RNA isolation and reverse transcription quantitative PCR (RT-qPCR) analysis

RNA extraction was performed on 2 replicates of 5 seeds of G. max and G. soja each at 5 different time intervals, i.e., 0, 12, 24, 48 and 72 h of imbibition using RaFlex™ total RNA Isolation kit (Merck Millipore) as per manufacturer’s protocol. RNA quantity and purity was ascertained using NANODROP 2000 (Thermo Scientific) and formamide denaturing gel electrophoresis.For RT-qPCR, 1 μg of total RNA was reverse-transcribed using RevertAid First strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer’s instructions. First strand cDNA was further diluted to 1:10 ratio for real-time expression studies performed on Roche Light Cycler 480 using SyBr GreenER qPCR Supermix (Invitrogen). Gene expressions from each cDNA sample were normalized to the endogenous reference gene i.e., 18SrRNA. Forward and reverse primers used for RT-qPCR analysis are listed in Additional file 14: Table S3. Relative expression levels were calculated using the comparative 2Δ(Ct) method [71] and standard errors were calculated.