Transcriptome Analysis of Rice Tissues

The collected samples were immediately frozen in liquid nitrogen, and then stored at −80°C before use. Total RNAs of the various tissues were isolated with the TRIzol reagent (Invitrogen). After the DNase treatment, about 5 μg of RNAs were used for the cDNAs synthesis that utilized the M-MLV reverse transcriptase (Promega) in a 50-μl reaction mixture. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed with the 2 × SYBR Green Master Mix reagent (Bio-Rad) in a 96-well plate of the Bio-Rad CFX96 real time PCR system. Eight rice reference genes: TI (LOC_Os01g05490), ARF (LOC_Os05g41060), EF-1α (LOC_Os03g08020), UBC (LOC_Os02g42314), Profilin-2 (LOC_Os06g05880), Edf (LOC_Os08g27850), PtfS (LOC_Os07g34589), and Actin1 (LOC_Os03g50885), were used to find the stable internal standards by geNorm as described (Vandesompele et al., 2002 (link); Wang et al., 2016 (link)). The selected internal standards for the different experimental conditions were showed in Figure S1. All primers used for qRT-PCR analysis are listed in Table S3, with good PCR efficiencies (80–100%) detected by using ten-times diluted gradients of the total cDNAs.