qRT-PCR
To determine the relative transcript levels of selected genes, qRT-PCR
was performed with specific primers (Table S1) according to the
manufacturer’s instructions for the BIO-RAD Real-time PCR system (Foster
City, CA, USA) and the SYBR Premix Ex Taq II system (TaKaRa). Total RNA
preparation and real-time RT-PCR were carried out following procedures
described in our previous studies (Cai et al., 2015; F. Dang et al.,
2013; Zhang et al., 2015). Total RNA was isolated from the pepper
samples using TRIzol Reagent according to manufacturer’s protocol
(Invitrogen, Canada). The mRNA was reverse-transcribed into cDNA using
the reverse transcription system (Takara Biotechnology, Japan). Four
replicates of each treatment were performed. Data were analyzed by the
Livak method (Livak & Schmittgen, 2001) and expressed as a normalized
relative expression level (2-ΔΔCT) of the respective
genes. The relative transcript level of each sample was normalized toCaActin (GQ339766) and 18S ribosomal RNA (EF564281),
respectively.