Myiq real time system

At least 30 barley seeds were used for RNA extraction. Total RNA from germinated seeds was extracted with an RNAprep Pure Plant kit (Tiangen, Beijing, China). The first strand of cDNA was synthesized from 1.5 μg of total RNA in a 30 μl reaction volume with a FastQuant RT kit (Tiangen, Beijing, China). qRT‐PCR was performed in 96‐well blocks on a MyiQ real‐time system (Bio‐Rad, California, USA) using the 2 × SYBR Premix UrTaq II (Nobelab, Beijing, China). Three biological replicates were included for each sample. α‐Tubulin was used as an internal control to normalize expression of the target genes. The primers for qRT‐PCR were used in previous studies (Shen et al., 2020 (link); Sheng et al., 2018 (link)), and their details are shown in Supplemental Table S1.

Rice tissue samples of ZH11 for spatial expression analysis of Osl2 and GWD1 were collected from 14‐day‐old whole seedlings or the leaf, seed, root, stem and leaf sheath of mature rice plant (10 DAF). Leaf samples for temporal expression analysis of Osl2 were collected from ZH11 mature rice plants 0, 5, 10, 15 and 20 DAF respectively. Leaf samples for expression analysis of GWD1 in its transgenic rice or mutants were from homozygous plants 10 DAF (T₂ generation). Then, total RNA was extracted from the collected rice samples using the RNAsimple Total RNA Kit (Tiangen, Beijing, China), after which it was treated with DNase I (Qiagen, Hilden, Germany) to remove residual genomic DNA. 1 μg samples of total RNA were reverse‐transcribed using the SuperScript™ First‐Strand cDNA Synthesis System (Invitrogen, Carlsbad, CA). qRT‐PCR assays were then performed on a MyiQ Real‐Time system (Bio‐Rad, CA) using Actin as the reference gene for normalization of gene expression. The primer pairs used for expression analysis of Osl2 and GWD1 were Osl2qRT‐F/Osl2qRT‐R and GWD1qRT‐F/GWD1qRT‐R respectively. Each experiment consisted of three biological replicates, and the primer sequences are given in Supplementary Table S4.