Quantitative Expression Analysis of CamTERFs in Capsicum

Total RNA was isolated from the four to six euphylla stages of capsicum 6421 using the RNAiso Plus reagent (TaKaRa, Dalian, China) and then treated with RNase-free DNase I (Promega). Subsequently, 0.5 μg RNA was used for first-strand cDNA synthesis using a HiScript II 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. qPCR was performed using LightCycler 96 (Roche, Basel, Switzerland) with the SYBR Green Premix Ex Taq™ II quantitative PCR system (TaKaRa), and the primers of eight subclasses of genes are listed in Table S5. At least three biological replicates were included. Briefly, after an initial denaturation step at 95 °C for 10 min, the amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 15 s and an annealing temperature of 60 °C for 30 s. The ΔC_t method was used to calculate the relative expression levels of CamTERFs [53 (link),54 (link)]. ΔC_t = [Gene expression − mean (Actin expression)]/3.