Wheat Gene and miRNA Expression Analysis

The total RNA of all samples was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufactures’ instructions, and approximately 1 µg RNA was reverse transcribed with PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Shiga, Japan) and miRcute Plus miRNA cDNA T (Tingen, Beijing, China) for genes and miRNAs, respectively, according to the manufacturer protocol. qRT-RCR assays for TaGH9 and miRNAs were conducted on an Eco Real-time PCR system (Illumina, CA, USA) using TB Green Premix Ex Taq (Tli RNaseH Plus) kit (TaKaRa, Shiga, Japan) and SYBR^® miRcute Plus miRNA PreMix (Tingen, Beijing, China), respectively, as described previously [28 (link),75 (link)]. Wheat 18S gene served as the reference gene for TaGH9 analysis, and U6 was used as a reference gene for miRNA analysis. The relative expression levels were calculated according to the 2^–ΔΔCt method for relative expression with three biological replicates and three technical replicates [76 (link)]. The primers used in this study were designed using the Primer premier 5.0 (Primer, CAN, UK) program and are listed in Table S4.