First-strand cDNA synthesis and Real-time quantitative PCR
The expression of a set of ABA, stress, hormone and root-development related genes previously selected (Ferrández-Ayela et al. 2016; Martínez-Andújar et al. 2020b) was analysed in roots by real-time quantitative PCR (RT-qPCR). First strand cDNA was synthesised with one µg of purified RNA using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Hercules, CA, USA). The resulting cDNA was diluted by adding 40 µL of sterile distilled water.
Primers were designed to amplify 79 to 143 bp of the cDNA sequences as described previously (Ferrández-Ayela et al., 2016). To avoid amplifying genomic DNA, forward and reverse primers were designed to hybridize across consecutive exons, except in the case of SlNCED1 gene . RT-qPCR reactions were prepared with 5 μL of the SsoAdvanced SYBR Green Supermix (Bio-Rad, USA), 1 μM of specific primer pairs, 0.8 μL of cDNA and DNase-free water (up to 10 μL of total volume reaction). PCR amplifications were carried out in 96-well optical reaction plates on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Three biological and two technical replicates were performed per genotype and treatment. The thermal cycling program started with a step of 30 s at 95°C, followed by 40 cycles (5 s at 95°C, 10 s at 55°C and 20 s at 72°C), and a melt curve (from 65°C to 95°C, with increments of 1°C every 5 s). Dissociation kinetic analyses and agarose gel loading and sequencing of the PCR product was used to confirm its specificity.
Primer pair validation and relative quantification of gene expression levels were performed using the comparative Ct method (Schmittgen & Livak 2008). Data were represented as the relative gene expression normalized to the Ct value for the tomato housekeeping geneSlACTIN2 (Solyc04g011500) as previously described (Ferrández-Ayela et al., 2016). In each gene, mean fold-change values relative to the expression levels of WT were used for graphic representation. ΔCt values were analyzed using SPSS 21.0.0 (SPSS Inc., USA) by applying the Mann-Whitney U test for determining statistical differences between samples (P-value ≤ 0.05).