qRT-PCR validation

In order to confirm the results of the transcriptome sequencing, 9 genes related to ROS, ethylene, auxin signaling pathways, MAPK signal transduction pathway, and fruit softening metabolic pathways, were selected for qRT-PCR (quantitative real-time PCR) analysis. Primer sequence were designed by Primer Premier 5 and their specificity were validated. Synthetic primers were purchased from Synbio Technology Co., Ltd. (China) and shown in Supplementary Table S1. The reference gene FaActin primers were F: 5′-GGTGACGAGGCTCAATCCAA-3′, R: 5′-GGGCAACACGAAGCTCATTG-3′.

cDNA was synthesized from quantified total RNA by HiScript^® QRT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instruction. Synthesized cDNA (50 ng/μL) which were frozen at −20°C, were diluted 10-fold used as the template for qRT-PCR. qRT-PCR was performed using SuperReal PreMix Plus (SYBR Green) (Tiangen Biotech Co., Ltd., China) in CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, USA). The 10 μL reaction mixture contained 0.3 μL of forward primer (5 μM), 0.3 μL reverse primer (5 μM), 5 ng cDNA template, 5 μL 2 × SuperReal Premix Plus (SYBR Green), and 3.4 μL water. The qRT-PCR conditions were as follows: 15 min at 95°C, followed by 40 cycles of 10 s at 95°C and 20 s at 55°C, and 20 s at 72°C. The melting curves were measured from 65 to 95°C at 0.5°C increments. Relative expression levels of target gene were normalized to internal control FaActin, with CK_0h sample as the benchmark, and were calculated by 2^−ΔΔCt method (57). For each gene analyzed, a template-free negative control was included in each run, and three independent biological replicates were made for each gene in each sample.