RESULTS FOR FUSARIUM STRAIN IDENTIFICATION IN ARTIFICIAL-INOCULATED WHEAT GENOTYPES
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Transcript of RESULTS FOR FUSARIUM STRAIN IDENTIFICATION IN ARTIFICIAL-INOCULATED WHEAT GENOTYPES
RESULTS FOR FUSARIUM STRAIN IDENTIFICATION
IN
ARTIFICIAL-INOCULATED WHEAT GENOTYPES
Fusarium graminearum, Fusarium culmorum
Deoxynivalenol (DON) and Nivalenol (NIV)
two chemotaxonomic groups based on production of trichothecenes:
474 control 474 - Fc 12551 474 -Fg 13.05
483 control 483 - Fc 12551 483-Fg 13.05
488 control 488 – Fc 12551 488-Fg 13.05
501 control 501- Fc 12551 501-Fg 13.05
561 control 561- Fc 12551 561-Fg 13.05
BIOLOGICAL MATERIALS
parts
1 grains
2 chaf
3 culm
4 flag leaf
DNA extraction and purification method - CTAB method
100 mg ground material was 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.
The DNA was quantified by spectophotometric method using
NanoDrop 8000
All the DNA samples were diluted to 70 ng/ul
Primers for Fusarium identification and toxin genes identification
Specie Primers sequence Amplicon size (bp)
Fusarium graminearum 5’-CTCCGGATATGTTGCGTCAA-3’5’-GGTAGGTATCCGACATGGCAA-3’
450
Fusarium culmorum 5’-ATGGTGAACTCGTCGTGGC-3’
5’-CCCTTCTTACGCCAATCTCG-3’
570
Tri 7 DON biosynthetic gene
5’ TGCGTGGCAATATCTTCTTCCTA 3’
3’ GTGCTAATATTGTGCTAATATTGTGC 5’
381-445
Tri 13 DON biosynthetic gene
5’ CATCATGAGACTTGTGTCAGAGTTTGGG 3’
3’ GCTAGATCGATTGTTGCATTGAG 5’
282
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.
Fusarium graminearum
Fusarium culmorum
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Positive control
Fg 13.05
Positive control
Fc 12551
1- 5 control 474, 483, 488, 501, 561
6-10 - Fc 12551 474, 483, 488, 501, 561
11-15 - Fg 13.05 474, 483, 488, 501, 561
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1- 5 control 474, 483, 488, 501, 561
6-10 - Fc 12551 474, 483, 488, 501, 561
11-15 - Fg 13.05 474, 483, 488, 501, 561
16 Fg 13.05
17 Fc 12551
Tri 7 DON biosynthetic gene
Tri 13 DON biosynthetic gene
Fusarium infection
Toxin genes identificationGRAINS
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