Optimization of DNA Isolation Protocols and Fungicide Resistance Gene Primers across Erysiphe and Podosphaera Species

Science Center, Bent Corridor

Powdery mildew is a fungal plant pathogen that costs grape growers millions of dollars in fungicides every year in the United States. Widespread use of these chemicals has caused fungicide resistance to develop in some powdery mildews. Amplicon Sequencing (AmpSeq) is a high-throughput technology that is used to study these fungicide resistance genes through the development of molecular markers for each target trait. The purpose of this study was to improve AmpSeq protocols by identifying the best DNA isolation protocols and designing efficient primer sets for different powdery mildew taxa. NanoDrop spectrophotometry results indicated that a CTAB DNA isolation protocol yielded the most consistent DNA product. Primers for fungicide resistance genes CYP51, CYTb, and SDHb were designed using polymorphisms found in previously generated AmpSeq sequences, as well as from sequences found in the National Center for Biotechnology Information (NCBI) database. The newly designed primers were tested for functionality across grape, hop, and strawberry powdery mildew species using quantitative PCR (qPCR). Primers designed using Podosphaera sequences showed increased amplification among Podosphaera samples compared to the original primers designed for Erysiphe necator, and vice versa. Our results will influence experimental and primer design in future runs of AmpSeq, allowing for greater success in identification of fungicide resistant alleles in Erysiphe and Podosphaera.