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Fusarium graminearum Double (Triple) Mutants Generation Using Sexual Crosses

YG Yan Guo
Dong Zhang Dong Zhang
Wei-Hua Tang Wei-Hua Tang

Published: Aug 5, 2018 DOI: 10.21769/BioProtoc.2961 Views: 4055

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Abstract

Fusarium graminearum is a destructive phytopathogen that infects major cereal crops such as wheat, maize and barley. Double or triple mutants are often very useful in the phenotypic and genetic analysis of genes that function redundantly or within similar pathways. When single gene mutants are available, double or triple mutants can be generated by crossing heterothallic strains or multiple rounds of protoplast transformation. When individual mutants carry different antibiotic resistance, it is convenient to use the sexual crossing to generate desired recombinant strains. Here, we present a protocol for generating double or triple mutants by sexual crossing in one homothallic strain with further antibiotic resistance and genomic DNA PCR screening of recombinant progenies.

Keywords: Fusarium graminearum search Sexual crossing search Double mutants search Triple mutants search

Background

The ascomycete fungus Fusarium graminearum is a devastating phytopathogen that causes head blight, ear rot, stalk rot and crown in cereals. It can produce perithecia on carrot agar in vitro. This assay can be used for studying perithecia development, ascospore discharge and sexual recombination (Nicholson, 2007).

F. graminearum is homothallic and has both MAT1-1 and MAT1-2-1 locus; each of these locus deletion mutants is sterile in self-crosses (Zheng et al., 2013). To generate double gene mutants, one single gene mutant has traditionally been outcrossed with MAT deletion mutants and further outcrossed with another single mutant (Bowden and Leslie, 1999; Lee et al., 2011; Son et al., 2012). Another strategy of double or triple mutants construct is deleting a gene in the other mutant strain with protoplast transformation (Oide et al., 2007 and 2014).

Here, we adapt and simplify this method, and present the details of the protocol to generate double and triple mutants using sexual crosses in one homothallic strain with further antibiotic resistance screening.

Materials and Reagents

  1. 60 mm x 15 mm diameter plates (Sigma-Aldrich, catalog number: P5481-500EA)
    Manufacturer: Excel Scientific, catalog number: D-901 .
  2. Sterile glass spreading rod (CHEMGLASS, catalog number: CLS-1350-01 )
  3. 1.5 ml sterile centrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
  4. Sterile toothpicks (Purchased from Carrefour Supermarket)
  5. 90 mm diameter plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 263991 )
  6. Fungal strain: F. graminearum PH-1 (NRRL 31084) 
  7. Sterile ddH2O
  8. Fresh carrot (Purchased from Carrefour Supermarket)
  9. TWEEN 60 (Sigma-Aldrich, catalog number: P1629 )
  10. Ampicillin sodium Salt (YEASEN, catalog number: 60203ES60 )
  11. Yeast extract (Oxoid, catalog number: LP0021 )
  12. Casamino acids (Sigma-Aldrich, catalog number: 22090 )
  13. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
  14. Hygromycin B (Sigma-Aldrich, Roche Diagnostics, catalog number: 10843555001 )
  15. G418 sulfate (Santa Cruz Biotechnology, catalog number: sc-29065 )
  16. V8 juice (Campbell Soup, 051000000675)
  17. CaCO3 (Sigma-Aldrich, catalog number: C4830 ) 
  18. Nourseothricin sulfate (Goldbio, catalog number: N-500-100 )
  19. Fresh carrot agar (see Recipes, store at 4 °C)
  20. 2.5% TWEEN 60 (see Recipes, store at RT)
  21. TB3 plate (see Recipes, store at 4 °C)
  22. V8 juice agar (see Recipes, store at 4 °C)

Equipment

  1. 1 L Erlenmeyer Flask (Fisher Scientific, catalog number: S63274)
    Manufacturer: Corning, Pyrex®, model: 49801L/EMD .
  2. 500 ml Erlenmeyer Flask (Fisher Scientific, catalog number: S63273)
    Manufacturer: Corning, Pyrex®, model: 4980500/EMD .
  3. Hemocytometer (0.10 mm, 1/400 mm2) (QIUJING, model: XB-K-25 )
  4. Biological safety cabinet (ESCO Micro, model: FHC1200A )
  5. Autoclave (Zealway Instrument, model: GI54DWS )
  6. Homogenizer (Ronghua Instrument Manufacturing, model: JJ-2B )
  7. UV light (Philips Lighting, model: TL-D 15W/BLB 1SL/25, catalog number: 928024810803 )
  8. Induction chamber (JIANGNAN INSTRUMENT, model: GXZ-300 )
  9. Vortexer (Bio-Rad Laboratories, model: BR-2000 Vortexer )
  10. Mold incubator (Yiheng, model: MJ-150I )
  11. Microscope (Olympus, model: BX51 )

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbial genetics > Mutagenesis

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