Quantitative Real-Time PCR Analysis of Gene Expression

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.