BMC Genomics 2004, 5:54 http://www.biomedcentral.com/1471-2164/5/54 Background Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. Expressed sequence tags (EST) are particularly useful genomic resources because they enable multiple lines of research and can be generated for any organism: ESTs allow the identification of molecular probes for developmental studies, provide clones for DNA microchip construction, reveal candidate genes for mutant phenotypes, and facilitate studies of genome structure and evolution. Furthermore, ESTs provide raw material from which strain-specific polymorphisms can be identified for use in population and quantitative genetic analyses. The utility of such resources can be tai- lored to target novel characteristics of organisms when ESTs are isolated from cell types and tissues that are actively being used by a particular research community, so as to bias the collection of sequences towards genes of spe- cial interest. Finally, EST resources produced for model organisms can greatly facilitate comparative and evolu- tionary studies when their uses are extended to other, closely related taxa. Salamanders (urodele amphibians) are traditional model organisms whose popularity was unsurpassed early in the 20th century. At their pinnacle, salamanders were the pri- mary model for early vertebrate development. Embryo- logical studies in particular revealed many basic mechanisms of development, including organizer and inducer regions of developing embryos [1]. Salamanders continue to be important vertebrate model organisms for regeneration because they have by far the greatest capacity to regenerate complex body parts in the adult phase. In contrast to mammals, which are not able to regenerate entire structures or organ systems upon injury or amputa- tion, adult salamanders regenerate their limbs, tail, lens, retina, spinal cord, heart musculature, and jaw [2-7]. In addition, salamanders are the model of choice in a diver- sity of areas, including vision, embryogenesis, heart devel- opment, olfaction, chromosome structure, evolution, ecology, science education, and conservation biology [8- 15]. All of these disciplines are in need of genomic resources as fewer than 4100 salamander nucleotide sequences had been deposited in GenBank as of 3/10/04. Here we describe results from an EST project for two ambystomatid salamanders: the Mexican axolotl, Ambystoma mexicanum and the eastern tiger salamander, A. tigrinum tigrinum. These two species are members of the Tiger Salamander Complex [16], a group of closely related species and subspecies that are widely distributed in North America. Phylogenetic reconstruction suggests that these species probably arose from a common ancestor about 10–15 million years ago [16]. Ambystoma mexica- num has a long research history of over 100 years and is now principally supplied to the research community by the Axolotl Colony [17], while A. t. tigrinum is obtained from natural populations in the eastern United States. Although closely related with equally large genomes (32 × 109 bp)[18], these two species and others of the Complex differ dramatically in life history: A. mexicanum is a paedo- morphic species that retains many larval features and lives in water throughout it's life cycle while A. t. tigrinum undergoes a metamorphosis that is typical of many amphibians. Like many other traditional model organ- isms of the last century, interest in these two species declined during the rise of genetic models like the fly, zebrafish, and mouse [19]. However, "early" model organisms such as salamanders are beginning to re-attract attention as genome resources can rapidly be developed to exploit the unique features that originally identified their utility for research. We make this point below by showing how the development of ESTs for these two spe- cies is enabling research in several areas. Furthermore, we emphasize the value of developing resources in model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research programs. Results and Discussion Selection of libraries for EST sequencing Eleven cDNA libraries were constructed using a variety of tissues (Table 1). Pilot sequencing of randomly selected clones revealed that the majority of the non-normalized libraries were moderate to highly redundant for relatively few transcripts. For example, hemoglobin-like transcripts represented 15–25% of the sampled clones from cDNA libraries V1, V2, and V6. Accordingly, we chose to focus our sequencing efforts on the non-normalized MATH library as well as the normalized AG library, which had lower levels of redundancy (5.5 and 0.25% globins, respectively). By concentrating our sequencing efforts on these two libraries we obtained transcripts deriving Table 1: Tissues selected to make cDNA libraries. Tissue cDNAs sequenced limb blastema limb blastema tail blastema brain liver spleen heart gill stage 22 embryo liver, gonad, lung, kidney, heart, gill 1029 16244 1422 3196 792 337 38 3039 96 19871 ID GARD MATH V1 V2 V3 V4 V5 V6 V7 AG Further information is found in Methods and Materials. Page 2 of 17 (page number not for citation purposes)