Rnaprep pure plant kit

Various floral organs including lemmas, paleae, stamens and pistils of NIP and Oat-like rice, and grains of NIP, Oat-like rice, OsMADS1^Olr-overexpression and OsMADS1-RNAi plants were collected from field-grown rice plants and subsequently stored at − 80 °C after freezing in liquid nitrogen. Total RNA was extracted from all samples using the RNA prep pure Plant kit (TransGen Biotech, Beijing, China). First-strand cDNAs were synthesized from 2 μg total RNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China). The qRT-PCR was performed using SYBR Green Mix (CWBIO, Beijing, China) and run on a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc. USA). The OsActin gene in rice was used as an endogenous control. The primers used in the qRT-PCR analysis are listed in Additional file 1: Table S6. To differentiate between the mutated OsMADS1^Olr and wild-type OsMADS1 transcripts by a combined RT-PCR amplification and sequencing method, cDNA fragments spanning the mutation site in OsMADS1^Olr or the corresponding wild-type site in OsMADS1 were amplified respectively from NIP, OE-2 and OE-10 plants with a designed OsMADS1-RT primer pair (Additional file 1: Table S5), and sequenced subsequently.

A heatmap was created using TBtools to exhibit the expression patterns of PtrDBBs. RNAseq data of PtrDBB genes in 14 tissues were collected according to Rodgers-Melnick et al. [51 (link)]. The NCBI Primer-BLAST tool was used to design the primers of the 12 PtrDBB genes, which could amplify the 100–200 bp PCR products (Additional file: Table S4). Total RNA was isolated from the samples of each tissue using an RNAprep Pure Plant Kit (TransGen Biotech, Beijing, China) according to the user manual. Total RNAs were used for complementary cDNA synthesis using SuperScript III transcriptase (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. qRT-PCR analysis was performed on a Bio-Rad CFX96 using the Light Cycler 480 SYBR Green Master Mix (TaKaRa, Dalian, China). The PCR reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The quantitative RT-PCR data were analyzed using the 2^−ΔΔCt method. The mean expression values and SE values were calculated from the results of three independent experiments.

Total RNA of all samples was extracted using RNAprep Pure Plant Kit (Transgen), and then reverse-transcribed into cDNA. The cDNA was used as template for expression analysis. qRT–PCR were performed using the Roche LightCycler ®480 system (Roche, Germany). The wheat gene 18SrRNA was used as an endogenous control. The gene expression profile were calculated using the 2^–ΔΔCT method. All qRT–PCR primers are listed in Table S14.
The bayesian phylogenetic tree was constructed by the amino acid sequences of the PK_Tyr_Ser-Thr domain using MrBayes v3.2.7. (A) Subfamilies I-IV. Detailed information is provided in Supplementary Fig. S1a. (B) Expanded subfamily II and other compressed subfamilies. Our two studied TKL_CTR1-DRK-2 sequences (TraesCS4D02G010200.1 and KQJ91573) in T. aestivum and B. distachyon were circled by red boxes.