Aphid-Induced Wheat Transcriptome Analysis

Leaves from five plants per biological replication were collected and fixed in liquid nitrogen 1 and 3 days after population with aphids. Total wheat RNA was extracted using TRIzol™ Reagent (Merck KGaA, Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s instructions. cDNA synthesis was carried out as described previously [33 (link)]. Primers for qRT-PCR were designed using a web-based primer designing tool from IDT (http://eu.idtdna.com/Scitools/Applications/Primerquest, accessed on 10 November 2022) (USA). The sequences of all the primers are presented in Table S2 (Supplementary Materials). Quantitative PCR was performed by polymerase chain reaction in real time using a set of predefined reagents EvaGreenI (Synthol, Moscow, Russia) and CFX Connect real-time PCR Detection System device (BioRad Laboratories, Hercules, CA, USA). To standardize the data, wheat gene TaRLI (RNaseLinhibitor-like) (Table S2, Supplementary Materials) was used as an internal reference for the real-time qPCR analysis. The quantification of gene expression was performed using CFX Connect real-time PCR Detection System (BioRad Laboratories, Hercules, CA, USA). In order to quantify the relative gene expression using the delta-delta Ct method was performed as described earlier [33 (link)]. Three independent biological and three technical replications were performed for each experiment.