Event Title

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

Presenter Information

Sophie Everbach, Oberlin College

Location

Science Center, Bent Corridor

Start Date

10-27-2017 6:00 PM

End Date

10-27-2017 6:40 PM

Research Program

Cornell University Summer Research Scholars Program, New York State Agricultural Experiment Station, Geneva, NY

Poster Number

31

Abstract

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.

Major

Biology; History

Project Mentor(s)

Breanne Kisselstein, David Gadoury and Lance Cadle-Davidson, Plant Pathology & Plant Microbe-Biology, Cornell University

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Oct 27th, 6:00 PM Oct 27th, 6:40 PM

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.