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.