Quantitative Real-Time PCR (qRT-PCR)

To validate the RNA-seq gene expression patterns, 14 DEGs were selected and investigated by qRT-PCR using the same RNA samples for the RNA-seq library construction. We used the housekeeping gene GmUKN1 (Glyma.12G020500) as an internal control to normalize the level of gene expression (Hu et al., 2009; Guan et al., 2014). Primer pairs were designed for qRT-PCR using the online website NCBI primer-BLAST³. Primer sequences and gene annotations are listed in Supplementary Table S1. First-strand cDNA was synthesized from 500 ng/10 μl total RNA by using a PrimeScript RT Reagent Kit (TaKaRa, Japan). qRT-PCRs were performed in a LightCycler^® 480 II (Roche, Germany) in a final volume of 25 μl containing 12.5 μl SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Japan), 2 μl (200 ng) of cDNA, 1 μl (10 mM) of the forward and reverse primers, and 8.5 μl of ddH₂O. PCR conditions were set at 95°C pre-denaturation for 3 min and followed by 40 cycles of 95°C denaturation for 15 s, 60°C annealing for 15 s, and 72°C extension for 15 s. Relative gene expression level was calculated using the 2^-ΔΔCT method (Livak and Schmittgen, 2001). All qRT-PCR were performed in three technical replicates. The correlation of RNA-seq data with qRT-PCR analysis was calculated using SAS (v9.3) proc corr based on log₂ fold changes.