Watermelon ClHSP70 Gene Expression Analysis

Total RNA was extracted from samples by Total RNA kit (TIANGEN, Beijing, China), and reverse-transcribed by One-Step gDNA Removal and cDNA Synthesis Super Mix kit (TransGen, Beijing, China) according to the manufacturer’s instructions. The final cDNA was used as the template for subsequent quantitative real-time PCR (qRT-PCR).
Specific primers for detection of ClHSP70 gene expression (Supplementary Table S3) were designed using Primer 5 software. The yellow-leaf-specific proein8 (ClYLS8) and β-actin (ClACT) genes from watermelon were used as the reference genes (Kong et al., 2014 (link)). qRT-PCR was conducted using ChamQ universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with a 20 μL system on the qTOWER³ Series-Real-Time Thermal Cyclers (Analytik Jena, Thüringen, Germany). The experimental procedure of qRT-PCR carried out in accordance with the instruction manual of SYBR qPCR Master Mix, including an initial activation of 95°C for 30 s, then 40 cycles of 95°C for 10 s, 60°C for 30 s. The 2^−△△CT method was used to calculate the relative transcript levels of ClHSP70s (Schmittgen and Livek, 2008 (link)).