This study is an integrated, collaborative effort that relies on expertise in molecular evolution and genomics (Debashish Bhattacharya), high quality cDNA library construction (Debashish Bhattacharya, Marcelo Bento Soares), an established Expressed sequence tag (EST) sequencing and bioinformatics pipeline (Marcelo Bento Soares, Debashish Bhattacharya), and expertise in the physiology and culture of Alexandrium tamarense and studies of toxin production (Donald Anderson). This study will result in a valuable molecular resource for scientists working to understand the ecology and toxicity of Harmful algal bloom (HAB) species like Alexandrium tamarense and will provide the detailed insights into the genome of these fascinating protists.
To generate a comprehensive unigene set for Alexandrium tamarense using serially subtracted libraries enriched for rare mRNAs.
We will construct a non−normalized cDNA library from Alexandrium tamarense that is derived from pooled cDNA isolated from cells that are nitrate−depleted, phosphate−depleted, generating newly−formed cysts, germinating cysts, synchronized in G1 phase, and in the middle of the dark period. A total of 1,000 ESTs will be sequenced at the 3’ terminus from this library to identify highly genome evolution in the context of Horizontal gene transfer, expressed genes. Thereafter, unique cDNAs that have already been generated using a nutrient−replete culture of Alexandrium tamarense (8,996 ESTs) will be used as a driver to subtract from the pooled cDNA library. A total of 9,000 cDNAs in the subtracted library will be sequenced at the 3’ terminus. These EST data will be made freely available to the scientific community with a dedicated Web server in the Bhattacharya lab (Alexandrium EST) and will also be released on a weekly basis to GenBank (dbEST) to facilitate the broadest distribution. Until now, a total of 10,885 Alexandrium tamarense ESTs have been released to dbEST.
To generate a transcriptome tag database for Alexandrium tamarense.
Massively Parallel Signature Sequencing (MPSS), which allows the generation of gene expression data via acquisition of transcriptome tags, will be used to examine Alexandrium tamarense cDNAs isolated under four different growth conditions (nutrient−repleted, nitrate−depleted, phosphate−depleted and a synchronized culture in G1 producing toxin). A total of at least 350,000 tags will be collected from each condition and identified by comparison to the EST data. Genes that are uniquely expressed within the MPSS data sets under different nutrients conditions or life−history stages will be identified as possible candidate genes that are involved in these functions (e.g., toxin production, nutrient uptake).