The high resolution melting PCR protocol for rapid identification of single nucleotide substitutions in cytochrome c oxidase subunit II of Globodera pallida populations assigned to three pathotypes as an attempt of their differentiation
Marta Budziszewska 1, A-F  
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Department of Molecular Biology and Biotechnology, Institute of Plant Protection-National Research Institute, Poland
A - Research concept and design; B - Collection and/or assembly of data; C - Data analysis and interpretation; D - Writing the article; E - Critical revision of the article; F - Final approval of article
Marta Budziszewska   

Department of Molecular Biology and Biotechnology, Institute of Plant Protection-National Research Institute, W. Wegorka 20,, 60-318, Poznan, Poland
Submission date: 2021-05-21
Acceptance date: 2021-07-26
Online publication date: 2021-09-08
The potato cyst nematode (PCN), Globodera pallida, originates from South America and is considered one of the most severe agricultural pests of potato crops and other Solanaceae plants globally. Based on their virulence and the same ability to reproduce on various potato cultivars, the populations of G. pallida are divided into three pathotypes, Pa1- Pa3. In this study, the comparative sequence analyses of the fragment of mitochondrial cytochrome c oxidase subunit II (mtCOII) gene for eight populations of G. pallida, representing three pathotypes: Pa1, Pa2 and Pa3, indicated genetic diversity between them. However, we did not identify significant mutations distinguishing Pa2 from Pa3. Interestingly, two single nucleotide substitution, T441C and A468G, were characteristic only for populations assigned to Pa1. On this basis, we developed the high resolution melting (HRM) PCR protocol. As a result, the melting curves obtained for samples of Pa1 populations were diverse from those obtained for populations designed as Pa2 and Pa3, allowing their differentiation. Thus, the developed here HRM protocol enables a rapid, very sensitive and low-cost screening assay for SNPs identification in mtCOII of G. pallida pathotypes. In effect, it might also be a helpful molecular tool in pathotypes differentiation. However, the further verification of the correlation of those single nucleotide mutations occurrence in mtCOII in particular pathotypes should be carried out on a much larger number of samples of G. pallida, to state that these mutations are characteristic only for this pathotype.
I wish to thank Dr. Renata Dobosz from the Department of Entomology and Animal Pests IPP–NRI (Poznan, Poland) for kindly providing cysts of G. pallida populations of three pathotypes. I also wish to thank Dr A. Obrępalska-Stęplowska from the Department of Molecular Biology and Biotechnology IPP-NRI (Poznań, Poland) for her valuable suggestions during preparation of the manuscript.
Natasza Borodynko-Filas
The authors have declared that no conflict of interests exist.