The pathogen strain identification based on toxin biosynthetic genes in the Romanian wheat genotypes

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The pathogen strain identification based on toxin
biosynthetic genes in the Romanian wheat genotypes
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Fungal control DNA
Culture media Potato Extract Glucose Agar
DNA extraction FastDNA SPIN Kit for Soil
Quiagen Permits a rapid isolation of PCR-ready
genomic DNA from fungal samples.
Cereal samples preparation
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DNA extraction and purification method - CTAB
method

• 100 mg ground material was pre-treated with
  Celulace (Promega) for 2 hours at 65C then mixed
  with 300 µl water and then 700 µl CTAB buffer was
  added together with 20 µl RNase solution (10
  mg/ml) before incubation at 65 C for 30 min. And
  then 10 µl proteinase K solution (20 mg/ml) was
  added before an incubation at 65 C for 30 min.
• samples were centrifuged at 12,000 g for 10 min
  and the supernatant was transferred to a tube
  with 500 µl chloroform, vortexed and centrifuged
  at 12,000 g for 15 min.
• the upper layer was transferred to a new tube
  and 2 volumes of CTAB precipitation solution were
  added and the samples were incubated at RT for 60
  min before centrifugation at 12,000 g for 5 min.
  
• the pellet was dissolved in 350 µl 1.2 M NaCl
  and 350 µl chloroform was added before vortexing
  and centrifugation at 12,000 g for 10 min.
• the upper layer was precipitated with 0.6
  volumes of isopropanol and incubated at RT for 20
  min and centrifuged at 12,000 g for 10 min.
• the pellet was washed in 70 ethanol, vacuum
  dried and resuspended in 100 µl MQ water.

Quantification of the extracted DNA by
spectrophotometric method
Thermo Scientific NanoDrop 8000
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PCR analyses
Amplification was performed in a Corbett
RESEARCH Thermal Cycler, folowing the indications
from literature.

• PCR reagents
• Go Taq Green Master Mix PCR kit from Promega
  2X.
• 20 pmol of each primer.
• different concentrations of DNA template.
• in a final volume 25 µl.

Amplicons were analyzed by electrophoresis on 2
agarose gel (Promega, USA) and visualized in
Ethidium Bromide (0.4 ng/ml) presence.
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Romanian wheat samples
DON 7 biosynthetic gene
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Romanian wheat samples
DON 13 biosynthetic gene
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Romanian wheat samples
NIV 13 biosynthetic gene
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Romanian wheat samples
NIV 13 biosynthetic gene
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Corn samples
Fusarium graminearum
15 artificially infected samples
14 GM corn samples
Fusarium culmorum
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