RNA Isolation, Quantification, and cDNA Synthesis

For gene expression quantification, leaves of the parental lines and each F₅ RIL individual were inoculated with U. pisi Up-CO-01 under growth chamber conditions. One to three biological replicates of 15-days-old L. cicera seedlings were inoculated using the same procedure described previously. Inoculated leaves were collected at 37 h after inoculation (hai) to be consistent with the time-point used in the already mentioned L. cicera RIL parental lines rust response transcriptomic studies (Santos et al., 2018). This time-point corresponds to the infection stage between fungus growth prior to stoma penetration and the early stages of infection, till colony development and if applicable, the presence of host cell necrosis observed at a microscopic level (Vaz Patto and Rubiales, 2009). Collected leaves were immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNA was extracted from about 100 mg of inoculated leaves using the GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s instructions. The extracted RNA was treated with Turbo DNase I kit (Ambion, Austin, TX, United States), according to the manufacturer’s instructions. RNA concentrations were measured by a Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, United States) using a Qubit dsRNA BR Assay kit. The RNA purity was checked by measuring the ratios of absorbance at 260/280 nm and 230/280 nm using a NanoDrop device (Thermo Scientific, Passau, Germany).

The cDNA was synthesised from 1.5 μg of total RNA from each sample following the manufacturer’s instructions from the iScript cDNA Synthesis Kit (Biorad, Hercules, CA, United States).