Abstract
BackgroundSheepshead minnow (Cyprinodon variegatus) are small fish capable of withstanding exposure to very low levels of dissolved oxygen, as well as extreme temperatures and salinities. It is an important model in understanding the impacts and biological response to hypoxia and co-occurring compounding stressors such as polycyclic aromatic hydrocarbons, endocrine disrupting chemicals, metals and herbicides. Here, we initiated a project to sequence and analyze over 10,000 ESTs generated from the Sheepshead minnow (Cyprinodon variegatus) as a resource for investigating stressor responses.ResultsWe sequenced 10,858 EST clones using a normalized cDNA library made from larval, embryonic and adult suppression subtractive hybridization-PCR (SSH) libraries. Post- sequencing processing led to 8,099 high quality sequences. Clustering analysis of these ESTs indentified 4,223 unique sequences containing 1,053 contigs and 3,170 singletons. BLASTX searches produced 1,394 significant (E-value < 10-5) hits and further Gene Ontology (GO) analysis annotated 388 of these genes. All the EST sequences were deposited by Expressed Sequence Tags database (dbEST) in GenBank (GenBank: GE329585 to GE337683). Gene discovery and annotations are presented and discussed. This set of ESTs represents a significant proportion of the Sheepshead minnow (Cyprinodon variegatus) transcriptome, and provides a material basis for the development of microarrays useful for further gene expression studies in association with stressors such as hypoxia, cadmium, chromium and pyrene.
Highlights
Sheepshead minnow (Cyprinodon variegatus) are small fish capable of withstanding exposure to very low levels of dissolved oxygen, as well as extreme temperatures and salinities
We have produced 8,099 high quality Sheepshead minnow (Cyprinodon variegatus) Expressed sequence tag (EST) sequences from multiple developmental stages
This should represent a significant fraction of the Sheepshead minnow genes, the exact gene number of the fish is unknown at present
Summary
Sheepshead minnow (Cyprinodon variegatus) are small fish capable of withstanding exposure to very low levels of dissolved oxygen, as well as extreme temperatures and salinities. It is an important model in understanding the impacts and biological response to hypoxia and co-occurring compounding stressors such as polycyclic aromatic hydrocarbons, endocrine disrupting chemicals, metals and herbicides. The objectives of this study were to create cDNA libraries suitable for the analysis of ESTs and to generate an EST resource for Sheepshead minnow to allow cDNA-based design of microarrays in order to provide genomic resources for the analysis of differentially expressed genes. To facilitate gene identification and functional genomics studies, the EST set has been annotated using the structured vocabulary provided by the Gene Ontology Consortium (2001), based on molecular studies of gene function and metabolic pathway analysis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database
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