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Chiasma Counting in Maize Male Meiocytes

Gaganpreet K. Sidhu Gaganpreet K. Sidhu
WP Wojciech P. Pawlowski

Published: May 5, 2020 DOI: 10.21769/BioProtoc.3604 Views: 2732

Edited by: Marisa Rosa

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Abstract

This protocol combines a classical chromosome spreading technique with 3-dimensional image reconstruction for visualization of bivalent configurations during meiosis to aid chiasma counting.

Keywords: Chromosomes search Meiosis search Recombination search Chiasma search Maize search

Background

Chiasmata are cytological manifestations of crossovers. Chiasma counting provides a fast and reliable assessment of meiotic recombination rates. Chiasmata are best quantified in maize at diakinesis in meiotic prophase I, as at this stage, they are most conspicuous. Advantages of cytological assessment of crossover rates are that it is fast, inexpensive, and can be performed on genetically homozygous individuals. This protocol improves on the classical chromosome spreading technique by combining it with modern 3-dimensional imaging, which improves the accuracy of chiasma counting, resulting in an error rate of 10% or less (Sidhu et al., 2015; Kianian et al., 2018).

Materials and reagents

  1. 10 or 20 μl pipette tips
  2. Acetocarmine solution (VWR, catalog number: 470300-054)
  3. 15-ml Falcon tubes (Corning, catalog number: 352096)
  4. Coplin jar
  5. Dissecting needle (Fisher Scientific, catalog number: 13-820-024)
  6. Diamond marking pen
  7. Glass microscope slides (Millipore Sigma, catalog number: S8902) and cover slips (Millipore Sigma, catalog number: S1815)
  8. Nail polish
  9. Plastic Petri dish (Fisher Scientific, catalog number: FB0875713)
  10. 95:5 (v/v) acetic acid:methanol solution (Millipore Sigma, catalog numbers: A6283 and 322415)
  11. 90% acetic acid (Millipore Sigma, catalog number: A6283)
  12. 70% ethanol (Millipore Sigma, catalog number: E7023)
  13. Sodium citrate (Millipore Sigma, catalog number: 1613859)
  14. EDTA (Millipore Sigma, catalog number: E9884)
  15. Onozuka R-10 cellulase (GoldBio, catalog number: C8001)
  16. Macerozyme (GoldBio, catalog number: M8002)
  17. Cytohelicase (Millipore Sigma, catalog number: C8274)
  18. Citric acid (Millipore Sigma, catalog number: 251275)
  19. DAPI powder (4,6-diamidino-2-phenylindole) (Millipore Sigma, catalog number: 10236276001)
  20. NaCl (Millipore Sigma, catalog number: S9888)
  21. KCl (Millipore Sigma, catalog number: P3911)
  22. Na2HPO4 (Millipore Sigma, catalog number: NIST2186II)
  23. KH2PO4 (Millipore Sigma, catalog number: NIST200B)
  24. DABCO (1,4 diazabicyclo-[2,2,2] octane) (EMS, catalog number: 17989-50)
  25. Tris (Millipore Sigma, catalog number: 10708976001)
  26. Glycerol (Millipore Sigma, catalog number: G7893)
  27. Citric acid buffer pH 5.5 (see Recipes)
  28. Structural weakening buffer (see Recipes)
  29. DAPI (see Recipes)
  30. 1x PBS (see Recipes)
  31. DABCO solution (see Recipes)

Equipment

  1. Fine-tip forceps (Fisher Scientific, catalog number: 22-327379 )
  2. Alcohol lamp (Fisher Scientific, catalog number: S41898B )
  3. Dissecting microscope
  4. Fluorescence microscope
  5. UV crosslinker

Procedure

Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant cell biology > Cell imaging

Plant Science > Plant molecular biology > Genetic analysis

Cell Biology > Cell imaging > Fluorescence

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