Quantitative real-time PCR (qRT-PCR) validation
In order to confirm the reliability and accuracy of the RNA-Seq analysis, qRT-PCR was performed on a set of 6 genes randomly selected from DEGs. Firstly, total RNA was extracted from approximately 3g of frozen tissues using E.Z.N.A®. Plant RNA Kit (OMEGA, R6827) by following the producer’s instructions. Genomic DNA was removed to obtain DNA-free RNA which was reversely transcribed to first-strand cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, RR047A). Based on sequences from the peach genome database (Jung et al., 2008), gene specific primers were designed (Supplementary Table S1 ) and evaluated using NCBI Primer-BLAST (Ye et al., 2012). Translation initiation factor 1A (PpeIF-1A, ppa002870m) was used as the reference gene for data normalization according to a previous publication (Kou et al., 2017). With diluted cDNA, qRT-PCR was performed using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, A25741) on QuantStudio™ 6 Flex Real-Time PCR Systems (Life Technologies) with previously described primer pairs. Samples for each condition were examined in 3 technical replicates and the mean Ct was calculated. The 2−ΔΔCt method was used to assess the relative gene expression level between two temperature conditions at every time point.
Results
Changes of firmness and ethylene production in peach fruits
To measure the internal physiological variation in peach fruits, the changes of firmness and ethylene production were examined at 6 time points for CT and HT storage conditions, respectively. Interestingly, the peach fruits at HT showed a much higher level of firmness than the fruits at CT, and this was accompanied by a significantly lower level of ethylene production of 0.05 μl/g/h in HT samples (Figure 1 ). After 3 days, firmness showed almost steady trend in both treatments. Besides, CT samples stayed in high ethylene production rate of more than 0.2 μl/g/h on all days. These data indicated that firmness and ethylene production were correlated with each other, but ethylene may not be the only influential factor on firmness.