Construction and characterization of stage-specific subtractive expressed sequence tag (EST) libraries of the pinewood nematode Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus (Steinner & Buhrer) Nickle, has two different life stages according to several environmental factors such as temperature, humidity, nutrition, and density: dispersal and propagative stages. The dispersal stage is closely related to migration to other host pines whereas the propagative stage is coupled to the direct cause of pine wilt. To establish expressed sequence tag (EST) database of two life cycles of B. xylophilus, subtractive EST libraries were constructed using suppressed subtractive hybridization. From 3,072 and 3,840 sequences of dispersal- and propagative-specific stage cDNA libraries, 1,927 and 2,604 clusters were generated, respectively, which were annotated by BLASTx and Gene Ontology (GO). A total of 1,112 (57.7%) and 1,215 (46.7%) clusters from the dispersal- and propagative-specific stage cDNA libraries respectively had the matched BLASTx hits (E £ 10^-2), among which 913 (47.4%) and 960 (36.9%) were classified into three categories in GO. From abundant ESTs and GO database, a few respective stage-specific genes were searched for the diagnosis and their relative transcripts levels were estimated using the quantitative real-time PCR.