8 protocols
The microarray data were analyzed using the statistical package R, version 2.14.0 (R Development Core Team, 2005) with various Bioconductor packages (http://www.bioconductor.org, Gentleman et al., 2004). Microarray quality controls were performed using the arrayQualityMetrics package (Kauffmann et al., 2009). Expression intensities were background corrected, quantile-normalized and summarized using the RMA function of the oligo package (Carvalho & Irizarry, 2010). Value are expressed in log2.
The slide was scanned using an MS200 from Tecan, after which resulting images were imported into NimbleScan software for grid alignment and expression data analyses. Raw data are in .xys.
The microarrays used were the grape whole genome NimbleGen microarrays (Roche, probeset design 090918 Vitus exp HX12). The microarray hybridizations were done for the 12 samples (three scion/rootstock combinations with four biological replicates) by the Plateforme Biopuces, Institut National des Sciences Appliquées, Toulouse, France according to the manufacturer’s instructions.
2 ug of RNA were used for cDNA synthesis according to the NimbleGen Gene Expression protocol. cDNA samples were labeled with Cy3 using a NimbleGen One-Color DNA labeling kit.
Total RNA was extracted using the Spectrum Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer’s instructions.
After the formation of a successful graft union, plants were transferred to a greenhouse for one month for rooting (the greenhouse is designed to preferentially heat the root system).After callusing and rooting, grafted plants were planted in 7 L pots filled with calcareous clay soil and cultivated in an experimental plot outside (at the end of May). The plants were trained to one stem, side branches and inflorescences were removed. The plants were supplied with a nutrient solution several times a day. The composition of the nutrient solution was: 2.5 mM KNO3, 0.25 mM MgSO4.7H2O, 0.62 mM NH4NO3, 1 mM (NH4)H2PO4, 9.1 mM MnCl2.4H2O, 46.3 mM H3BO3, 2.4 mM ZnSO4.H2O, 0.5 mM CuSO4 and 0.013 mM (NH4)6Mo7O24. Iron was supplied as 8.5 mg/L Sequestrene 138 (EDDHA 5.9% Fe). Tap water introduced 0.69 mM Mg2+, 0.22 mM K+ and 0.22 mM SO42-, and final pH was 6.0. The climatic conditions from 1st June until 30th September 2010 were: average air temperature 20.6 °C; the average daily mean global radiation was 1962 joules/cm²; the average relative humidity 68.1 %.
Vitis vinifera cv. ‘Cabernet-Sauvignon N’ (CS, clone 15), V. riparia cv. ‘Riparia Gloire de Montpellier’ (RG, clone 1030) and the V. berlandieri x V. rupestris hybrid cv. ‘1103 Paulsen’ (1103P, clone 198) hardwood was collected from a vineyard in January and stored as one meter long stems in a cold chamber (4 °C) until grafting in March.The scion CS was grafted onto RG and 1103P as well as auto-grafted onto CS rootstocks. Stored stems were taken out of the cold chamber the day before grafting and soaked in water at room temperature in order to rehydrate. One bud cuttings were made for scions and two-node de-budded cuttings were made for rootstocks shortly before grafting. Mechanical omega grafting was performed on scion/rootstock pairs of approximately the same diameter. Grafts were briefly dipped into melted wax and settled for callusing for 21 days at 28°C, 100% humidity.
Four pools of 5 shoot apex zones (approximately 4 mm in length) were harvested at the end of the night for each scion/rootstock combination in July (4 months after grafting) and immediately snap frozen in liquid nitrogen.