Quantitative Gene Expression Analysis in Cucurbit

The seed coat samples from different developmental stages (18 and 26 DAP) and other tissue samples, including roots, stems, leaves, and male flowers were collected from both parental lines. RNA was isolated using the plant total RNA purification kit (TIANGEN, China) according to the manufacturer’s instructions and then the first-strand cDNA was synthesized using a cDNA synthesis kit (Takara, Japan).
The gene-specific primers of the candidate genes and reference gene Actin (Kong et al., 2015 (link)) for quantitative real-time PCR (qRT-PCR) were designed based on the Cucurbit Genomic Database (http://cucurbitgenomics.org), using the software Primer Premier 5. The expression levels of the candidate genes were performed using a LightCycler480 RT-PCR system (Roche, Swiss) with a Real Master Mix (SYBR Green) kit (Toyobo, Japan). Amplification was carried out in a 20 µl reaction mixture containing 1 µl cDNA, 1 µl forward and reverse primers (10 µM), 10 µl 2 × SYBR Green real-time PCR mixes, with nuclease-free water added to a total reaction of 20 µl. Three biological and technical replicates were used for qRT-PCR. Average relative expression levels for each sample were calculated. The expression level was analyzed by the 2^−△△Ct method (Livak and Schmittgen, 2001 (link)), and the primer sequences used in this study are listed in Table S3.