Salt tolerance response in root of citrus rootstocks

Roots from Citrus reshni × Citrus trifoliata hybrids (107 and 90) were collected from the NG experiment as described by Asins et al. (2023) after one year of salinity treatment. Six out of twelve repetitions were randomly selected to establish control and salinity treatment (15 mM NaCl) in a greenhouse under natural light conditions into pots (4 L) using vermiculite as a substrate. Two full sibs from the Citrus reshni × Citrus trifoliata progeny differing in salt tolerance and the C. reshni allele at LCl-6 QTL were selected: 107 (salt tolerant) and 90 (salt sensitive). The nutrient solution (electrical conductivity: 1.68, pH: 7.13) contained the following concentration of macronutrients (in mM): NO3- 8.45; H2PO4- 0.74; SO42- 1.84; K+ 6.08; Ca2+ 4.25; Mg2+ 1.33; plus the following concentration of micronutrients (in µM): Fe2+ 66, Mn2+ 1.33; Zn2+ 2.3; B 17, Cu2+ 2.3, supplemented with either 0 for control (1.67 dS/m) or 15 mM NaCl for the saline treatment (4.67 dS/m) until completion of the experiment. Plants were watered three times a week with 200 ml at each irrigation event. Total RNA was extracted as described by Romero-Aranda et al. (2021) using an initial purification step with CTAB extraction buffer, followed by separation in chloroform:isoamyl alcohol (24:1, v:v) and LiCl precipitation. The resuspended RNA was further purified using the AurumTM total RNA mini kit (Bio-Rad Laboratories S.A., Madrid, Spain), together with RNAse-free DNase in-column treatment (Promega Biotech Ibérica SL) according to the manufacturer's instructions. Three replicates for each sample were sequenced in an Illumina NextSeq 500 sequencer.