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.