RNA extraction and quantitative RT-PCR analysis

Total RNA was extracted from soybean tissues according to Wang and Vodkin⁶⁴. The RNA samples were quantified using a NanoDrop spectrophotometer (Thermo Scientific, USA), and their integrity was checked. Total RNA (1 μg) from each sample was used for cDNA synthesis using the QuantiTect® Reverse Transcription Kit (Qiagen, USA). For quantitative RT-PCR, SsoFast^TM EvaGreen® Supermix (Bio-Rad, USA) was used with the CFX96 real-time PCR detection system (Bio-Rad, USA). Quantitative analysis of GmCYP1 and isoflavonoid biosynthetic gene expression was carried out using the primers listed in Table S2. The amplicons were cloned into a pGEM-T Easy vector (Promega, USA), and its sequence verified. SOYBEAN UBIQUITIN-3 (SUBI-3) or CON4 was used as a reference gene for data normalization and to calculate the relative mRNA levels. The data were analyzed using CFX manager (Bio-Rad, USA).