Download EXPRESSED SEQUENCE TAGS FROM IMMUNE TISSUES OF

EXPRESSED SEQUENCE TAGS FROM IMMUNE TISSUES OF OLIVE FLOUNDER
Paralichthys olivaceus CHALLENGED WITH PATHOGENES
Woo-Jin Kim*, Bo-Hye Nam, Hee Jeong Kong, Young-Ok Kim, Eun-Ha Shin, Hyung Soo
Kim, Bong-Seok Kim, Sang-Jun Lee
Biotechnology Research Division, National Fisheries and Research and Development Institute,
Busan 619-902, Korea. [email protected]
The olive flounder (Paralichthys olivaceus) is an economically important and the most widely
cultured marine species in Korea. Edwardsiella tarda and viral haemorrhagic septicaemia
virus (VHSV) are two pathogens that affect olive flounder culture causing serious economic
losses to the olive flounder industry. Little is known about the molecular mechanisms for
disease resistance and host pathogen interactions in this species. In this study, tens of
thousands of expressed sequence tags (ESTs) for functional genomic studies and potential
markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms
(SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated
genes in response to these pathogens and it constitutes the basis for subsequent and more
accurate microarray analysis. An important factor that determines the usefulness of generated
data for biologists is efficient data access. Public searchable databases play a crucial role in
providing such service.
A total of 33,501 ESTs sequenced from twenty-seven different cDNA libraries of olive
flounder infected with E. tarda and VHSV and from healthy fish were analyzed. Three
immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points
in the infection process for library construction. The sequences were processed into 25,644
high-quality sequences, which constituted the source for the olive flounder EST database.
Clustering and assembly of these sequences revealed 13,457 different putative transcripts,
consisting of 3,650 contigs and 9,807 singletons. BLAST searches with public databases
detected significant similarity (e-value ≤ 1e-10) in 6,796 (50.5%) sequences and 3,418 of
them (25.4%) could be functionally annotated. Eight hundred five of these genes (25.9%)
encoded for defence/immune related proteins. Some ESTs showed significant differences in
the number of transcripts when comparing the libraries, suggesting regulation in response to
these pathogens. A total of 585 microsatellites, with 520 having sufficient flanking sequences
for primer design, and 3,474 putative SNPs were identified from these EST resources.
We determined whether SNPs in lipopolysaccharide binding protein/ bactericidal permeability
increasing protein (LPB/BPI) gene, which play an important role in innate immune system,
are associated with resistance to E. tarda disease in olive flounder. By direct DNA sequencing
in 24 individuals, we identified 10 SNPs in LPB/BPI. 6 polymorphic sites were selected for
genotyping in E. tarda disease-susceptible (n=48) and resistant olive flounder individuals
(n=48). Statistical analysis revealed that 816G>A polymorphism showed putative association
with E. tarda disease resistance (P<0.05). Polymorphic SNP markers developed in this study
are applicable in genetic mapping and genetic association studies of olive flounder.