2.4. Expression analyses.
The total RNA of three biological replicates samples was extracted from 0.5 g of fruit powder according to Yu et al. (2012). Each biological replicate sample contained a fruit mixture of at least 50 fruits. The cDNA was prepared by reverse transcription of 1 μg of total RNA using the Maxima cDNA Kit with dsDNase kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer instructions. Quantification was performed by real‐time quantitative RT‐PCR (qPCR) using iCycler iQ™ Real-Time PCR Detection System (BIORAD) and quantified using Real Time Detection System Software (version 2.0). The amplification reactions were carried out in a final volume of 12 μL containing 6 μl of NZY qPCR Green Master Mix (2x) (NZYTech, Ltd), 1 μL of each primer (10 μM) and 1 μL of the cDNA. The PCR profile used was 2 min at 50 °C, 95 °C for 10 min, followed by 40 cycles of 20 s at 95 °C and 30 s at 55 or 60 ºC. Three technical replicates were made from each of the genes studied. Gene expression was determined by 2−ΔΔCT method using a F. vesca Actin-97-like (XM_004307470) as housekeeping gene. The specific primers used are described in Supplementary Table S1 and PCR amplicons were sequenced to confirm specificity.