Tb green master mix reagent

To validate the RNA-seq results, water-stressed tomato leaf samples from each sampling time point were subjected to qRT-PCR analysis. Total RNA was provided by Gene Denovo Biological Technology Co., Ltd. (Guangzhou, China). The cDNA was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, China), following the protocol of the manufacturer. Gene-specific qRT-PCR primers were designed using Primer-BLASÉ in National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) (accessed on 10 January 2019) for 13 selected genes. qRT-PCR was performed using a LightCycler-480 RT-PCR system (Roche, Basel, Switzerland). Each reaction mixture contained 5 µL 2 × TB Green Master Mix Reagent (Takara, China), 1 µL cDNA sample, and 100 nM gene-specific primer in a final volume of 10 µL. PCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles of heating at 95 °C for 5 s and annealing at 60 °C for 30 s. A template-free control for each primer pair was set for each cycle. All PCR reactions were normalized using the Ct value corresponding to the tomato UBI gene. Measurements of three biological and three technical replicates were used.

The total RNA of tomato leaves was extracted by using a E.Z.N.A. Plant RNA extraction Kit (Omega Bio-tek, Inc., GA, USA), which includes a genomic DNA elimination step. Total RNA from stem samples was provided by Gene Denovo Biological Technology Co., Ltd. (Guangzhou, China). The cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Guangzhou, China), according to the manufacturer’s instructions. We selected 15 DEGs from RNA-seq data for RT-qPCR analysis to verify the results of RNA-seq. RT-qPCR was performed in a 10 μL reaction volume containing 5 μL of 2 × TB Green Master Mix Reagent (Takara, Guangzhou, China), 1 μL of cDNAs and 4 μL of gene-specific primers (Table S5), which were designed using Primer-BLAST in National Center for Biotechnology Information (NCBI). The expression levels of housekeeping gene SlUBI was used as reference for calculating the relative expression of target gene using the 2^−∆∆Ct method [21 (link)]. RT-qPCR analysis was based on three biological replications and three technical replications.