|
| Status |
Public on Sep 30, 2015 |
| Title |
Fg_Wheat_4d_BR1_cy3 vs mock |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Mock_Wheat_BR1
|
| Organism |
Triticum aestivum |
| Characteristics |
time: 2 days
biorep: BR1
growth condition: Greenhouse
cultivar: Roblin
|
| Treatment protocol |
All three plant species were inoculated with F. graminearum strain DAOM180378 (15ADON-producer). Field-grown maize (Zea mays) inbred B73 ears were sib-crossed and developing kernels (eleven days post-fertilization) were inoculated through the husks with 0.1 ml of macroconidia suspension (5 x 105 spores/ml) (between 8 am and 10 am) and fields were irrigated for 15 min every afternoon during the inoculation period. The spring wheat (Triticum aestivum) cultivar Roblin and barley (Hordeum vulgare) cultivar Encore were grown in a controlled-environment cabinet with 16h light (20°C) and 8h dark (16°C). At mid-anthesis, each biological replicate of six wheat heads were point inoculated by pipetting 10 µl of a 10,000 spores/mL F. graminearum macroconidia suspension between lemma and plea of two primary florets in each spikelet. All fully developed spikelets on each head were inoculated. In the case of barley, at mid-anthesis, each fully developed floret of only two central rows was inoculated and florets from laterals rows were stripped from the rachis. In each inoculated barley floret, the tip of the lemma that includes the awn was excised and 1000 F. graminearum macroconidia were pipetted inside the lemma. A mock inoculation using water was carried out in parallel. Inoculated wheat and barley plants were misted overhead for 20 sec every 30 min for 2 days to maintain high humidity during the initial infection period and then moved to a non-misted bench in the same cabinet. Infected ears or spikelets were harvested 1, 2 and 4 days post inoculation, flash-frozen in liquid nitrogen, and stored at -80oC.
|
| Growth protocol |
Maize inbred B73 was grown in the field on the Central Experimental Farm, Ottawa, ON in 2004 and 2006. Wheat cultivar Roblin and barley cultivar Encore were grown in the greenhouse under conditons of 16 h light (20oC) and 8h dark (16oC).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a guanidine isothiocyanate-cesium chloride method (Ohan and Heikkila 1995).
|
| Label |
Cy5
|
| Label protocol |
500ng of total RNA was labelled using reagents and procedure described in the Two-color Quick Amp Labeling protocol version 5.7 (Agilent Technologies) except only half the amounts of reagents were used in Step 2 of the labelling reaction.
|
| |
|
| Channel 2 |
| Source name |
Fg_Wheat_4d_BR1
|
| Organism |
Triticum |
| Characteristics |
time: 4 days
biorep: BR1
growth condition: Greenhouse
cultivar: Roblin
|
| Treatment protocol |
All three plant species were inoculated with F. graminearum strain DAOM180378 (15ADON-producer). Field-grown maize (Zea mays) inbred B73 ears were sib-crossed and developing kernels (eleven days post-fertilization) were inoculated through the husks with 0.1 ml of macroconidia suspension (5 x 105 spores/ml) (between 8 am and 10 am) and fields were irrigated for 15 min every afternoon during the inoculation period. The spring wheat (Triticum aestivum) cultivar Roblin and barley (Hordeum vulgare) cultivar Encore were grown in a controlled-environment cabinet with 16h light (20°C) and 8h dark (16°C). At mid-anthesis, each biological replicate of six wheat heads were point inoculated by pipetting 10 µl of a 10,000 spores/mL F. graminearum macroconidia suspension between lemma and plea of two primary florets in each spikelet. All fully developed spikelets on each head were inoculated. In the case of barley, at mid-anthesis, each fully developed floret of only two central rows was inoculated and florets from laterals rows were stripped from the rachis. In each inoculated barley floret, the tip of the lemma that includes the awn was excised and 1000 F. graminearum macroconidia were pipetted inside the lemma. A mock inoculation using water was carried out in parallel. Inoculated wheat and barley plants were misted overhead for 20 sec every 30 min for 2 days to maintain high humidity during the initial infection period and then moved to a non-misted bench in the same cabinet. Infected ears or spikelets were harvested 1, 2 and 4 days post inoculation, flash-frozen in liquid nitrogen, and stored at -80oC.
|
| Growth protocol |
Maize inbred B73 was grown in the field on the Central Experimental Farm, Ottawa, ON in 2004 and 2006. Wheat cultivar Roblin and barley cultivar Encore were grown in the greenhouse under conditons of 16 h light (20oC) and 8h dark (16oC).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a guanidine isothiocyanate-cesium chloride method (Ohan and Heikkila 1995).
|
| Label |
Cy3
|
| Label protocol |
500ng of total RNA was labelled using reagents and procedure described in the Two-color Quick Amp Labeling protocol version 5.7 (Agilent Technologies) except only half the amounts of reagents were used in Step 2 of the labelling reaction.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were incubated overnight at 65ºC in a Robbins 400 oven outfitted with a Hybridization Oven Rotator (Agilent G2530-60029). Slides were washed as recommended by Agilent: Wash with Stabilization and Drying Solution.
|
| Scan protocol |
Slides were scanned on the Genepix 4200a (Molecular Devices) at 5µ resolution. Images were quantified using Genepix Pro 6.0 (Molecular Devices)
|
| Description |
Wheat infected with Fusarium graminearum for 96hrs vs mock infected wheat
|
| Data processing |
Array results files (.gpr) were imported in Acuity 4.0 and normalized by ratio-based method using spike-in control (+)E1A_r60_1, which has a cy5:cy3 ratio of 1:1. There are 20 copies of (+)E1A_r60_1 on each array. Two datasets were formed with all arrays and the datatypes were changed to F532-B532 and F635-B635 respectively to remove false signals from dust or other contaminants. After the removal of false signals, for each dataset, features that did not have intensities higher than 250 in at least 1 of 8 replicates were removed. A new dataset was formed with the remaining features from each , and the datatype was changed to Ratio of Medians. Spike-in controls were removed. Cy3 test array data were swapped to cy5. Technical replicates were combined.
|
| |
|
| Submission date |
May 09, 2012 |
| Last update date |
Sep 30, 2015 |
| Contact name |
Linda J Harris |
| E-mail(s) |
Linda.Harris@agr.gc.ca
|
| Phone |
613-759-1314
|
| Organization name |
Agriculture & Agri-Food Canada (AAFC)
|
| Department |
Eastern Cereal & Oilseed Research Centre
|
| Lab |
Building #21
|
| Street address |
960 Carling Ave.
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
K1A 0C6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11046 |
| Series (1) |
| GSE37886 |
Transcriptional profiling of the plant pathogen Fusarium graminearum on three cereal hosts. |
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