4.7. Gene Expression Using Quantitative Real-Time PCR

According to the QTL mapping analysis, the gene related to PHS resistance was located in 564.6–573.6 Mb on chromosome 3D based on the IWGSC_RefSeq_v1.0 wheat genome. Combined with the transcriptome results, 12 DEGs were screened in the same interval. The high expression of TraesCS3D01G466100 was positively correlated with PHS resistance, and it was selected for the next experiment.

Real-time PCR was carried out to determine the expression levels of genes. The qRT-PCR was conducted on an ABI LightCyler QuantStudio 6 instrument, using the HiScript Reverse Transcriptase (RNase H) and 5 × HiScript Buffer (Vazyme, Nanjing, China). Each reaction was performed in a 20 µL mixture containing 10 µL of SYBR Green Master Mix; three biological replications were performed. The actin gene served as the internal control, and the specific primers were designed in Primer Premier 5.0 (Table 4). Data are presented here as relative transcript levels and calculated using the 2^−ΔΔCt method [79].

Primers used for the qRT-PCR and cDNA cloning.