Ssofast kit

The heatmaps and dendograms were generated using R‐statistical software with pheatmap 1.0.12 from CRAN (https://CRAN.R‐project.org/package=pheatmap) with gene expression data obtained from Phytomine (https://phytozome.jgi.doe.gov/phytomine). Quantitative real‐time PCR was performed with total RNA extracted from 100 mg of S. italica plant material used in stress assays (see above) using the Plant RNA Purification Kit (Qiagen, https://www.qiagen.com/). Equal amounts of RNA were used for cDNA synthesis with the iScript cDNA Synthesis Kit (Bio‐Rad, https://www.bio‐rad.com/) and oligo(dT) primers. The subsequent qPCR reaction was performed on a Bio‐Rad CFX96 real‐time system using the SsoFast kit and target‐specific oligonucleotides (Table S4). The log fold‐change in gene expression was calculated using the ΔΔC_t method with S. italica elongation factor 1α (SiEF1α) (XM_004984777) (Kumar et al., 2013) as the reference gene and triplicate measurements with three biological replicates. Target specificity was confirmed by sequence verification of representative amplicons. Statistical significance for gene expression was analyzed using the Welch two‐sample t‐test (P‐value < 0.05).

Total RNA was isolated from needles of plants grown for six months using PureLink® Plant RNA Reagent (ThermoFisher, Waltham, MA, USA) using approximately 100 mg tissue according to manufacturer’s instructions. RNA integrity and concentration was measured using Bioanalyzer 2100 RNA Nano chip assays (Agilent, Santa Clara, CA, USA) following the manufacturer’s protocol. Equal RNA amounts were used for cDNA synthesis with the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA). qRT-PCR reactions were performed on a Bio-Rad CFX96 Real-time system using the SsoFast kit (Bio-Rad, Hercules, CA, USA) in triplicate. Relative transcript abundance was calculated using efficiency corrected ΔC_T and ΔΔC_T values based on ELF-1α as the reference gene. Target-specific oligonucleotides were as follows: ELF-1α-f—5′-CCCTTCCTCACTCCAACTGCATA; ELF-1α-r—5′-TCGGCGGTGGCAGAGTTTACATTA; or PgβGLU1-f—5′-TTGGATCCTCTGAAGGT GT; PgβGLU1-r—5′-TCCCTCCCTTATGGCTTC. Target specificity was confirmed by sequence verification of representative amplicons.

Total RNA was isolated as previously described using approximately 100 mg tissue [75 (link)]. RNA integrity and concentration was measured using Bioanalyzer 2100 RNA Nano chip assays (Agilent) following the manufacturer’s protocol. Equal RNA amounts were used for cDNA synthesis with the Superscript III reverse transcriptase (ThermoFisher) and oligo(dT) primers. The subsequent qPCR reaction was performed on a Bio-Rad CFX96 Real-time system using the SsoFast kit (www.bio-rad.com) and target-specific oligonucleotides (Additional file 9: Table S2). Relative transcript abundance was calculated using efficiency corrected ΔCT using the amplification efficiency values (E-values) generated from primer efficiency calculations for each gene pair. Relative gene expression values were calculated based on M. vulgare Elongation Factor 1α as reference gene and triplicate measurements with three biological replicates. Target specificity was confirmed by sequence verification of representative amplicons.