qRT-PCR

Real time qRT-PCR was carried out as described in previous study (Li et al., 2019). Since the homoeoalleles of most tri-genes exhibited similar expression levels (Pfeifer et al., 2014), we used universal primers to analyze the expressions of TaARF homoeoallele genes. A total of 20 pairs of primers were designed based on the consensus sequences of homoeoalleles for every wheat ARF member, and the primers were listed in Table S1. The β-actin gene was used as an internal control and each reaction was performed with three biological replicates. The relative expressions of TaARFs were calculated by 2^−ΔΔCT methods (Livak & Schmittgen, 2001).