The Porcelain Crab Transcriptome and PCAD, the Porcelain Crab Microarray and Sequence Database

Abstract

BackgroundWith the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes.Methodology/Principal FindingsA set of ∼30K unique sequences (UniSeqs) representing ∼19K clusters were generated from ∼98K high quality ESTs from a set of tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66% of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases.Conclusions/SignificanceThe EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in EST library sequencing approaches, and thus represent a rich resource for studies of environmental genomics.

The proteomic response of cheliped myofibril tissue in the eurythermal porcelain crabPetrolisthes cinctipesto heat shock following acclimation to daily temperature fluctuations

“…The number of arginine kinase isoforms varied from five (cluster I) to nine. The diversity of arginine kinase isoforms can, in part, be explained by the existence of four different arginine kinase transcripts in the Petrolisthes EST library (Tagmount et al, 2010). They may be separate isoforms for the cytosolic and mitochondrial compartment (Uda et al, 2006).…”

“…Furthermore, the trancriptome of several tissues of one species, Petrolisthes cinctipes, has been characterized in response to acute temperature stress (Tagmount et al, 2010;Teranishi and Stillman, 2007). Petrolisthes cinctipes occupies the mid-to high-intertidal zone and has one of the highest thermal tolerances of the temperate Petrolisthes congeners (Stillman and Somero, 2000).…”

Changes in extreme cold tolerance, membrane composition and cardiac transcriptome during the first day of thermal acclimation in the porcelain crabPetrolisthes cinctipes

“…A pooled total RNA sample was prepared for each group by mixing equal quantities of total RNA from five individuals in each group in order to have the same amount of biological diversity within each pooled RNA sample. Linear amplification of RNA by in vitro transcription (Van Gelder et al, 1990) was performed as follows. First-strand cDNA was synthesized by heating 3g of pooled total RNA with 500ng of OligodT-T7 primer (5Ј-GCATTAGC -GGCCGCGAAATTAATACGACTCACTATAGGGAGATTTTT -TTTTTTTTTTTTTTTTV-3Ј) (Baugh et al, 2001) 10ϫ DNA ligase buffer were added and incubated at room temperature for 15min.…”

“…Custom microarrays were printed on poly-L-lysine coated glass slides (Erie Scientific, Portsmouth, NH, USA) at the UCSF Center for Advanced Technology using 24,748 PCR-amplified cDNAs, each representing a unique consensus sequence from a library of 61,440 cloned cDNAs Tagmount et al, 2010). Before use, spots were rehydrated by incubating slides in humidified air, snap dried at 100°C and cross-linked by 200mJ UV exposure in a cross linker (Spectronics Co., Westbury, NY, USA).…”

“…Gene homology for each microarray feature was determined from consensus sequences submitted to the following searches: BLASTx (GenBank, UniProt, SwissProt, Daphnia pulex v1.1 Filtered Models protein set), InterPro Scan (13 different protein identification algorithms) and tBLASTx (Daphnia pulex v1.1 transcript set) Tagmount et al, 2010). Using all of the available data, we ascribed gene identity for each feature where there was a match with a score of ≤1e -5 .…”