|HANSEN, ZACHARIAH - Cornell University - New York|
|TABIMA, JAVIER - Oregon State University|
|JUDELSON, HOWARD - University Of California|
|Grunwald, Niklaus - Nik|
|SMART, CHRISTINE - Cornell University - New York|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2015
Publication Date: 3/10/2016
Citation: Hansen, Z.R., Knaus, B.J., Tabima, J.F., Press, C.M., Judelson, H.S., Grunwald, N.J., Smart, C.D. 2016. SNP-based differentiation of Phytophthora infestans clonal lineages using locked nucleic acid probes and high resolution melt analysis. Plant Disease. 100(7):1297-1306. doi: 10.1094/PDIS-11-15-1247-RE.
Interpretive Summary: Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, reproduces as distinct genetic clones in the US. Each clone can differ in resistance to fungicide or other phenotypic characteristics. Distinguishing clones using PCR assays could be a novel tool allowing more rapid identification of clones compared to traditional genotyping techniques. We used available whole genome sequences to find candidate regions that distinguish clones and developed and validated PCR assays.
Technical Abstract: Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic differences can exist between lineages. Whole P. infestans genomes were aligned and single nucleotide polymorphisms (SNPs) identified as targets for the development of clonal lineage specific molecular diagnostic tools. Informative SNPs were used to develop high-resolution melt (HRM) assays and locked nucleic acid (LNA) probes to differentiate lineage US-23, the predominant lineage in the Eastern US for the past several years, from three other US lineages. Three different primer pairs targeting one to three SNPs were capable of separating lineage US-23 from lineages US-8, US-11, and US-24 using HRM analysis. A fourth HRM primer pair targeted a highly variable genomic region containing nine polymorphisms within 63 base pairs. These primers separated US-23, US-11, and US-8 plus US-24 into three separate groups following HRM analysis, but did not separate US-8 from US-24. Additionally, two LNA probes were designed to target a portion of the P. infestans genome containing two SNPs diagnostic for US-23. A single multiplex quantitative PCR assay containing both differentially-labeled LNA probes differentiated individuals belonging to lineage US-23 from those belonging to US-8, US-11, and US-24.