Total RNAs were extracted using a CTAB extraction buffer from 3 g of frozen fruit flesh tissue ground in liquid nitrogen, as described in Nobile et al., [15 (link)] and Rienth et al., [115 (link)]. mRNAs were amplified, labelled and co-hybridized according to Celton et al., [36 (link)] as following: aRNAs were produced with Message AmpII aRNA amplification kit (Ambion) from 200 ng of total RNA. Then, 5 μg of each aRNAs were retrotranscribed and labelled with either Cyanine-3 or Cyanine-5 fluorescent dye (Interchim, Montluçon, France). Labelled samples were combined as 30 pmol for each dye and co-hybridized to the Nimblegen microarray AryANE v1.0 containing 135,000 60-mers oligonucleotide probes as described in Celton et al., [36 (link)]. Deva software (Nimblegen) was used to extract pair-data files from the scanned images, obtained using the MS200 microarray scanner (Roche Nimblegen).
Genotypes were associated in 4 pairs (I062/V083, V034/W029, H097/I095, I016/H074) for competitive hybridizations at each time point (60 DAF, 110 DAF, H, 1 M and 2 M). Two independent biological repeats were performed with fruits from PH or P12 plots and technical replicates with dye swap were included for a total of 38 arrays.
Genotypes were associated in 4 pairs (I062/V083, V034/W029, H097/I095, I016/H074) for competitive hybridizations at each time point (60 DAF, 110 DAF, H, 1 M and 2 M). Two independent biological repeats were performed with fruits from PH or P12 plots and technical replicates with dye swap were included for a total of 38 arrays.