Mgcl2

Multiplex PCR primers 3CON, 3NA, 3D3A and 3D15A [9 (link)] were employed to determine trichothecene chemotype of the F. graminearum isolates. This multiplex assay generates an 840 bp fragment for the NIV chemotype, 610 bp fragment for the 15ADON chemotype and 243 bp for the 3ADON chemotype. The PCR amplification reaction (25 µL) consisted of 20 ng template DNA, 2.5 µL 10× PCR buffer containing MgCl₂ (FroggaBio, Concord, Canada), 1 μL of dNTP (2.5 mM each), 0.25 μL of each primer (10 mM) and 0.5 U Taq DNA polymerase (FroggaBio, Concord, Canada). The PCR thermal cycling conditions consisted of an initial denaturation at 94 °C for 4 min; followed by 35 cycles of 1 min at 94 °C, 40 s at 58 °C, 40 s at 72 °C; and a final extension of 72 °C for 6 min. PCR amplicons were separated on a 1.5 % agarose gel in 1× TAE buffer stained with RedSafe (FroggaBio, Concord, Canada) and sizes were estimated with a 100 bp DNA ladder.

Genomic DNA was extracted according to Fernando et al. (2006). Fusarium graminearum isolates were confirmed using molecular markers (Fg16F and Fg16R) specific to F. graminearum [55 (link)], which produces an amplification of 450 bp. The PCR amplification reaction (25 µL) consisted of 20 ng template DNA, 2.5 µL 10× PCR buffer containing MgCl₂ (FroggaBio, Concord, ON, Canada), 1 μL of dNTP (2.5 mM each), 0.25 μL of each primer (10 mM) and 0.5 U Taq DNA polymerase (FroggaBio, Concord, ON, Canada). Thermal cycling conditions consisted of an initial denaturation at 95 °C for 3 min; followed by 35 cycles of 30 s at 95 °C, 1 min at 56.7 °C, 1 min at 72 °C; and a final extension of 72 °C for 5 min. PCR products were run on a 1% agarose gel.