Comparative Genomic Analyses among Citrus-Associated strains of Xylella fastidiosa

With the aid of a biochip, carrying representative sequences from approximately 2200 sequences from the genome of isolate 9a5c from X. fastidiosa (Xf), microarray-based comparisons have been performed with 6 different Xf isolates obtained from citrus plants (Table 1). Four of these isolates (56a, 9.12c, 187b, and 36f) were obtained from CVC-affected trees and are representatives of the most prevalent Xf haplotypes found in sweet orange orchards across the state of São Paulo, while isolate CV21 was obtained from a non-symptomatic tree from the same region. Isolate Fb7, on the other hand, was obtained from a sweet orange tree that displayed symptoms of “Pecosita”, a disease similar to CVC that occurs in some citrus-growing regions of Argentina. Keywords: Comparative Genomic Hybridization DNA from the 9a5c isolate was used as a reference in competitive hybridization experiments against DNA from 6 different Xf isolates obtained from citrus plants. Equimolecular amounts of each DNA have been labelled with either Cy3- or Cy5-dCTP and hybridized with a 9a5c Xf biochip. Statistical validation of fold change variations was performed with the aid of the Significance Analysis of Microarrays (SAM) method Tusher and coworkers, using the software developed by B. Narasimham and available at http://www-stat.stanford.edu/~tibs/SAM/index.html. Spots that showed a Reference/Test ratio <1:2 were considered to be present in greater copy number in the test over the reference strain, as proposed by Smoot and coworkers, while spots that showed an average Reference/Test ratio >5:1 were considered to be missing in the test strain. The application of these criteria in a direct sequence comparison between strains 9a5c and Temecula-1, which have been completely sequenced, provided an estimated error rate below 0.3%