cultivar: Goldrich developmental stage: immature fruit (S1)
Treatment protocol
Apricot fruit were harvested 45, 80 and 87 days after full bloom. These harvest days corresponds to immature-green stage (6 weeks before fully-ripe stage), mature-firm-ripe stage (change of peel color, 1 week before fully-ripe stage and onset of climacteric phase) and at fully-ripe stage (full climacteric phase) and designated as S1, S2 and S3 stages,respectively. Samples of 30 fruits were collected from each developmental stage and evaluated for color parameters (L, a*, b* values) using a Minolta colorimeter (model CM2500d), tissue firmness using an Effegi penetrometer, employed with an 8 mm probe and ethylene evolution as described by Tonutti et al., 1997 (J Am Soc Hort Sci 122:642–647). Fruit from each developmental stage were divided into three sub-lots (10 fruit for each) and mesocarp tissue from each sub-lot was removed, immediately frozen in liquid nitrogen and subsequently maintained at -80°C until needed. Peach fruits (cv Fantasia) were harvested at distinct developmental stages, designated as S1, S2, S3, S4, as described by Trainotti et al., 2007 (J Exp Bot 58:3299-3308) and stored at -80°C for subsequent molecular analysis.
Growth protocol
Fruit from apricot cv ‘Goldrich’ and peach cv 'Fantasia’ was used in the current study. By comparing the position of anchor markers in maps constructed with different Prunus species, it has been demonstrated that the genomes of the diploid Prunus species (including peach and apricot) are essentially collinear thus, at the genome level, the Prunus genus can be treated as a single genetic entity thus the platform developed for massive analysis of peach fruit transcriptome can be extended to apricot.
Extracted molecule
total RNA
Extraction protocol
Frozen fruit (3 g) was ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Bonghi and co-workers (1998, Planta. 102:346-352). Fifty μg of total RNA were treated with 10 units of RQ1 RNase-Free DNAse (Promega) and 1 unit of RNAguard (RNase INHIBITOR) (Amersham) for 30 min, and then purified by phenol-chlorophorm according to the manufacturer’s instructions. The concentration of RNA was quantified by measuring the absorbance at 260 nm and its integrity was checked electrophoretically on agarose gels.
Label
Cy5,Cy3
Label protocol
Total RNA (20 μg) was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labeling System (Invitrogen, USA) following manufacturer instruction. The amino-modified cDNA was coupled to a monoreactive N-hydroxysuccinimide (NHS)-ester fluorescent dyes: the red-fluorescent cyanine5 (Cy5) and the green-fluorescent cyanine3 (Cy3) (GE Healthcare, USA). A final purification step using Microcon-PCR centrifugal filter devices (Millipore, USA) removed any unincorporated dye. The purity and yield of the labelled cDNA was calculated from the OD values obtained by means of a spectrophotometer using the formulas reported in the SuperScriptTM Indirect cDNA Labeling System instruction manual.
cultivar: Goldrich developmental stage: fully ripe fruit (S3)
Treatment protocol
Apricot fruit were harvested 45, 80 and 87 days after full bloom. These harvest days corresponds to immature-green stage (6 weeks before fully-ripe stage), mature-firm-ripe stage (change of peel color, 1 week before fully-ripe stage and onset of climacteric phase) and at fully-ripe stage (full climacteric phase) and designated as S1, S2 and S3 stages,respectively. Samples of 30 fruits were collected from each developmental stage and evaluated for color parameters (L, a*, b* values) using a Minolta colorimeter (model CM2500d), tissue firmness using an Effegi penetrometer, employed with an 8 mm probe and ethylene evolution as described by Tonutti et al., 1997 (J Am Soc Hort Sci 122:642–647). Fruit from each developmental stage were divided into three sub-lots (10 fruit for each) and mesocarp tissue from each sub-lot was removed, immediately frozen in liquid nitrogen and subsequently maintained at -80°C until needed. Peach fruits (cv Fantasia) were harvested at distinct developmental stages, designated as S1, S2, S3, S4, as described by Trainotti et al., 2007 (J Exp Bot 58:3299-3308) and stored at -80°C for subsequent molecular analysis.
Growth protocol
Fruit from apricot cv ‘Goldrich’ and peach cv 'Fantasia’ was used in the current study. By comparing the position of anchor markers in maps constructed with different Prunus species, it has been demonstrated that the genomes of the diploid Prunus species (including peach and apricot) are essentially collinear thus, at the genome level, the Prunus genus can be treated as a single genetic entity thus the platform developed for massive analysis of peach fruit transcriptome can be extended to apricot.
Extracted molecule
total RNA
Extraction protocol
Frozen fruit (3 g) was ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Bonghi and co-workers (1998, Planta. 102:346-352). Fifty μg of total RNA were treated with 10 units of RQ1 RNase-Free DNAse (Promega) and 1 unit of RNAguard (RNase INHIBITOR) (Amersham) for 30 min, and then purified by phenol-chlorophorm according to the manufacturer’s instructions. The concentration of RNA was quantified by measuring the absorbance at 260 nm and its integrity was checked electrophoretically on agarose gels.
Label
Cy5,Cy3
Label protocol
Total RNA (20 μg) was converted into target cDNA by reverse transcription using the SuperScriptTM Indirect cDNA Labeling System (Invitrogen, USA) following manufacturer instruction. The amino-modified cDNA was coupled to a monoreactive N-hydroxysuccinimide (NHS)-ester fluorescent dyes: the red-fluorescent cyanine5 (Cy5) and the green-fluorescent cyanine3 (Cy3) (GE Healthcare, USA). A final purification step using Microcon-PCR centrifugal filter devices (Millipore, USA) removed any unincorporated dye. The purity and yield of the labelled cDNA was calculated from the OD values obtained by means of a spectrophotometer using the formulas reported in the SuperScriptTM Indirect cDNA Labeling System instruction manual.
Hybridization protocol
Pre-hybridisations were carried out by soaking whole glass slides in a solution containing 5X SSC, 0.1% SDS, 5X Denhardt’s solution and 100ng/μL DNA carrier at 48°C for at least 2 h. Then the slides were washed once with a 0.2X SSC solution and dried by centrifuging for 4 min at 1000 rpm. Hybridisations were carried out in 250 µL of hybridisation solution (5X SSC, 0.1% SDS, 25% formamide) containing 100-150 pmol of Cy3- and Cy5-labelled target cDNAs. The glass slides were placed in a hybridisation chamber (Corning, USA) kept on a water bath at 48°C at least 36 h. Then the slides were briefly rinsed with 1X SSC 0.1% SDS and washed once the same solution for 5 min. Three additional washes (one with 0.2XSSC 0.1% SDS and two with 0.2X SSC, for 5 min each) at room temperature were performed before drying the glass slides with a brief centrifugation. The probe design and these hybridisation/washing conditions allow the detection of specific genes even within gene families.
Scan protocol
The microarray was scanned with a two channel confocal microarray scanner (ScanArray® Lite, Perkin Elmer, USA) using its dedicated software (ScanArray Express 3.0.0., Perkin Elmer). The laser power and the photomultiplier tube (PMT) were set between 75 and 85 % of maximum. The excitation/emission settings were 543/570 nm for Cy3 and 633/670 nm for Cy5. After laser focusing and balancing of the two channels, scans were conducted at a resolution of 5 μm. For any scan, two separate 16-bit TIFF images were produced.
Description
Analysis used to compare transcripts level in fully ripe fruit and immature-green fruit.
Data processing
Software from the TM4 (www.tm4.org) package developed at TIGR (www.tigr.org, Saeed et al., 2003) was used to analyze microarray data. Images were processed using the Spotfinder 2.2.3. software by means of the Otsu algorithm. Spots were also visually examined to delete the non-uniform ones. The expression data extracted by Spotfinder were normalized by MIDAS 2.18 using the LOWESS (Locally Weighted Regression Scatter Plot Smoothing, Cleveland, 1979) algorithm with the “block mode”, keeping as reference the Cy3 channel. The “IA” and “IB” values have been used to determine log2(IB/IA) reported in the “VALUE” column. Normalized split data were loaded in MeV 4.3 and subjected to SAM (Significance Analysis of Microarrays, Tusher et al., 2001) analyses. Since each comparison was repeated at least twice, and a “percentage cutoff filter”, set to 75%, was used to eliminate those gene with less than 3 data points, there were at least 3 values for each gene to be used in the SAM analysis. Lists of clones with significant changes in expression were identified at delta values that gave a false discovery rate (FDR) of about 5%.
Please see 'readme.txt' file on the Series record for the sample labeling scheme for each supplementary file.
