*4.7. Total RNA Extraction and qPCR Analysis*

To study the expression patterns of the MADS-box genes involved in flower organ identity and fruit development in tomato, total RNA was extracted from the sepals (Se), petals (Pe), stamens (St), and carpels (Ca) of tomato and different developmental stages of tomato fruits at different developmental stages, including IMG (immature green), MG (mature green), B (breaker), B+4 (4 days after breaker), and B+7 (7 days after breaker) using RNAiso Plus (Takara) in accordance with the instructions. After DNase digestion (Promega, Madison, WI, USA), cDNA was synthesized with oligo(dT)20 as a primer for RNA reverse-transcription using M-MLV reverse transcriptase (Promega, Madison, WI, USA). For gene expression quantification, qPCR analysis was performed with the CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) using the GoTaq qPCR Master Mix (Promega, Madison, WI, USA). First, 1.0 µL of mixture primers, 1.0 µL of cDNA, and 3.0 µL of ddH2O were used. NRT (no reverse transcription control) and NTC (no template control) experiments were performed to eliminate the genomic DNA and environment effects. The tomato *SlCAC* gene was used as an internal standard [96], and the 2−∆∆CT method was used to perform the relative gene expression levels analysis [97]. In addition, all the experiments were performed in three biological triplicates with three technical replicates. The standard curves were run at the same time. All the primers used were designed by Primer 5.0 software and are shown in Table S3.
