AN IN SILICO WORKFLOW FOR THE SELECTION OF NOVEL DNA MARKERS FOR DETECTION OF PHYTOPATHOGENIC BACTERIA, USING AGROBACTERIUM TUMEFACIENS AS A MODEL

P. Albuquerque, A.S. Rodrigues, C.M.R. Caridade, A.R.S. Marcal, F. Tavares

Abstract


DNA-based methods for bacterial detection are increasingly acknowledged as specific, reliable and costeffective alternatives or complements to the classical culture-based methods, facilitating early detection and presumptive diagnosis, in particular when symptomless plants and quarantine procedures are involved. The massive number of non-redundant bacterial DNA sequences available in gene banks nowadays, and the accessibility to resourceful bioinformatics tools to analyse those sequences, provide an excellent and extensive source for new genus-, species-, or biovar-specific molecular markers. In this study, we propose an in silico workflow framed by four successive criteria and using two web-based bioinformatics applications, CUPID and Insignia, to identify specific chromosomal loci for phytopathogenic bacteria belonging to the genus Agrobacterium (herein called ‘agrobacteria’), useful as DNAmarkers to detect pathogenic strains (belonging to several species) of the genus. The selection criteria for the markers are: (i) retrieve the sequence overlaps between CUPID and Insignia databases using a C++ program developed by us in order to increase marker consistency; (ii) the chosen markers should undergo a BLAST (basic local alignment search tool) analysis to confirm their specificity; (iii) an evolutionary and comparative genomic analysis should allow to infer the likelihood of the putative markers being exclusive for the target taxon, and simultaneously ubiquitous within the taxon diversity; (iv) markers should be chosen regarding their suitability for a particular detection technique, e.g. PCR or hybridization profiling.

Keywords


CUPID; Insignia; DNA markers; detection; bioinformatics

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DOI: http://dx.doi.org/10.4454/jpp.v94i1sup.014

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EDIZIONI ETS, Pisa, Italy