Total RNA isolation, reverse transcription, and gene expression analysis
Total RNA was isolated from frozen plant samples using the TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China) according to the manufacturer’s instructions. RNA purity and concentration were determined using an Epoch Microplate spectrophotometer (BioTek, Winooski, VT, USA), whereas the integrity of the RNA was evaluated by 1% agarose gel electrophoresis. Following the manufacturer’s recommendations, TaKaRa PrimerScript ™ RT Master Mix (Takara, Dalian, China) was used for reverse transcription and total RNA (1 μg) was used for cDNA synthesis. qRT-PCR was performed using a Quantstudio 6 Flex real-time PCR system (Thermo Fisher, Carlsbad, California, California, USA) and 2x FAST qPCR Master Mixture (with OOX II) (DiNing, Dalian, China). Every qRT-PCR sample contained 1 μl of cDNA, including those for antioxidant related genes, osmoprotectants related genes and symbiosis-related genes (Table S1), 10 μl of EvaGreen 2× qPCR MasterMix (DiNing, Dalian, China), 2 μl of primer and 7 μl of sterilized water. The qRT-PCR cycle parameters were as follows: 10 min at 95°C, and then 40 cycles of 30 s at 95°C and 1 min at 60°C. Three independent replicates were performed for each sample. The Gmactin andGm60s were used as endogenous Control genes were expressed as 2−ΔΔCt using the comparative threshold cycle (Ct) method (Livak & Schmittgen, 2001).