|Developing a molecular diagnostic for Fusarium oxysporum f.sp. cubense tropical race 4 through Diversity Array Technology genotyping|
N. I. ORDONEZ ROMAN (1), M. Salacinas (1), C. Schoen (1), O. Mendes (1), A. Kilian (2), G. Kema (1). (1) Wageningen University and Research, Wageningen, Netherlands; (2) Diversity Arrays Technology Pty Ltd, Canberra, Australia
Early and reliable detection of <i>Fusarium oxysporum</i> f.sp. <i>cubense</i> (Foc) tropical race 4 (TR4) is required for effective monitoring of this banana (<i>Musa</i>) pathogen. To date, TR4 - originally identified in Taiwan - has already spread throughout Southeast Asia and was recently discovered outside this region. Traditional morphological Foc characterization is not feasible, as microscopic structures are similar within strains belonging to the <i>F. oxysporum</i> complex. Vegetative Compatibility Group (VCG) analyses, yet proven to be largely consistent, are time consuming and not always possible. Rapid molecular based methods are preferential due to their accuracy. The detection of unique genomic regions is a vital step for development of strain-specific probes. In this study, the Diversity Array Technology Sequencing (DArTseq) approach was used for the discovery of unique genome regions for primer design. DNA was extracted from 29 strains representing the overall VCG diversity of Foc. Using a stringency of call rate >0.66, reproducibility = 100 and Q value > 2.7 we selected 15,900 markers that included unique DArTseq markers for three TR4 strains-VCG01213. Alignments of the DArTseq sequences with the reference genome of <i>Foc</i> TR4 II-5 allowed up- and downstream extension of the ~69 base pair sequences that enabled the design of <i>Foc</i> TR4 specific primers for the rapid detection of TR4 strains in different sample matrices from a range of geographically different locations.