Validating Gene Expression via qRT-PCR

For the verification of gene expression, cDNA were performed for qRT-PCR with a 10 µL reaction mixture containing 1 µL of cDNA, 1 µL of each reverse and forward primer, 3 µL of RNAse free water and 5 µL of SYBR green mixture (SYBR green, Toyobo, New York, NY, USA). The cDNA was synthesized using the HiScript II Q Select RT Supermix for qPCR (+gDNA wiper) cDNA synthesis kit according to the manufacturer instructions (Vazyme Biotech, Nanjing, China). qRT-PCR was performed by using a Real time PCR cycler (Eppendorf AG 22331, Hamburg, Germany) with the thermal cycling conditions as follows: 50 ℃ for 2 min, 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 15 s, and 60 ℃ for 60 s. The cycle threshold (Ct) values were automatically calculated. The relative gene expression level was quantified using the 2^−ΔΔCt method and housekeeping gene EF1-α of F. graminearum was used as the reference [34 (link)]. All primers were designed using the online NCBI primers tool software (https://www.ncbi.nlm.nih.gov/tools/primer-blast, accessed on 20 May 2021) and listed in Table S1.