The pinewood nematode, Bursaphelenchus xylophilus (Steinner & Buhrer) Nickle, has two different life stages according to several environmental factors: dispersal stage and propagative stages. The dispersal stage is closely related to the 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 (SSH). 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 Gene ontology. From GO database, some respective stage-specific genes were searched and estimated the relative transcripts level according to stages using the quantitative real-time PCR.

To successful molecular breeding, identification and functional characterization of breeding related genes and development of molecular breeding techniques using DNA markers are essential. Although the development of a useful marker is difficult in the aspect of time, cost and effort, many markers are being developed to be used in molecular breeding and developed markers have been used in many fields. Single nucleotide polymorphisms (SNPs) markers were widely used for genomic research and breeding, but has hardly been validated for screening functional genes in olive flounder. We identified single nucleotide polymorphisms (SNPs) from expressed sequence tag (EST) database in olive flounder; out of a total 4,327 ESTs, 693 contigs and 514 SNPs were detected in total EST, and these substitutions include 297 transitions and 217 transversions. As a result, 144 SNP markers were developed on the basis of 514 SNP to selection of useful gene region, and then applied to each of eight wild and culture olive flounder (total 16 samples). In our experimental result, only 32 markers had detected polymorphism in sample, also identified 21 transitions and 11 transversions, whereas indel was not detected in polymorphic SNPs. Heterozygosity of wild and cultured olive flounder using the 32 SNP markers is 0.34 and 0.29, respectively. In conclusion, we identified SNP and polymorphism in olive flounder using newly designed marker, it supports that developed markers are suitable for SNP detection and diversity analysis in olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.

Molecular markers are useful for selecting to include superior character genetic like as strong immune system and rapid growth in fish. The marker is also very important part of breeding technology in Olive flounder (Paralichthys olivaceus). Single nucleotide polymorphisms (SNPs) marker is already in use widely for genomic research and breeding. But this SNPs marker hardly has been validated for screening functional genes in Olive flounder. We study identify single nucleotide polymorphisms (SNPs) on Expressed sequence tag (EST) database, develop usable SNP marker and apply to wild sample and cultured of olive flounder. As a result, Out of total 4.327 ESTs, 693contigs and 514 SNP from total contigs were detected while these substitutions include 297 transitions and 217 transversions. 144 developed markers were applied in 16 samples (wild 8, culture 8), Out of total marker, only 32 markers had detected polymorphic in sample. Polymorphism of 32 markers was observed in the variety genes region involved in immunity and protein synthesis. And the 32 marker were identified 21 transitions, 11 transversions, and indel was not detected in polymorphic SNPs. The analysis on heterozygosity by sample showed 0.34 in wild sample and 0.29 in cultured sample. In conclusion, we was identified SNP and Polymorphism by designed new marker, it supports that development marker is suitable for SNP detection and diversity analysis in Olive flounder. The outcome of this study can be basic data for researches for immunity gene and characteristic with SNP.