RNA extraction and Quantitative real time PCR
RNA extraction was performed on 3 replicates of 30 seeds each that were ground in liquid nitrogen using mortar and pestle. Total RNA was extracted using the NucleoSpin RNA Plant Kit (Macherey Nagel) according to the manufacturer instruction. cDNA synthesis was carried out with the iScript Ready-to-Use cDNA Supermix (Bio-Rad Laboratories, Inc) according to the manufacturer instructions. RT- qPCR was performed with SsoAdvanced ™ Universal SYBR® Green Supermix (Bio-Rad Laboratories). Quantification of transcript levels were performed on a CFX96 Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA) with primers shown in Table S1. Reference genes were MtTCTP and ACTIN11commonly used in the laboratory for M. truncatula qRT-PCR analysis (Zinsmeister et al., 2016). The relative expression (RE) was normalized with the geometric mean of the 2 reference genes previously mentioned and was calculated according to the following formula:RE= 2^ΔCt (where ΔCt = geometric mean of the Ct reference genes - Ct target gene) with Ct is the value of the detection cycle of the transcript. Each point represents the average of three independent biological replicates of 30 seeds each.