Real-Time qPCR Analysis of Defense Gene Expression

Real-time amplifications were performed using a CFX Connect Real Time Detection System (Bio-Rad) in 20 µl reactions containing Standard Taq Reaction Buffer (New England BioLabs), 0.25 mM MgCl₂, 0.2 mM of each dNTP, 2× EvaGreen (Biotium), 1 µM of gene specific primers ( Table 1 ), and 0.5 U Hot Start Taq DNA Polymerase (New England BioLabs). Thermocycles were as follows: initial denaturation at 94°C for 5 min, 40 cycles of a denaturation step at 94°C for 20 s, annealing step at 60°C for 30 s, an extension step at 68°C for 30 s, and 80°C for 30 s. The fluorescence was read at the 80°C step of each cycle. After each run, a melting curve was generated between 65° and 95°C. Ct values of defense genes were normalized to the ubiquitin gene expression, and their relative expressions in each sample were determined against gene expressions in control plants without nematodes, using the 2^–ΔΔCt method (Pfaffl, 2001).

Primer pairs used in this study.