Real-time quantitative PCR assay
Plants total RNA was isolated by Trizol (Nakayama et al. 2017) and cDNA from mRNA and miRNA synthese were performed by HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and Mir-X miRNA First-Strand Synthesis Kit (Takara, USA). qRT-PCR analyses were performed by using the ChamQTM Universal SYBR qPCR Master MIX (Vazyme). The qPCR primers of wheat and Arabidopsis thaliana genes were listed in Table S1. Each reaction was performed in a final volume of 20 μl in a Stratagene MX3000PTM System (Genetimes, Shanghai, China). The program referred to instruction of qPCR reagent and data were quantified using the comparative 2-ΔΔCTmethod after the PCR program. All the gene expression experiments were three biological and three technical repeats. Subsequently, the mean of the technical replicates for each biological replicate were calculated, which values were used for statistical analysis.