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| Status |
Public on Aug 08, 2015 |
| Title |
Berry_MB_Bc_48hpi_rep_b |
| Sample type |
RNA |
| |
|
| Source name |
berry
|
| Organism |
Vitis vinifera |
| Characteristics |
tissue: berry
developmental stage: harvesting stage
hours post inoculation: 48
|
| Treatment protocol |
The equatorial area of each berry was inoculated with 3 inoculum droplets spotted in triangle, each of 10 µl with 5000 conidia in potato dextrose browth
|
| Growth protocol |
All grapevine berries used in this study belongs to the cultivar Marselan and were collected from one experimental INRA vineyard near Bordeaux between 2009 and 2011. The B. cinerea inoculum was produced by growing the strain B05-10 (Quidde et al., 1998) on solid medium (1.5 % malt extract, 0.5 % glucose, 0.1 % tryptone, 0.1 % casein hydrolysate, 0.1 % yeast extract and 0.02 % tRNA) first for five days in the dark, followed by five days under black light (300–400 nm) to promote fungal sporulation (OSRAM L36-73; 10 h/14 h day/night).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with a modified Reid et al. (2006) protocol. Pre-warmed (60ºC) extraction buffer consisting of 300 mM Tris-HCl (pH 8), 25 mM EDTA, 2 M NaCl, 1 mM aurintricarboxylic acid, 2 % (m/v) hexadecyl-trimethyl-ammonium bromide, 2 % (m/v) polyvinylpolypyrrolidone, 0.05 % (m/v) spermidine trihydrochloride, and 2 % (v/v) ß-mercaptoethanol was added to frozen berry peel powder at 15 mL/g and shaken vigorously. Samples were incubated in a 60ºC water bath for 10 min and shaken every 2 min. An equal volume of chloroform/3-methyl-1-butanol (24:1) was added and samples were mixed vigorously, followed by centrifugation at 3500 g for 15 min at 4ºC. The water phase was collected and subjected to a repeated chloroform extraction. Nucleic acids in the water phase were precipitated in 0.3 M sodium acetate (pH 5.2) and by adding 0.6 vol of isopropyl alcohol. After 1 h at -20ºC the precipitates were pelleted by centrifugation at 3500 g for 30 min at 4ºC and the pellet was dissolved in 1 mL TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA). LiCl was added to 2.5 M and large RNAs were precipitated overnight at 4ºC, pelleted by centrifugation at 20 000 g for 30 min at 4ºC. The pellet was washed with 70% ethanol and dissolved in water. Remaining traces of DNA were removed by DNaseI treatment (DNA-free, Invitrogen) and the RNA quality was analyzed using a RNA 6000 nano kit (Agilent Technologies, Waldbronn, Germany).
|
| Label |
Cy3
|
| Label protocol |
cDNA samples were labeled with Cy3 using a NimbleGen One-Color DNA labeling kit
|
| |
|
| Hybridization protocol |
Samples were hybridised to slides using a NimbleGen hybridization system.
|
| Scan protocol |
The slide was scanned using an Axon GenePix® 4000A scanner, after which resulting images were imported into NimbleScan software for grid alignment and expression data analyses.
|
| Description |
Grapevine berry at harvesting stage 48h post inoculation with Botrytis cinerea B05-10
|
| Data processing |
The data were normalized using quantile normalization and gene calls generated using a robust multi-chip average (RMA) algorithm (Irizarry). The statistically significant transcripts from the grapevine microarray data were detected using ANAIS with the FDR-test for multiple tests correction of ANOVA p-values (Simon & Biot, 2010).
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| |
|
| Submission date |
Feb 17, 2015 |
| Last update date |
Aug 08, 2015 |
| Contact name |
Alberto Ferrarini |
| E-mail(s) |
alberto.ferrarini@univr.it
|
| Phone |
+39-045-802-7058
|
| Organization name |
University of Verona
|
| Department |
Scientific and Technological Department
|
| Lab |
Plant Functional Genomics Centre
|
| Street address |
Strada le Grazie, 15
|
| City |
Verona |
| State/province |
Veneto |
| ZIP/Postal code |
37134 |
| Country |
Italy |
| |
|
| Platform ID |
GPL13936 |
| Series (1) |
| GSE65969 |
Analysis of the molecular dialogue between gray mold (Botrytis cinerea) and grapevine (Vitis vinifera) reveals a clear shift in defense mechanisms during berry ripening |
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