Quantitative Analysis of TaPPH-7A Gene Expression

A primer pair RT-F/R (Table 3) was designed to use for analyzing the expression of TaPPH-7A in different wheat genotypes. Quantitative real-time PCR (qRT-PCR) was performed in triplicate with Roche LightCycler® 96 using the SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Japan). The qRT-PCR reaction system with specific primer contains 10 μL 2 × SYBR Premix Ex Taq™, 0.4 μL 50 × Rox Reference Dye II, 0.4 μL (5 μM) of each primer (Table 3), 1 μL cDNA template, and 7.8 μL ddH₂O. The reaction procedure was as follows: denaturation at 95 °C for 2 min; followed by 45 cycles at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. TaActin was used as the endogenous control of normalizing expression levels of different samples. Gene relative expression levels were calculated using the 2^-△△CT method [63 (link)]. The statistical analysis of ΔΔC_T according to the method described by Zhang et al. [35 (link)].