Validating Transcriptome Data through qRT-PCR

To verify the transcriptome results, 4 genes for the culm neck were randomly selected, and gene-specific primers were designed using Primer Premier 5 software (http://www.premierbiosoft.com/ (accessed on 24 September 2023)); their sequences are listed in Table S9. Total RNA was extracted from the culm necks at three different developmental stages (CNS3, CNS4, and CNS5), as mentioned above. The first strand of cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Transgene). Quantitative real-time PCR (qRT-PCR) was performed on an ABI StepOne Plus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using the ChamQTM Universal SYBR quantitative real-time polymerase chain reaction Master Mix Kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions. To ensure reliable normalization, the tonoplast intrinsic protein 41 (TIP41) gene was used as an internal control [75 (link)]. The relative abundance of each gene was calculated from the 2^ΔCq values between the target gene and the reference gene, with three replicates for each gene [76 (link)].