2.6 qRT-PCR analysis
Total RNA was isolated from the berry samples using the same method as described above. First-strand cDNA was synthesized using Superscript IV reverse transcriptase (Invitrogen) from 1μg total RNA according to manufacturer’s instructions. MJ-MiniOpticon Real-time PCR system (Bio-Rad) was used for qRT-PCR analysis with SsoFast™ EvaGreen Supermix (Bio-Rad) in 15μl volume reaction. The PCR conditions were as follows: initial denaturation at 95°C for 30 sec followed by 40 cycles at 95°C for 5 sec, and 60°C for 10 sec. Subsequent melting curve analysis, ranging from 65°C to 95°C with an increment of 0.5°C per cycle, was used to assure amplification of only one product. All analyses were performed with three biological replicates and two technical replicates. The results were analyzed using CFX Connect software (Bio-Rad) using 2(−ΔΔCt) method, and the relative expression levels were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) or Actin . Primer sequences of the genes are listed in Table S1.