Rotor gene 3000 real time system

Leave samples used for qRT-PCR were the same as those used for sequencing. Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi accessed on 29 December 2021) was used to design the primer sequences with the expected product size of 80–200 bp. The primers are listed in the Supplemental Table S5. Total RNA was extracted from the leaves by using Tranzol (TransGen Biotech, Beijing, China). The HiScript II One Step RT-PCR Kit (Vazyme Biotech, Beijing, China) was used to perform reverse transcription with 2μg of RNA according to the manufacturer’s instructions. The qRT-PCR was carried out using SYBR^® Green PCR Master Mix (Roche, CH, Switzerland) in a Rotor-Gene 3000 Real Time system (Qiagen, Hilden, Germany). qRT-PCR was performed on an ABI 75000 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA) with the following amplification program: 40 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. The melting curve was recorded after 40 cycles to verify the primer specificity. The relative quantization of gene expressions was calculated using the 2^−△△CT method [42 (link)]. Three biological replicates were established for each treatment, with six plants per replicate.

In order to validate the reliability of DEGs obtained from RNA-seq, four DEGs were randomly selected from leaves and roots, respectively, for qRT-PCR analysis. RLI (Ta2776) was used as an internal control. Gene-specific primer pairs were designed using Primer Premier 5.0 software (Premier Biosoft International), and primer information is shown in Table S6. Total RNA from each tissue was extracted as described above. Two micrograms RNA was reverse-transcribed into cDNA using the iScriptTM advanced cDNA Synthesis Kit (Promega, Madison, WI, USA) following RNase-free DNase I (Promega, Madison, WI, USA) treatment. Standard curve for each gene was prepared with several dilutions of cDNA. The qRT-PCR was carried out using SYBR^® Green PCR Master Mix (Roche, CH) in a Rotor-Gene 3000 Real Time system (Qiagen, Hilden, Germany). Quantitative PCR reactions cycling conditions were performed as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s. The relative expression value of the different genes was calculated using 2^−△△Ct method [52 ]. The experiment was performed using three biological replicates.