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Title: A qPCR-HRM assay (high-resolution melting-curve analysis) to discriminate between pathotypes of Synchytrium endobioticum

Jaroslaw Przetakiewicz

National Research Institute, Poland

Biography

Dr. Jarosław Przetakiewicz graduated from Nicolaus Copernicus University in Toruń, Poland – biology department – in 1998. His M. Sc. degree thesis concerned identification of aminoacides connected with phosphorylation in proteins using immunoprecipitation methods in oats. In 1998 Dr J. Przetakiewicz started to work in Plant Breeding and Acclimatization Institute – National Research Institute. This work fructified Ph.D. degree in 2003. Title of thesis: “Production of tetraploid somatic hybrids of potato (Solanum tuberosum L.) from selected diploid lines”. From 2004 he is working for Department of Plant Pathology in Laboratory of Quarantine Organisms. His research is connected with improving of detection and pathotype determination of S. endobioticum and resistance tests of potato genotypes to different pathotypes of the fungus.

Abstract

Synchytrium endobioticum (Shilb.) Perc. is an obligate biotrophic, soil-borne fungus which causes the potato wart disease. S. endobioticum originated from Andean zone in South America and is currently present almost all over the world. The thick-walled winter sporangia are the dormant structures of the fungus and can survive for more than 40 years in the absence of host even under adverse environmental conditions. Since the discovery of pathotype 2(G1) in Germany, more than 40 pathotypes have been reported in Europe. HRM is a recently developed molecular technique. It is a cost-efficient, closed-tube system that allows high-throughput analysis without any post-PCR processing. PCR amplification and HRM were performed using specific primers of SNP sites (L1, L2, L3, L4, L5, L8 & L9) and DNA extracts of 10 different pathotypes of S. endobioticum [1(D1), 2(G1), 2(Ch1), 3(M1), 6(O1), 8(F1), 18(T1), 39(P1), #PL5/2010 & #PL2/20015]. Testing the primers  set L1 on all pathotypes showed that HRM discriminates clearly between pathotype-1(D1) and non pathotype-1(D1). The second PCR test (L3) showed that HRM discriminates clearly between pathotype 1(D1)/8(F1)/18(T1) and other pathotypes of S. endobioticum. The third PCR test (L4) showed that HRM discriminates clearly between pathotypes 1(D1)/3(M1) and 2(G1)/6(O1)/ 8(F1)/18(T1) and 39(P1)/ #PL5/2010/#PL2/20015. This study presents the first results on HRM method to discriminate different pathotypes of S. endobioticum and confirms results obtained by Bonants et al. (2015) by the use of TaqMan PCR assays. HRM is more cost-effective than other genotyping technologies, such as TaqMan SNP typing, because no additional probes are required.