Parental lines FUKU and MUT were grown in a greenhouse (February to June 2018) under a long-day photoperiod (14.5 h/9.5 h) until the end of March. Developing seed was collected randomly from several plants at 40, 55, and 70 days after flowering (DAF). Total RNA was isolated from developing seed (50–100 mg) with a Total RNA Extraction Kit Mini Plant (SciTrove, Tokyo, Japan) with additional rDNase I (Takara Bio Inc., Shiga, Japan) treatment. cDNA was synthesized from 1 μg total RNA with ReverTra Ace (Toyobo, Osaka, Japan). A 5-μL aliquot (approx. 25 ng) of cDNA was used as a template. Quantitative real-time PCR was conducted as follows in a LightCycler 96 system: 95°C for 5 min for activation of enzyme, 95°C for 15 s, 60°C for 15 s, 72°C for 20 s, for a total of 40 amplification cycles. EvaGreen Dye (20× in water; Biotium, Inc., Fremont, CA, USA) was used as the fluorescent dye, and dNTPs (Takara), homemade recombinant Taq polymerase, the PCR buffer mentioned in a previous study (Yamagata et al. 2018 (link)), and 1 pmol of each gene-specific primer were used to evaluate transcript levels of target genes. Expression relative to GmACT2/7 (Glyma.19G147900), the internal reference gene, was calculated by using the 2-ΔΔCt method (Schmittgen and Livak 2008 (link)). Data from four technical replicates were analyzed. The primers used are listed in Supplemental Table 1.