Transstart top green qpcr supermix 2

The samples TPs, STs and YSs of Guomai 301 were prepared at 5 time points for real-time PCR. The intervals of the TP samples were seven days from 30 December 2016 to 27 January 2017. The intervals of the ST and YS samples were seven days from 1th March 2017 to 29 March 2017. Among them, the samples prepared at the first time point were used for RNA-seq. All primers were designed using Primer Premier 5.0 (http://www.premierbiosoft.com/primerdesign/index.html). The information of primers was listed in Table S1. Reverse transcription was performed using TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech, Beijing, China). Real time - quantitative reverse transcription PCR (qRT-PCR) was performed using TransStart^® Top Green Qpcr SuperMix (2×) (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol on the CFX Connect^TM Real-Time System (Bio-Rad, Hercules, CA, USA). The ingredients of the reaction system were strictly carried out according to the instructions. The wheat actin gene was used as an internal control gene. The qRT-PCR reactions were performed in 20 µL volumes [6 (link)]. The gene expression levels were calculated according to the 2^−ΔΔCT method [71 (link)].

The six samples of Shengnong 1 and NWMS1 at the S1, S2, and S3 stages were prepared for real-time PCR. The experimental samples were consistent with the samples of RNA-seq. All primers were designed using Primer Premier 5.0 software (www.premierbiosoft.com/primerdesign/index.html). The information of primers is listed in Table S6. Reverse transcription was performed using TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech, Beijing, China). Real time qRT-PCR was performed using TransStart^® Top Green Qpcr SuperMix (2×) (TransGen Biotech, Beijing, China), according to the manufacturer’s protocol on CFX ConnectTM Real-Time System (Bio-Rad, Hercules, CA, USA). The ingredients of the reaction system were strictly carried out according to the specified instructions. The reaction of quantitative RT-PCR and semi-quantitative RT-PCR were performed in 20 µL volumes [88 (link)]. The gene expression levels were calculated according to the 2^−∆∆Ct method [95 (link)]. The wheat actin gene was used as an internal control.

The tiller primordia and leaf samples of the mutant dmc and guomai 301 were prepared at 10 time points for real-time qRT-PCR; the intervals of the 10 time points were seven days from 23 December 2016 to 24 February 2017. Twelve DEGs were selected to test and verify the sequencing data by qRT-PCR. The primers were designed using Primer Premier 5.0 (Table S13). The wheat actin gene was used as an internal control gene. The qRT-PCR reactions were performed in 20 µL volumes containing 10 µL TransStart^® Top Green Qpcr SuperMix (2×) (TransGen Biotech, Beijing, China), 2 µL primer mix (10 µM), 1 µL cDNA (50 ng) and 7 µL ddH₂O. The PCR parameters were: 94 °C for 30 s, then 42 cycles of 94 °C for 5 s, 60 °C for 30 s. All qRT-PCR reactions were replicated three times. The gene expression levels were calculated according to the 2^−ΔΔCt method [85 (link)].