Abi bigdye terminator version 3.1 cycle sequencing kit

Eight commonly used housekeeping genes were chosen as candidates (Table 1). Primers for qRT-PCR were designed based on the sequences extracted from Online Resource for Community of Annotation of Eukaryotes (http://bioinformatics.psb.ugent.be/orcae/, accessed on June 28^th 2016). The cDNA from the susceptible strain (feeding on lima beans) was used as a template for PCR that was performed in a Peltier-Effect thermal cycler (MJ Research, Inc., Canada). PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Pittsburgh, PA) under the following cycling parameters: 94°C for 3 min 50 s, 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with final extension for 10 min at 72°C. PCR products were purified with DNA Clean & Concentrator (Zymo Research, Irvine, CA) following the manufacturer's protocol. Each individual PCR product was sequenced using ABI BigDye Terminator Version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) on an ABI 3730 at the Molecular Biology and Genomics Core at Washington State University. The obtained sequences were analyzed with BioEdit 7.0.1 software (Ibis Biosciences, Carlsbad, CA) to confirm sequence identities.