Gene expression analysis
Harvested nodules and roots were frozen in liquid nitrogen and ground.
Total RNA extraction was performed using the RNAzol ® RT reagent
following the manufacturer’s recommendations (Sigma-Aldrich, USA). After
the DNAse treatment with RQ1 DNAse (Promega, USA), 1 µg or total RNA was
used for the cDNA synthesis (GoScript ™ Reverse Transcription System,
Promega). Quantitative real-time PCRs were performed in the Agilent
AriaMx System using the GoTaq® qPCR Master Mix (Promega, USA). Two
reference genes (MtC27 and a38) were used to normalize the transcript
level. The complete list of primers is reported in Table S1. Data were
quantified using the Aria Software and analyzed with RqPCRBase, an R
package working on R computing environment for analysis of quantitative
real-time PCR data (Rancurel et al., 2019).