Watermelon Gene Expression Quantification

Gene transcript levels were analyzed using quantitative real-time PCR (qRT‒PCR) with a BIO-RAD CFX ConnectTM Optics Module (Bio-Rad, USA). cDNA was synthesized from RNA using the PrimeScript^® RT reagent kit with gDNA Eraser (TaKaRa, Japan). All gene-specific primers in this study (Table S2) synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). qRT‒PCR was performed using SYBR® Premix Ex Taq^TM (Perfect Real Time) (TaKaRa, Japan) and contained 12.5 µL 2 × SYBR Premix Ex Taq^TM, 2 µL cDNA solution, 2 µL mix solution of target gene primers and 8.5 µL ddH₂O in a final volume of 25 µL. The amplification was carried out under the following conditions: 50 °C for 2 min followed by an initial denaturation step at 95 °C for 30 s, 40 cycles at 95 °C for 5 s, 50 °C for 15 s, and 72 °C for 30 s. Relative gene expression levels of target genes were calculated by the 2^−∆∆Ct comparative threshold cycle (Ct) method ^{38 (link)}. The Ct values of the triplicate reactions were gathered using Bio-Rad CFX Manager V1.6.541.1028 software. The watermelon Actin gene was used as an internal reference and PCR analysis for each gene was performed with three biological and three technical replicates.