Oysters were challenged with the Dermo parasite.  Biological samples from oysters with several tissues with and without infection were for the construction of normalized libraries.  mRNA was extracted and isolated.  A normalized and subtracted C. virginica cDNA library was constructed from pooled RNA isolated from hemocytes, mantle, gill, gonad and digestive tract, muscle, and a whole juvenile oyster.  A total of 6,528 clones were sequenced from this library generating 5,542 high-quality EST sequences.  Cluster analysis indicated the presence of 635 contigs and 4,053 singletons, generating a total of 4,688 unique sequences. About 46% (2,174) of the unique ESTs had significant hits (E value ≤ 1e-05) to the non-redundant protein database; 1,104 of which were annotated using Gene Ontology (GO) terms.  A total of 35 microsatellites were identified from the ESTs, with 18 having sufficient flanking sequences for primer design.  A total of 6,533 putative SNPs were also identified using all existing and the newly generated EST resources of the eastern oysters.  These genome resources provide the material basis for microarray development, marker validation, and genetic linkage and QTL analysis.  Bioinformatic analysis for the design of microarrays have been completed.  We are working with Niblegen in the processes of microarray construction.  Challenges have been completed, and the samples have been collected for use as probes from both a resistant and a susceptible line at various time points.  RNA has been extracted for the making of probes.

Objectives

To determine molecular indicators of oysters after infection with dermo in relation to genetic background in resistance.

Methodology

SSH subtraction cDNAs will be used to sequence ESTs that will be used to develop microarray technology. The microarrays will be used to analyze differentially expressed genes in oysters after infection with Dermo in resistance lines and susceptible lines. The results will then be compared for the analysis of molecular indicators for resistance.

Rationale

Genome expression is responsible for performance traits such as disease resistance. We have identified oyster lines with various levels of resistance. Their genomic expression signatures are expected to be different accounting for the observed difference in resistance. This project will use microarray technology to reveal the genome expression differences.

Phenotypes such as resistance or susceptibility are controlled by genes and their expression.  Upon infection, a set of genes would be expected to be induced by infection, and many of them are produced for the purpose of defense.  In this regard, the genes produced from a resistance line may be different from the genes expressed in a susceptible line.  By comparing the differences in response, and between lines with different genetic background, genes important for defense and resistance may be identified.

Results

This project has produced a high quality normalized cDNA library that can be further exploited for long term genome research of oysters.  This project also generated a large number of ESTs, and the microarray technology in oysters. Though basic, such products will bring oyster disease research to a new level that will have a positive impact to the oyster industry.