|
| Status |
Public on Mar 31, 2014 |
| Title |
2013 infected grapes with B. cinerea, EL33,biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
green berries
|
| Organism |
Vitis vinifera |
| Characteristics |
genotype/variation: Trincadeira
tissue: pulp and skin
treatment: infected with B. cinerea
Stage: green stage (EL 33)
|
| Treatment protocol |
The Botrytis cinerea isolate used in this experiment was obtained from diseased grapevine plants and maintained in potato dextrose agar (PDA, Difco, Detroit, E.U.A.), at 5°C. Conidia production was achieved by exposing inoculated Petri dishes with PDA to continuous fluorescent light, at 24°C. Conidia were harvested from 14- to 20-day-old cultures and collected by rubbing with phosphate buffer (0.03M KH2PO4), filtered through cheesecloth to remove mycelia, and the concentration determined and adjusted to 105 conidia ml-1. Infections were made on berries by spraying the conidia suspension at developmental stage of pepper-corn size (EL 29). Control clusters were sprayed with phosphate buffer. Following inoculation, the clusters were incubated by enclosing them in individual plastic bags for a period of one week. Collection of samples was performed in two stages of development at pre-véraison (EL33) and véraison (EL35) (Coombe, 1995). For each time point and each treatment (infected and mock) three-four biological replicates were collected. Trincadeira berries were harvested around 10 a.m. and were immediately frozen in liquid nitrogen and transported to the lab in dry ice and kept at -80ºC.
|
| Growth protocol |
Field experiments were conducted in an experimental vineyard with 15 year old grapevines Vitis vinifera L. of cv. Trincadeira, at the Instituto Superior de Agronomia, University of Lisbon, Portugal.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was carried out according to Fortes et al. (2011 BMC Plant Biology). A DNAse treatment was performed according to suppliers’ instructions (Invitrogen, San Diego, CA, USA). Samples were then extracted in phenol/chloroform/isoamylalcohol (75:24:1,v/v/v), precipitated with sodium acetate and ethanol, washed in 70% ethanol and dissolved in DEPC-treated water. RNA was further purified using RNeasy Plant Mini kit according to the manufacturer’s instructions (Quiagen, Valencia, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from 10 µg of total RNA using cDNA Synthesis System Kit (NimbleGen-Roche). The cDNA preparation (1 µg) was amplified and labelled with Cy3-random nonamers using One color labeling kit (NimbleGen-Roche).
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| |
|
| Hybridization protocol |
If the quality control was correct, then 4 µg of labelled cDNA were hybridized. Hybridization solution (NimbleGen Hybridization kit) was added to each labelled cDNA and hybridization was performed for 16 h at 42ºC in a HS 4 Hybridization station (NimbleGen-Roche).
|
| Scan protocol |
Hybridized microarrays were washed with Wash buffer kit (NimbleGen-Roche) and scanned at 532 nm and 2 µm resolution in a DNA Microarray Scanner with Surescan High-Resolution Technology (Agilent technologies).
|
| Description |
Gene expression data from skin and pulp of infected grapes at green stage (EL 33)
|
| Data processing |
After evaluation of hybridization quality by Experimental metrics report implemented in NimbleScan Software version 2.6 (NimbleGen-Roche), probeset signal values from all the microarray hybridizations were background corrected and normalized together using Robust Microarray Average (RMA) (Irizarry et al., 2003) in NimbleScan Software, which produces a calls file for each sample with normalized expression data for each gene.
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| |
|
| Submission date |
Nov 20, 2013 |
| Last update date |
Mar 31, 2014 |
| Contact name |
Ana Margarida Fortes |
| E-mail(s) |
[email protected]
|
| Phone |
+351 21 7500382
|
| Organization name |
BIOFIG_FCUL
|
| Department |
Plant Biology
|
| Lab |
Biotechnology and Plant Molecular Biology
|
| Street address |
Campus da FCUL
|
| City |
Lisboa |
| ZIP/Postal code |
1749-016 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL17894 |
| Series (1) |
| GSE52586 |
Transcript and metabolite analysis of Vitis vinifera cv. Trincadeira berries infected with Botrytis cinerea reveals an activation of a non-sustained plant defense response |
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