Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 2, 2003
Publication Date: February 2, 2003
Citation: Rexroad III, C.E., Keele, J.W. 2003. Development of microarrays for transcriptome research in rainbow trout. VI Inter Congress of Comparative Physiology & Biochemistry. Feb 2-7, 2003. Vol 134. Supl 1. Abstract 8.2. p.S29. Technical Abstract: Current research efforts at the USDA/ARS National Center for Cool and Cold Water Aquaculture (NCCCWA) are centered on the development of a selective breeding program for improving rainbow trout aquaculture production efficiency. This involves characterizing multiple sets of germplasm with respect to phenotypes associated with growth, disease resistance, reproduction, and stress. In addition to characterizing such phenotypes, we are progressing toward identifying the genetic variation affecting these traits and understanding the basic gene expression patterns underlying them. The number of publicly available salmonid nucleotide sequences has been limited and lacking in functional annotation. To this end, NCCCWA initiated a rainbow trout Expressed Sequence Tag (EST) project with the goals of 1) identifying as many unique rainbow trout transcripts as possible, 2) functionally annotating ESTs using comparative genome information from public databases, 3) targeting EST sequences of interest for genetic mapping, and 4) identification of ESTs for utilization with technologies developed for high throughput studies of gene expression. The EST project was initiated with the construction of a single normalized cDNA library from rainbow trout brain, liver, gill, spleen, kidney, and white muscle tissue mRNA. The normalization process was observed to reduce the level of the GAPDH transcript in the library 85 fold. Five prime end sequencing of 61,188 clones from the library yielded 46,792 with PHRED>20 over 100 bp averaging read length 521.5 bp, 36,845 of those had PHRED>20 over 400 bp averaging read length 594.4 bp. Analysis of redundancy revealed that only 4.55% of the 46,792 clones had a match within those sequences yielding approximately 44,662 unique sequences. Comparative genome analysis included the following using GenBank: blastx on the protein database, blastn on the non-redundant database, blastn on the zebrafish genome, and blastn on dbEST parsing results for Atlantic salmon, catfish, zebrafish, medaka, pufferfish, human, chicken, and mouse. Using the results of these analyses, ESTs are being identified as candidates for spotting on microarrays based on uniqueness and association with functional annotation.