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.