - 5 V/cm.

References

Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Research 7: 431 - 444 (1999)

Reagents and Solutions

use deionized or double-distilled water in all recipes

Hoaglands Nutrient Solution (Gamborg and Wetter 1975)

Solution A

H3BO3 280 mg
MnSO4 . H2O 340 mg
CuSO4 . 5H2O 10 mg
ZnSO4 . 7H2O 22 mg
(NH4)6Mo7O24 . 4H2O 10 mg

Adjust volume to 100 ml with deionized H2O 
Store at 4°C

Solution B

H2SO4 0.5 ml

Adjust volume to 100 ml with deionized H2O 
Store at 4°C

Solution C

Na2EDTA 3.36 g
FeSO4 2.79 g

Adjust volume to approximately 400 ml 
Heat the solution to 70°C while stirring until the colour turns yellow-brown 
Cool down, adjust the volume to 500 ml 
Store at 4°C

Hoaglands Stock Solution (10x)

Ca(NO3)2 . 4H2O 4.7 g
MgSO4 . 7H2O 2.6 g
KNO3 3.3 g
NH4H2PO4 0.6 g
solution A 5 ml
solution B 0.5 ml

Adjust volume to 500 ml with deionized H2O 
Store at 4°C

Hoaglands Nutrient Solution (1x)

10x stock solution 100 ml
solution C 5 ml

Adjust volume to 1000 ml with deionized H2O 
Prepare just before use

Hoaglands Nutrient Solution (0.1x)

10x stock solution 10 ml
solution C 0.5 ml

Adjust volume to 1000 ml with deionized H2O 
Prepare just before use

Amiprophos-Methyl Solutions 
Stock Solutions (20 mM)

amiprophos-methyl 60.86 mg

Adjust volume to 10 ml with cold acetone 
Store at - 20°C in 1 ml aliquots 

Treatment Solution 
Prepare the treatment solution by adding specified volume of amiprophos-methyl stock solution to Hoaglands Nutrient Solution

B5 nutrient medium (Gamborg et al. 1968)

Solution A

H3BO3 300 mg
MnSO4 . H2O 758 mg
ZnSO4 . 7H2O 200 mg

Adjust volume to 1000 ml with deionized H2O 
Store at 4°C

Solution B

Na2MoO4 . 2H2O 25 mg
CoCl2 . 6 H2O 2.5 mg
KI 75 mg
CuSO4 . 5 H2O 2.5 mg

Adjust volume to 100 ml with deionized H2O 
Store at 4°C

Solution C

thiamine 100 mg
pyridoxine 10 mg
nicotinic acid 10 mg

Adjust volume to 100 ml with deionized H2O 
Store at 4°C

Solution D

m-inositol 100 mg

Adjust volume to 100 ml with deionized H2O 
Store at 4°C

Solution E

Na2EDTA 3.36 g
FeSO4 2.79 g

Adjust volume to approximately 400 ml 
Heat the solution to 70°C while stirring until the color turns yellow-brown 
Cool down and adjust the volume to 500 ml 
Store at 4°C

B5 Stock Solution (10x)

KNO3 30 g
MgSO4 . 7H2O 5 g
(NH4)SO4 1.34 g
CaCl2 . 2H2O 1.5 g
NaH2PO4 . H2O 1.5 g
solution A 100 ml
solution B 10 ml

Adjust volume to 500 ml with deionized H2O 
Store at 4°C

B5 Nutrient Solution (1x)

10x stock solution 100 ml
solution C 10 ml
solution D 10 ml
solution E 5 ml

Adjust volume to 1000 ml with deionized H2O 
Adjust the final pH to 5.5 
Sterilise by autoclaving

Tris Buffer

10 mM Tris 0.606 g
10 mM Na2EDTA 1.861 g
100 mM NaCl 2.922 g

Adjust volume to 500 ml with deionized H2O 
Adjust the final pH to 7.5 using 1N NaOH 
Prepare just before use

Formaldehyde Fixative

10 mM Tris 0.303 g
10 mM Na2EDTA 0.931 g
100 mM NaCl 1.461 g
0.1% v/v Triton X-100 250 µl

Adjust volume to 200 ml with deionized H2O 
Adjust the final pH to 7.5 using 1N NaOH 
Add formaldehyde solution (37%, Catalog No. 1.04003, Merck, Darmstadt, Germany):

Species Volume Conc.
Field Bean 27 ml 4%
Pea 20 ml 3%
Chickpea 13.5 ml 2%
Vicia sativa 13.5 ml 2%
Barley 13.5 ml 2%
Oat 13.5 ml 2%
Rye 13.5 ml 2%
Wheat 13.5 ml 2%
Durum Wheat 13.5 ml 2%

Finally, adjust volume to 250 ml with deionized H2O 
Prepare the fixative just before use

LB01 Lysis Buffer (Dolezel et al. 1992)

15 mM Tris 0.363 g
2 mM Na2EDTA 0.149 g
0.5 mM spermine . 4HCl 0.035 g
80 mM KCl 1.193 g
20 mM NaCl 0.234 g
0.1 % v/v Triton X-100 200 µl

Adjust volume to 200 ml with deionized H2O 
Adjust the final pH*) 
Filter through a 0.22 ľm filter to remove small particles 
Add 220 ľl ß-mercaptoethanol and mix well 
Store at - 20°C in 10 ml aliquots 
*) Please note that the original LB01 buffer was adjusted to pH 7.5. However, we have found later that the optimal pH differs for different applications. To distinguish the variants, we use the following abbreviations: 
LB01/7.5  pH adjusted to 7.5 using 1N HCl 
LB01/9  pH adjusted to 9 using 1M NaOH

LB01T Lysis Buffer (for sorting)

22.5 mM Tris 0.545 g
3 mM Na2EDTA 0.223 g
0.75 mM spermine . 4HCl 0.052 g
120 mM KCl 1.790 g
30 mM NaCl 0.351 g
0.15% v/v Triton X-100 300 µl

Adjust volume to 200 ml with deionized H2O 
Adjust the final pH to 7.5 using 1N HCl 
Filter through a 0.22 ľm filter to remove small particles 
Add 330 ľl ß-mercaptoethanol and mix well 
Store at - 20°C in 5 ml aliquots

3:1 Fixative 
Mix 3 volumes of 96% ethanol with 1 volume of glacial acetic acid 
Prepare the fixative just before use

Schiff's reagent

1N HCl 30 ml
Parafuchsin (Serva, C.I.42500) 2 g
K2S2O5 3.8 g
deionized H2O 170 ml

Stir for 2 hours in a tightly closed bottle and leave to stay overnight 
Add 2 g of active charcoal 
mix 1 min and filter through a paper filter moistened with 1N HCl 
Repeat the filtration if the solution is not colourless 
Store in a tightly closed bottle at 4°C

Fructose syrup

fructose 30 g
deionized H2O 20 ml

Incubate the mixture at 37°C overnight 
Add a crystal of thymol 
store at 4°C

5N HCl

HCl - 35% (v/v) 419.8 ml

Adjust volume to 1000 ml with deionized H2O

45% (v/v) acetic acid

acetic acid - 99% (v/v) 227 ml

Adjust volume to 500 ml with deionized H2O

DAPI Stock Solution (0.1 mg/ml)

Dissolve 5 mg DAPI in 50 ml deionized H2O by stirring for 60 min. 
Filter through a 0.22 ľm filter to remove small particles 
Store at - 20°C in 0.5 ml aliquots

Mithramycin Stock Solution (1 mg/ml) 

Dissolve 50 mg mithramycin A in 50 ml deionized H2O by stirring for 60 min. 
Filter through a 0.22 ľm filter to remove small particles 
Store at - 20°C in 0.5 ml aliquots

Sheath Fluid SF50

50 mM NaCl 7.31 g

Adjust volume to 2500 ml with deionized H2O 
Sterilise by autoclaving

Magnesium Sulfate Stock Solution (100 mM)

Dissolve 1.23 g of MgSO4 . 7H2O in 50 ml of deionized H2O. 
Filter through a 0.22 ľm filter to remove small particles 
Store at 4°C

PRINS Buffer

10 mM Tris base 0.605 g
50 mM KCl 1.864 g
2mM MgCl2.6H2O 0.203 g

Adjust volume to 500 ml with deionized H2O 
Adjust the final pH to 8.0 using 1N HCl 
Sterilise by autoclaving 
Store at 4°C

PRINS Reaction Mix

10x DNA polymerase buffer* 5 µl
25 mM MgCl2 (4 mM final) 5 µl
2 mM dCTP, dGTP (0.1 mM each final) 2.5 µl
0.2 mM fluorescein-12-dUTP (2 ľM final) 2 µl
0.2 mM fluorescein-15-dATP (2 ľM final) 2 µl
0.2 mM dTTP (34 ľM final) 4.25 µl
0.2 mM dATP (34 ľM final) 4.25 µl
20 ľM forward primer (2 ľM final) 5 µl
20 ľM reverse primer (2 ľM final) 5 µl
5U/ľl Taq DNA polymerase (3 U / 50 ľl final) 1.5 µl

*) The buffer contains 15 mM MgCl2 
Add sterile deionized H2O to 55 ľl (includes 5 ľl for evaporation) 
Actual composition of the mix (e.g., MgCl2, concentration and ratio of labelled and unlabeled nucleotides, primer concentration) should be optimised for given primer pair and species

Stop Buffer for PRINS

0.5 M NaCl 2.923 g
50 mM Na2EDTA 1.861 g

Adjust volume to 100 ml with deionized H2O 
Adjust the final pH to 8.0 using 1N NaOH 
Sterilise by autoclaving 
Store at 4°C

Wash Buffer for PRINS

100 mM maleic acid 1.161 g
150 mM NaCl 0.876 g
0.05% v/v Tween-20 0.5 ml

Adjust volume to 100 ml with deionized H2O 
Adjust the final pH to 7.5 using 1N NaOH 
Sterilise by autoclaving 
Store at 4°C

PCR Premix

(for a 50 ľl reaction mixture)

10x Taq DNA polymerase buffer* 5 µl
25 mM MgCl2 (1.5 mM final) 3 µl
10 mM dNTPs (0.2 mM each final) 1 µl
50 ľM forward primer (1 ľM final) 1 µl
50 ľM reverse primer (1 ľM final) 1 µl
5U/ľl Taq DNA polymerase (2.5 U/50 ľl final) 0.5 µl
sterile deionized H2O 18.5 µl

*) The buffer does not contain MgCl2 
Mix well and centrifuge briefly 
Prepare shortly before the use

TAE buffer 

Stock Solution (50 x)

Tris (2M final) 242 g
glacial acetic acid (1 M final) 57.1 ml
0.5 M EDTA (pH 8.0), (100 mM final) 200 ml

Adjust volume to 1000 ml with deionized H2O 
Store at room temperature 

Working Solution (1x) 
Dilute TAE stock solution 1:50 in deionized H2O (final concentrations: 40 mM Tris, 20 mM acetic acid, 2 mM EDTA)

Loading Buffer

0.5M EDTA (pH 8.0) (100 mM final) 2 ml
SDS (1% w/v final) 100 mg
bromophenol blue (0.05% w/v final) 5 mg
xylenecyanol (0.05% w/v final) 5 mg
gycerol (50% v/v final) 5 ml

Adjust volume to 10 ml with deionized H2O 
Store at room temperature

Ethidium Bromide Solution

Stock Solution (0.5 mg/ml)

Dissolve 5 mg ethidium bromide in 10 ml deionized H2O by stirring for 60 min

Ethidium Bromide Working Solution (0.5 µg/ml)

Dilute stock solution 1:1000 in deionized H2O 
The solution may be used several times 
Store at room temperature

References 

  • Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992) 
  • Gamborg OL, Wetter LR. Plant Tissue Culture Methods. Saskatoon: National Research Council of Canada (1975) 
  • Gamborg OL, Miller RA, Ojima K. Nutrient requirement of suspension cultures of soybean root cells. Experimental Cell Research 50: 151 - 158 (1968)

Suppliers of Reagents, Consumables and Equipment

Plant DNA Standards

It is generally agreed that flow cytometric estimation of nuclear DNA amount in absolute units should be performed using internal standard (the nuclei of a standard are isolated, stained and analysed simultaneously with the nuclei of a sample).

DNA content of the sample is then calculated:

2C DNA Amount (pg) = sample G1 peak mean × standard 2C DNA amount (pg) standard G1 peak mean


To estimate nuclear DNA content in plants, most laboratories prefer to use plant DNA standards. When choosing a standard, it is advisable to select a taxon whose DNA amount is not very different from that of a sample (this will decrease a risk of errors due to nonlinearity of the instrument). Several species are thus needed to cover the large range of genome size known for plants. Unfortunately, there is no general agreement on DNA standards for plant flow cytometry. This laboratory has been using the following plant cultivars:

List of DNA standards suitable for plant DNA flow cytometry

Species Cultivar 2C DNA Content (pg)* 1C Genome Size (Mbp)** Reference
Allium cepa Alice 34.89 17 061 Dolezel et al. (1998)
Vicia faba ssp. faba var. equina Inovec 26.90 13 154 Dolezel et al. (1992)
Secale cereale Dankovske 16.19 7 917 Dolezel et al. (1998)
Pisum sativum Ctirad 9.09 4 445 Dolezel et al. (1998)
Zea mays CE-777 5.43 2 655 Lysak and Dolezel (1998)
Glycine max Polanka 2.50 1 223 Dolezel et al. (1994)
Lycopersicon esculentum Stupicke polni tyckove rane 1.96 958 Dolezel et al. (1992)
Raphanus sativus Saxa 1.11 543 Dolezel et al. (1998)

*) Nuclear DNA content was established using human male leukocytes (2C = 7.0 pg DNA; Tiersch et al. 1989) as a primary reference standard.
**) 1 pg DNA = 978 Mbp (Dolezel et al. 2003)

All cultivars fulfil the criteria for plant DNA standards as they are: a) suitable for flow cytometric analysis of DNA content; b) seed propagated; c) available as elite lines from breeders. Upon request, this laboratory can provide reasonable amounts of seeds free of charge. Please, send your requests to: dolezelatueb [dot] cas [dot] cz

References

  • Dolezel J, Bartos J, Voglmayr H, Greilhuber J. Nuclear DNA content and genome size of trout and human. Cytometry 51: 127, 128 (2003)  Abstract PDF
  • Dolezel J, Dolezelova M, Novak FJ. Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biologia Plantarum 36: 351, 357 (1994)
  • Dolezel J, Greilhuber J, Lucretti S, Meister A, Lysak M A, Nardi L, Obermayer R. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals Botany 82 (Suppl. A): 17 - 26 (1998)
  • Dolezel J, Sgorbati S, Lucretti S. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiologia Plantarum 85: 625, 631 (1992)
  • Lysak MA, Dolezel J. Estimation of nuclear DNA content in Sesleria (Poaceae). Caryologia 52: 123 - 132 (1998)
  • Tiersch TR, Chandler RW, Wachtel SSM, Ellias S. Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10: 706 - 710 (1989)