Real Time Quantitative Polymerase Chain Reaction

The PrimeScript RT Reagent Kit (Vazyme, Nanjing, China) was employed to extract RNA from seeds and prepare cDNA through reverse transcription. The CFX96 Touch Real-Time PCR system (Biorad) was utilized for RT-qPCR procedures by using the ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). Supplementary Table 2 presents all the primers utilized in this study, with GmTubulin being the internal control. The fold change of expression (FC) was determined with the formula of FC = EΔCt, where E stands for the average gene amplification efficiency, ΔCt indicates the difference in average Ct values for all biological replicates between two compared samples. The normalized results were displayed in a form of mean SD. The RT-PCR assays were performed with four independent biological replicates and three technical replicates.