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Discovery of molecular markers for the detection of geographic origin of the pinewood nematode and detection kit development

caso3.pngBursaphelenchus xylophilus is the causal agent of pine wilt disease. The initial burst of the disease occurred in 1999, in Setúbal, Portugal, and, in spite of containment measures, the nematode spread to the entire country. The nematode was first identified in the United States, later in Asia and now in Portugal. The nematode infects pines when the insect vector, Monochamus, feeds in young twigs. Once inside the pine, the nematode reproduces and disperses in the xylem, causing cavitation, cessation of water flow and pine wilt.

Through next generation DNA sequencing technologies, we sequenced the transcriptome of seven B.xylophilus isolates from four distinct geographic regions, four from Portugal, one from China, one from Japan and one from the United States.  The diversity of transcripts sequenced was high; thus we just compared the sequences of genes involved in B.xylophilus pathogenicity, namely cellulases, pectin lyases, expansins and venom allergen proteins, and other pathways such as ubiquitination, and fungal feeding (chitinases), detecting 136 SNPs.

We validated the SNPs in corresponding genes in DNA in a wider set of isolates and used these polymorphisms to measure the genetic distance between isolates. This analysis revealed that Portuguese isolates are distant from United States and the Japanese isolates, are more closely related to Chinese isolates and identical to a Korean isolate. Among detected SNPs, five sufficed to discriminate between the four geographic locations. To ease the detection of the differentiating SNPs we developed a highly sensitive and efficient identification kit, based in real-time PCR.


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