Central Potato Research Institute, India
Title: Molecular detection, characterization and localization of Potato virus M (PVM) by RT-PCR from infected potato plants and dormant potato tubers
Baswaraj Raigond has completed his PhD in the field of Plant Virology at an age of 30 years from University of Agricultural Sciences, Dharwad, India. He is working as Scientist since 2009 to till date in Central Potato Research Institute (CPRI), Shimla, India. He research focus is mainly on serological and molecular diagnostics, comparative genomics along with electron microscopic studies on potato viruses. He has published more than 10 publications in reputed journals and newsletters.
Potato virus M (PVM) is one of the important potato virus distributed worldwide. Hence, sensitive technique like RT-PCR was standardized by designing three sets of primers specific to coat protein (CP) gene of PVM. Primer showing a sharp amplicon of ~360 bp was selected and PCR mix and conditions were optimized for detection. The same amplicon was gel eluted, ligated in pPrime cloning vector and transformation of E. coli which resulted in several recombinant colonies. These colonies were confirmed by colony PCR and restriction digestion as well. The recombinant clones consisting of CP gene were sequenced and analyzed by BLAST and the results revealed that the query had similarity match of 96% and 94% with that of the sequences reported from Canada and Czech Republic respectively. After optimization of RT-PCR detection and characterization of CP gene, the protocol was validated by screening different cultivars of potato collected from different geographical locations of India. Wherein the protocol has successfully detected PVM in many of the cultivars tested from different locations. To localize the distribution of PVM in potato, different parts of potato plant (sprouts, roots, bark and leaves) along with dormant potato tubers were tested by RT-PCR. The results showed that all the tested parts are infected with PVM indicating its systemic nature of infection. Therefore, the optimized RT-PCR protocol can be employed for quick and reliable detection of PVM in a given sample and the variability in CP gene drives to have the complete genome sequencing and its variability study in India. Overall this study will aid in developing better management strategies against PVM.
Keywords: Potato, PVM, CP gene, RT-PCR, Localization.