Quantstudio design and analysis

Total RNA was extracted from frozen plant tissues using TRIzol RNA Isolation Reagent (Thermo Fisher Scientific). The middle part of the fifth leaf was used for analysis. The cDNA was reverse-transcribed from 2 μg of total RNA using RevertAid Reverse Transcriptase (Thermo Scientific). qPCR was performed using SYBR Green qPCR SuperMix (Thermo Fisher Scientific) on a QuantStudio™ 5 Real-Time PCR Cycler (Thermo Fisher Scientific) following the manufacturer’s instructions. The wheat TaWIN1 gene (Tenea et al., 2011 (link)) was selected as the internal control. The qPCR analyses were performed on three technical replicates and analyzed using QuantStudio TM Design and Analysis (Applied Biosystems, Thermo Fisher Scientific). The primers used for qPCR are listed in Supplementary Table S1.

Total RNA was isolated from fresh or frozen leaves of greenhouse plants of clonal rootstock 146-2 using the method described in [56 (link)]. To remove DNA residues, the RNA solution was treated with DNase I (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. The middle part of 5th leaf was used for analysis. The cDNA was reverse transcribed from 2 μg of total RNA using MMLV Reverse Transcriptase (Evrogen JSC, Moscow, Russia). RT–qPCR was performed using 5X qPCRmix-HS SYBR+LowROX (Evrogen JSC, Moscow, Russia) with a QuantStudio™ 5 Real-Time PCR Cycler (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The mitochondrial nad5 gene was selected as the internal control [57 (link)]. The qPCR analyses were performed with three technical replicates and analyzed using QuantStudio TM Design and Analysis (Applied Biosystems, Waltham, MA, USA; Thermo Fisher Scientific, Waltham, MA, USA). Primers used for qRT–PCR are listed in Supplementary Table S1.