Development and application of a salmonid EST database and cDNA microarray: data mining and interspecific hybridization characteristics

Abstract

We report 80,388 ESTs from 23 Atlantic salmon (Salmo salar) cDNA libraries (61,819 ESTs), 6 rainbow trout (Oncorhynchus mykiss) cDNA libraries (14,544 ESTs), 2 chinook salmon (Oncorhynchus tshawytscha) cDNA libraries (1317 ESTs), 2 sockeye salmon (Oncorhynchus nerka) cDNA libraries (1243 ESTs), and 2 lake whitefish (Coregonus clupeaformis) cDNA libraries (1465 ESTs). The majority of these are 3' sequences, allowing discrimination between paralogs arising from a recent genome duplication in the salmonid lineage. Sequence assembly reveals 28,710 different S. salar, 8981 O. mykiss, 1085 O. tshawytscha, 520 O. nerka, and 1176 C. clupeaformis putative transcripts. We annotate the submitted portion of our EST database by molecular function. Higher- and lower-molecular-weight fractions of libraries are shown to contain distinct gene sets, and higher rates of gene discovery are associated with higher-molecular weight libraries. Pyloric caecum library group annotations indicate this organ may function in redox control and as a barrier against systemic uptake of xenobiotics. A microarray is described, containing 7356 salmonid elements representing 3557 different cDNAs. Analyses of cross-species hybridizations to this cDNA microarray indicate that this resource may be used for studies involving all salmonids.

Figure 1

Open reading frame (ORF) and BLAST results. Numbers of assembled Salmo salar (A) and Oncorhynchus mykiss (B) ESTs with and without 200 base ORFs are given. Within each of these categories, proportions of assembled ESTs with and without significant (E < 10^–5) BLASTX hits against GenBank nonredundant protein database are shown, as are proportions of assembled ESTs with and without significant (E < 10^–5) BLASTN hits against the nonredundant nucleotide database. The lengths and putative identifications (gene names of best BLASTX hits) of the longest ORFs in each species are given.

Figure 2

Evolutionary relationships, genome sizes, and microarray hybridization characteristics of three salmonids relative to smelt. (A) Phylogenetic tree, based on morphological characters, showing evolutionary relationships among teleosts relevant to this study, and other fish orders with genome projects (Nelson 1994). (B) Phylogenetic tree, based on morphological characters, showing evolutionary relationships of select salmonids (Smith and Stearley 1989; Kido et al. 1991). Arrows indicate putative genome duplication events (Wolfe 2001). (C, D) Mean total signals on Atlantic salmon (AS) or rainbow trout (RT) chip elements/spots (Table 6) are converted to “smelt units” by dividing by 0.661E7 for AS chip elements, or 0.824E6 for RT chip elements. Genome sizes for AS (Salmo salar), RT (Oncorhynchus mykiss), and smelt (Osmerus eperlanus, close relative of Osmerus mordax used in this study) were measured by DNA flow cytometry (Vinogradov 1998). Genome size of lake whitefish (LW, Coregonus clupeaformis) was measured by Feulgen densitometry (Booke 1968). Error bars (C) show mean total signal SEM values (Table 6) converted to “smelt units” as above. n indicates number of microarrays hybridized with labeled target from each species.