BMC Genomics 2004, 5:54 http://www.biomedcentral.com/1471-2164/5/54 14,961 A. mexicanum limb regeneration ESTs TBLASTX vs 19,039 zfish regeneration ESTs potential regeneration homologues 1058 BLASTN vs 404,876 zfish non-regeneration ESTs candidate regeneration homologues 43 aBRFgLieagAsiunSlrsTtteszho3eiftbsBrfaLofAirsShAT.ENmSTeaxndidcaatTnaubBmaLsAerSseTgeXneseratricohnesEStTosidsenatricfyhebdest Results of BLASTN and TBLASTX searches to iden- tify best BLAST hits for A. mexicanum regeneration ESTs searched against zebrafish EST databases. A total of 14,961 A. mexicanum limb regeneration ESTs were assembled into 4485 contigs for this search. were filtered to retain only those alignments between sequences that were one another's reciprocal best BLAST hit. As expected, the number of reciprocal 'best hits' varied depending upon the E value threshold, although increasing the E threshold by several orders of magnitude had a dis- proportionately small effect on the overall total length of BLAST alignments. A threshold of E<10-80yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of E<10-20 yielded 2820 align- ments encompassing a total of 1.32 Mbp. The percent sequence identity of alignments was very high among presumptive orthologues, ranging from 84–100% at the more stringent E threshold of E<10-80. On average, A. mex- icanum and A. t. tigrinum transcripts are estimated to be 97% identical at the nucleotide level, including both pro- tein coding and UTR sequence. This estimate for nuclear sequence identity is surprisingly similar to estimates obtained from complete mtDNA reference sequences for these species (96%, unpublished data), and to estimates for partial mtDNA sequence data obtained from multiple nat- ural populations [16]. These results are consistent with the idea that mitochondrial mutation rates are lower in cold versus warm-blooded vertebrates [35]. From a resource per- spective, the high level of sequence identity observed between these species suggests that informatics will enable rapidly the development of probes between these and other species of the A. tigrinum complex. Extending EST resources to other ambystomatid species Relatively little DNA sequence has been obtained from species that are closely related to commonly used model organisms, and yet, such extensions would greatly facili- tate genetic studies of natural phenotypes, population structures, species boundaries, and conservatism and divergence of developmental mechanisms. Like many amphibian species that are threatened by extinction, many of these ambystomatid salamanders are currently in need of population genetic studies to inform conservation and management strategies [e.g. [13]]. We characterized SNPs from orthologous A. mexicanum and A. t. tigrinum ESTs and extended this information to develop informa- tive molecular markers for a related species, A. ordinarium. Ambystoma ordinarium is a stream dwelling paedomorph endemic to high elevation habitats in central Mexico [36]. This species is particularly interesting from an ecological and evolutionary standpoint because it harbors a high level of intraspecific mitochondrial variation, and as an independently derived stream paedomorph, is unique among the typically pond-breeding tiger salamanders. As a reference of molecular divergence, Ambystoma ordinarium shares approximately 98 and 97% mtDNA sequence identity with A. mexicanum and A. t. tigrinum respectively [16]. To identify informative markers for A. ordinarium, A. mexi- canum and A. t. tigrinum EST contigs were aligned to identify orthologous genes with species-specific sequence variations (SNPs or Insertion/Deletions = INDELs). Primer pairs cor- responding to 123 ESTs (Table 8) were screened by PCR using a pool of DNA template made from individuals of 10 A. ordinarium populations. Seventy-nine percent (N = 97) of the primer pairs yielded amplification products that were approximately the same size as corresponding A. mexica- num and A. t. tigrinum fragments, using only a single set of PCR conditions. To estimate the frequency of intraspecific DNA sequence polymorphism among this set of DNA marker loci, 43 loci were sequenced using a single individ- ual sampled randomly from each of the 10 populations, which span the geographic range of A. ordinarium. At least one polymorphic site was observed for 20 of the sequenced loci, with the frequency of polymorphisms dependent upon the size of the DNA fragment amplified. Our results suggest that the vast majority of primer sets designed for A. mexicanum / A. t. tigrinum EST orthologues can be used to Page 9 of 17 (page number not for citation purposes)