Cfx96 real time system machine

Total RNA was extracted from all the samples using TRIzol reagent (Invitrogen, Germany), according to the manufacturer’s instructions. The RNA extraction procedure and cDNA synthesis followed the same procedures as detailed in our recently published papers (Ali et al., 2018 (link), 2020a (link)). For qRT-PCR, specific primer sets were designed and checked for primer specificity in Arabidopsis genome (Supplementary Table 6). qRT-PCR was performed in CFX96 Real-Time System machine (Bio-RAD, Hercules, CA, United States) using SYBR Premix Ex Taq^TM II (TaKaRa). Relative expression levels were normalized by Arabidopsis ACTIN7 gene and calculated using the 2^–ΔΔCt method (Schmittgen and Livak, 2008 (link)). Data were presented as means and +SD of three replications obtained from three independent biological experiments.

Total RNA was extracted from the collected materials using TRIZOL reagent (Invitrogen, Germany) according to the manufacturer-recommended protocol. The first cDNA strand was generated using the PrimerScript RT reagent kit (Takara, Japan) according to the manufacturer's instructions. Specific primers used in the qRT-PCR were designed by Primer 5 Software, and each primer was checked against cucumber DNA database to ensure its specificity.
The qRT-PCR reactions were performed on the CFX96 Real Time System machine (Bio-RAD, USA), programmed to heat for 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 45 s at 55°C, and at the end, 1 cycle of 1 min at 95°C, 30 s at 50°C, and 30 s at 95°C. Two biological and three technical replicates for each sample were performed with 15 μL of reaction volume using the SYBR Premix Ex Taq kit (TOYOBO, Japan). The EF1a gene (accession number EF446145) of C. sativus was selected as an internal control (Wan et al., 2010 (link)). Relative gene expression was calculated using the 2−^ΔΔCt method. Heatmap was generated by Multiple Array Viewer using the relative expression data of each gene.

Quantitative real-time PCR (qPCR) was performed with arbitrarily selected genes to validate expression of randomly-selected transcripts. Primers were designed using the online Realtime PCR tool (Integrated DNA Technologies, Inc., San Diego, CA, United States¹⁰) and alfalfa sequences generated in this work. cDNA for qPCR analyses was made using the SuperScript^TM III First-Strand Synthesis System with oligo d(T) (Thermo Fisher Scientific) and the same RNA samples that were used for RNA sequencing. Amplification was conducted with a CFX96 Real-time system machine (Bio-Rad), with three biological replicates using the following parameters: 95°C for 10 min (one cycle), 95°C for 10 s and 60°C for 45 s (40 cycles). The Delta Delta C(T) method (2^-ΔΔCT) was used for analysis of relative expression (Livak and Schmittgen, 2001 (link)). The reference gene for alfalfa in all qPCR experiments was NP_001237047, a gene of unknown function with little variation in expression levels (Postnikova et al., 2013 (link)). For the qPCRs of P. penetrans transcripts, the 18S rDNA gene was used as reference (Vieira et al., 2018 (link)).

The total RNA was extracted from the collected materials using TRIZOL reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer-recommended protocol. The first cDNA strand was generated from 1 μg of total RNA using the PrimerScript RT reagent kit (Takara, Otsu, Japan) according to the manufacturer’s instructions. Specific primers used in the qRT-PCR were designed by Primer 5 Software, and each primer was searched in the tomato database to ensure its specificity. The qRT-PCR reactions were performed on the CFX96 Real Time System machine (Bio-RAD, Hercules, CA, USA), programmed to heat for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 45 s at 55 °C, and at the end, 1 cycle of 1 min at 95 °C, 30 s at 50 °C and 30 s at 95 °C. Two biological and three technical replicates for each sample were performed with 15 μL of reaction volume using the SYBR Premix Ex Taq kit (TOYOBO, Osaka, Japan). The tomato SlUbi3 gene (GenBank accession number X58253) was selected as an internal control [60 (link)]. The relative gene expression level was calculated using the 2^−ΔΔCt method. Heatmap was generated by Multiple Array Viewer using the relative expression data of each gene [58 ].

qRT-PCR was performed on a Bio-Rad CFX96 Real-time System machine using the Sofast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 5 s and 72 °C for 5 s, and a melt cycle from 65 °C to 95 °C. The primers used for real-time PCR (Table S1) were designed by Primer Premier 5.0 software. CpActin and CpTubulin were used as the internal reference for the gene expression analysis of wintersweet [47 (link)]. Bio-Rad Manager^TM Software (Version 1.1) was used to analyze real-time PCR data. There were three biological replicates for each experiment and three technical replicates for each sample.

Quantitative RT-PCR was performed as described (Tisch et al., 2011a (link)). All reactions were performed on a CFX96 Real-Time system machine (Bio-Rad) with the GoTaq qPCR Master Mix (Promega, Madison, WI, USA) and primers for cbh1 and L6e (reference gene) (Tisch et al., 2011a (link)). At least two biological replicates were analyzed with three technical replicates. Data was analyzed with qbase+ (Biogazelle) and the CFX Maestro (Bio-Rad) software (statistics including ANOVA).

For qRT-PCR, random hexamers were annealed to DNase-treated cDNA for reverse transcription using the PrimeScript^™ RT reagent Kit with a gDNA Eraser (TaKaRa, Dalian, China) at 37 °C for 30 s. Then, cDNA was diluted 1:3 with ddH₂O and stored at −20 °C. One microliter of diluted cDNA was used for qPCR in a Bio-Rad CFX96 Real Time System machine (Bio-Rad Laboratories Inc., Hercules, CA, USA). qRT-PCR was performed with 5Μ TB Green^® Premix Ex Taq^™ II (Tli RNaseH Plus) (TaKaRa) in a 10 μL total reaction volume. Forward and reverse gene-specific primers used were designed by Primer Premier 5 software (Premier, Ottawa, ON, Canada) and are listed in Supplementary Table S5. GAPDH was used as an internal control. Three technical replicates were used for each sample. The relative mRNA abundance of each tested gene was averaged for triplicate reactions, and the values were normalized according to the Ct of the internal control using the 2^−ΔΔCt method [54 (link)].