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. We developed ESTs for Mexican axolotl ( Ambystoma mexicanum ) and Eastern tiger salamander ( A. tigrinum tigrinum ), species with deep and diverse research histories.
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. We developed ESTs for Mexican axolotl ( Ambystoma mexicanum ) and Eastern tiger salamander ( A. tigrinum tigrinum ), species with deep and diverse research histories.Ambystoma mexicanumA. tigrinum tigrinumResults Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for  A. mexicanum  were processed to yield 35,413 and 20,599 high quality ESTs for  A. mexicanum  and  A. t. tigrinum , respectively. Because the  A. t. tigrinum  ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human –  Ambystoma  synteny group with marker loci, and extended PCR primer sets designed for  A. mexicanum  /  A. t. tigrinum  orthologues to a related tiger salamander species.
Results
Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for  A. mexicanum  were processed to yield 35,413 and 20,599 high quality ESTs for  A. mexicanum  and  A. t. tigrinum , respectively. Because the  A. t. tigrinum  ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human –  Ambystoma  synteny group with marker loci, and extended PCR primer sets designed for  A. mexicanum  /  A. t. tigrinum  orthologues to a related tiger salamander species.A. mexicanumA. mexicanumA. t. tigrinumA. t. tigrinumAmbystomaA. mexicanumA. t. tigrinumConclusions Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.
Conclusions
Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

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 tailored 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 special interest. Finally, EST resources produced for model organisms can greatly facilitate comparative and evolutionary 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 20 th  century. At their pinnacle, salamanders were the primary model for early vertebrate development. Embryological 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 amputation, 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 diversity of areas, including vision, embryogenesis, heart development, 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 mexicanum  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 × 10 9  bp)[ 18 ], these two species and others of the Complex differ dramatically in life history:  A. mexicanum  is a paedomorphic 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 organisms 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 species 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.
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 tailored 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 special interest. Finally, EST resources produced for model organisms can greatly facilitate comparative and evolutionary 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 20 th  century. At their pinnacle, salamanders were the primary model for early vertebrate development. Embryological 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 amputation, 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 diversity of areas, including vision, embryogenesis, heart development, 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 mexicanum  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 × 10 9  bp)[ 18 ], these two species and others of the Complex differ dramatically in life history:  A. mexicanum  is a paedomorphic 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 organisms 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 species 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.Ambystoma mexicanumA. tigrinum tigrinumAmbystoma mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumResults 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 primarily from regenerating larval tissues in  A. mexicanum  and several non-regenerating larval tissues in  A. t. tigrinum . Table 1 Tissues selected to make cDNA libraries. ID Tissue cDNAs sequenced GARD limb blastema 1029 MATH limb blastema 16244 V1 tail blastema 1422 V2 brain 3196 V3 liver 792 V4 spleen 337 V5 heart 38 V6 gill 3039 V7 stage 22 embryo 96 AG liver, gonad, lung, kidney, heart, gill 19871 Further information is found in Methods and Materials. EST sequencing and clustering A total of 46,064 cDNA clones were sequenced, yielding 39,982 high quality sequences for  A. mexicanum  and  A. t. tigrinum  (Table  2 ). Of these, 3,745 corresponded to mtDNA and were removed from the dataset; complete mtDNA genome data for these and other ambystomatid species will be reported elsewhere. The remaining nuclear ESTs for each species were clustered and assembled separately. We included in our  A. mexicanum  assembly an additional 16,030 high quality ESTs that were generated recently for regenerating tail and neurula stage embryos [ 20 ]. Thus, a total of 32,891 and 19,376 ESTs were clustered for  A. mexicanum  and  A. t. tigrinum , respectively. Using PaCE clustering and CAP3 assembly, a similar number of EST clusters and contigs were identified for each species (Table  2 ). Overall contig totals were 11,190 and 9,901 for  A. mexicanum  and  A. t. tigrinum  respectively. Thus, although 13,515 more  A. mexicanum  ESTs were assembled, a roughly equivalent number of contigs were obtained for both species. This indicates that EST development was more efficient for  A. t. tigrinum,  presumably because ESTs were obtained primarily from the normalized AG library; indeed, there were approximately twice as many ESTs on average per  A. mexicanum  contig (Table  2 ). Thus, our EST project yielded an approximately equivalent number of contigs for  A. mexicanum  and  A. t. tigrinum , and overall we identified > 21,000 different contigs. Assuming that 20% of the contigs correspond to redundant loci, which has been found generally in large EST projects [ 21 ], we identified transcripts for approximately 17,000 different ambystomatid loci. If ambystomatid salamanders have approximately the same number of loci as other vertebrates (e.g. [ 22 ]), we have isolated roughly half the expected number of genes in the genome. Table 2 EST summary and assembly results. A. mex A. t. tig cDNA clones sequenced 21830 24234 high-quality sequences 19383 20599 mt DNA sequence 2522 1223 seqs submitted to NCBI 16861 19376 sequences assembled 32891 a 19376  PaCE clusters 11381 10226  ESTs in contigs 25457 12676  contigs 3756 3201  singlets 7434 6700  putative transcripts 11190 9901 a Includes 16,030 ESTs from [20]. Identification of vertebrate sequences similar to  Ambystoma  contigs We searched all contigs against several vertebrate databases to identify sequences that exhibited significant sequence similarity. As our objective was to reliably annotate as many contigs as possible, we first searched against 19,804 sequences in the NCBI human RefSeq database (Figure  1 ), which is actively reviewed and curated by biologists. This search revealed 5619 and 4973 "best hit" matches for the  A. mexicanum  and  A. t. tigrinum  EST datasets at a BLASTX threshold of  E  = 10 -7 . The majority of contigs were supported at more stringent E-value thresholds (Table  3 ). Non-matching contigs were subsequently searched against the Non-Redundant (nr) Protein database and  Xenopus tropicalus  and  X. laevis  UNIGENE ESTs (Figure  1 ). These later two searches yielded a few hundred more 'best hit' matches, however a relatively large number of ESTs from both ambystomatid species were not similar to any sequences from the databases above. Presumably, these non-matching sequences were obtained from the non-coding regions of transcripts or they contain protein-coding sequences that are novel to salamander. Although the majority are probably of the former type, we did identify 3,273 sequences from the non-matching set that had open reading frames (ORFs) of at least 200 bp, and 911 of these were greater than 300 bp. Figure 1 Results of BLASTX and TBLASTX searches to identify best BLAST hits for  Ambystoma  contigs searched against NCBI human RefSeq, nr, and  Xenopus  Unigene databases . Table 3 Ambystoma  contig search of NCBI human RefSeq, nr, and  Xenopus  Unigene databases. A. mex A. t. tig # BLASTX Best Matches 6283 5545 < E -100 630 870 < E -50  > E -100 2015 1990 < E -20  > E -50 2153 1595 < E -10  > E -20 967 745 < E -7  > E -10 518 345 The distribution of ESTs among contigs can provide perspective on gene expression when clones are randomly sequenced from non-normalized cDNA libraries. In general, frequently sampled transcripts may be expressed at higher levels. We identified the 20 contigs from  A. mexicanum  and  A. t. tigrinum  that contained the most assembled ESTs (Table  4 ). The largest  A. t. tigrinum  contigs contained fewer ESTs than the largest  A. mexicanum  contigs, probably because fewer overall  A. t. tigrinum  clones were sequenced, with the majority selected from a normalized library. However, we note that the contig with the most ESTs was identified for  A. t. tigrinum:  delta globin. In both species, transcripts corresponding to globin genes were sampled more frequently than all other loci. This may reflect the fact that amphibians, unlike mammals, have nucleated red blood cells that are transcriptionally active. In addition to globin transcripts, a few other house-keeping genes were identified in common from both species, however the majority of the contigs were unique to each list. Overall, the strategy of sequencing cDNAs from a diverse collection of tissues (from normalized and non-normalized libraries) yielded different sets of highly redundant contigs. Only 25% and 28% of the  A. mexicanum  and  A. t. tigrinum  contigs, respectively, were identified in common (Figure  2 ). We also note that several hundred contigs were identified in common between Xenopus and  Ambystoma ; this will help facilitate comparative studies among these amphibian models. Table 4 Top 20 contigs with the most assembled ESTs. Contig ID # ESTs Best Human Match E-value MexCluster_4615_Contig1 415 (NM_000519) delta globin E -39 MexCluster_600_Contig1 354 (NM_182985) ring finger protein 36 isoform a E -110 MexCluster_6279_Contig1 337 (NM_000559) A-gamma globin E -32 MexCluster_10867_Contig1 320 (NM_000558) alpha 1 globin E -38 MexCluster_5357_Contig1 307 (NM_000558) alpha 1 globin E -37 MexCluster_9285_Contig3 285 (NM_001614) actin, gamma 1 propeptide 0 MexCluster_7987_Contig3 252 (NM_001402) eukaryotic translation elongation f1 0 MexCluster_9285_Contig1 240 (NM_001101) beta actin; beta cytoskeletal actin 0 MexCluster_9279_Contig3 218 (NM_000223) keratin 12 E -113 MexCluster_11203_Contig1 181 (NM_002032) ferritin, heavy polypeptide 1 E -70 MexCluster_8737_Contig2 152 (NM_058242) keratin 6C E -131 MexCluster_3193_Contig1 145 (NM_004499) heterogeneous nuclear ribonucleoprotein E -90 MexCluster_8737_Contig7 134 (NM_058242) keratin 6C E -131 MexCluster_5005_Contig3 132 (NM_031263) heterogeneous nuclear ribonucleoprotein E -124 MexCluster_6225_Contig1 125 (NM_001152) solute carrier family 25, member 5 E -151 MexCluster_1066_Contig1 122 [31015660] IMAGE:6953586 E -16 MexCluster_8737_Contig4 114 (NM_058242) keratin 6C; keratin, epidermal type II E -132 MexCluster_8187_Contig2 113 (NM_005507) cofilin 1 (non-muscle) E -65 MexCluster_2761_Contig1 109 (NM_001961) eukaryotic translation elongation factor2 0 MexCluster_9187_Contig1 105 (NM_007355) heat shock 90 kDa protein 1, beta 0 A. t. tigrinum TigCluster_6298_Contig1 654 (NM_000519) delta globin E -38 TigCluster_10099_Contig2 193 (NM_001614) actin, gamma 1 propeptide 0 TigCluster_6470_Contig1 167 (NM_000558) alpha 1 globin E -39 TigCluster_9728_Contig2 142 (NM_000477) albumin precursor E -140 TigCluster_6594_Contig1 117 (NM_001402) eukaryotic translation elongation f1 0 TigCluster_5960_Contig1 91 (NM_001101) beta actin; beta cytoskeletal actin 0 TigCluster_7383_Contig1 77 (NM_001614) actin, gamma 1 propeptide 0 TigCluster_6645_Contig1 76 (NM_001063) transferrin 0 TigCluster_7226_Contig4 74 (NM_006009) tubulin, alpha 3 E -160 TigCluster_7191_Contig1 67 (NM_019016) keratin 24 E -89 TigCluster_10121_Contig1 64 (NM_005141) fibrinogen, beta chain preproprotein 0 TigCluster_6705_Contig1 63 (NM_000558) alpha 1 globin E -39 TigCluster_7854_Contig1 62 (NM_021870) fibrinogen, gamma chain isoform E -121 TigCluster_6139_Contig1 52 (NM_001404) eukaryotic translation elongation f1 0 TigCluster_7226_Contig2 51 (NM_006009) tubulin, alpha 3 0 TigCluster_10231_Contig1 44 (NM_003018) surfactant, pulmonary-associated prot. E -08 TigCluster_6619_Contig1 36 (NM_000041) apolipoprotein E E -38 TigCluster_7232_Contig2 35 (NM_003651) cold shock domain protein A E -46 TigCluster_5768_Contig1 34 (NM_003380) vimentin E -177 TigCluster_9784_Contig3 32 |XP_218445.1| similar to RIKEN cDNA 1810065E05 E -15 Figure 2 Venn diagram of BLAST comparisons among amphibian EST projects.  Values provided are numbers of reciprocal best BLAST hits ( E< 10 -20 ) among quality masked  A. mexicanum  and  A. t. tigrinum  assemblies and a publicly available  X. tropicalis  EST assembly  Functional annotation For the 10,592 contigs that showed significant similarity to sequences from the human RefSeq database, we obtained Gene Ontology (23) information to describe ESTs in functional terms. Although there are hundreds of possible annotations, we chose a list of descriptors for molecular and biological processes that we believe are of interest for research programs currently utilizing salamanders as model organisms (Table  5 ). In all searches, we counted each match between a contig and a RefSeq sequence as identifying a different ambystomatid gene, even when different contigs matched the same RefSeq reference. In almost all cases, approximately the same number of matches was found per functional descriptor for both species. This was not simply because the same loci were being identified for both species, as only 20% of the total number of searched contigs shared sufficient identity (BLASTN;  E< 10 -80  or  E< 10 -20 ) to be potential homologues. In this sense, the sequencing effort between these two species was complementary in yielding a more diverse collection of ESTs that were highly similar to human gene sequences. Table 5 Functional annotation of contigs A. mex A. t. tig Molecular Function (0016209)  antioxidant (0016209) 25 29  binding (0005488) 3117 2578  chaparone (0003754) 100 85  enzyme regulation (003023) 193 223  motor (0003774) 73 75  signal transduction (0004871) 344 375  structural protein (0005198) 501 411  transcriptional reg. (0030528) 296 221  translational reg. (0045182) 94 59  bone remodeling (0046849) 8 8  circulation (0008015) 23 78  immune response (000695) 182 263  respiratory ex. (0009605) 254 288  respiratory in. (0009719) 72 58  stress (0006950) 263 320 Biological Process (0008150) Cellular (0009987)  activation (0001775) 4 6  aging and death (0008219) 158 148  communication (0007154) 701 696  differentiation (0030154) 31 20  extracellular mat. (0043062) 4 4  growth and main. (0008151) 1731 1445  migration (0016477) 8 14  motility (0006928) 163 154 Developmental (0007275)  aging (0007568) 32 21  embryonic (0009790) 6 1  growth (0040007) 2 2  morphogenesis (0009653) 350 272  pigment (0048066) 13 26  post embryonic (0009791) 8 13  reproduction (0000003) 42 27 Physiological (0007582)  coagulation (0050817) 22 73  death and aging (0016265) 159 148  homeostasis (0042592) 22 27  metabolism (0008152) 3059 2513  secretion (0046903) 9 16  sex differentiation (0007548) 3 2 Numbers in parentheses reference GO numbers [23]. Informatic searches for regeneration probes The value of a salamander model to regeneration research will ultimately rest on the ease in which data and results can be cross-referenced to other vertebrate models. For example, differences in the ability of mammals and salamanders to regenerate spinal cord may reflect differences in the way cells of the ependymal layer respond to injury. As is observed in salamanders, ependymal cells in adult mammals also proliferate and differentiate after spinal cord injury (SCI) [ 24 , 25 ]; immediately after contusion injury in adult rat, ependymal cell numbers increase and proliferation continues for at least 4 days [[ 26 ]; but see [ 27 ]]. Rat ependymal cells share some of the same gene expression and protein properties of embryonic stem cells [ 28 ], however no new neurons have been observed to derive from these cells  in vivo  after SCI [ 29 ]. Thus, although endogenous neural progenitors of the ependymal layer may have latent regenerative potential in adult mammals, this potential is not realized. Several recently completed microarray analyses of spinal cord injury in rat now make it possible to cross-reference information between amphibians and mammals. For example, we searched the complete list of significantly up and down regulated genes from Carmel et al. [ 30 ] and Song et al. [ 31 ] against all  Ambystoma  ESTs. Based upon amino acid sequence similarity of translated ESTs (TBLASTX;  E <10 -7 ), we identified DNA sequences corresponding to 69 of these 164 SCI rat genes (Table  6 ). It is likely that we have sequence corresponding to other presumptive orthologues from this list as many of our ESTs only contain a portion of the coding sequence or the untranslated regions (UTR), and in many cases our searches identified closely related gene family members. Thus, many of the genes that show interesting expression patterns after SCI in rat can now be examined in salamander. Table 6 Ambystoma  contigs that show sequence similarity to rat spinal cord injury genes. Ambystoma Contig ID RAT cDNA clone E-value MexCluster_7440_Contig1 gi|1150557|c-myc, exon 2 E -29 MexCluster_4624_Contig1 gi|1468968| brain acyl-CoA synthtase II E -09 TigCluster_4083_Contig1 E -09 TigSingletonClusters_Salamander_4_G20_ab1 gi|1552375| SKR6 gene, a CB1 cannabinoid recept. E -08 MexSingletonClusters_NT009B_B04 gi|17352488| cyclin ania-6a E -46 TigCluster_3719_Contig1 E -114 TigCluster_8423_Contig1 gi|1778068| binding zyginI E -102 TigCluster_7064_Contig1 gi|1836160| Ca2+/calmodulin-dependent E -20 MexCluster_3225_Contig1 gi|1906612| Rattus norvegicus CXC chemokine E -68 TigSingletonClusters_Salamander_13_F03_ab1 E -38 MexSingletonClusters_BL285B_A06 gi|203042| (Na+, K+)-ATPase-beta-2 subunit E -63 TigCluster_6994_Contig1 E -65 MexSingletonClusters_BL014B_F12 gi|203048| plasma membrane Ca2+ ATPase-isoform 2 E -112 TigSingletonClusters_Salamander_5_F07_ab1 E -92 MexCluster_1251_Contig1 gi|203167| GTP-binding protein (G-alpha-i1) E -110 TigSingletonClusters_Salamander_3_P14_ab1 E -152 TigSingletonClusters_Salamander_22_B01_ab1 gi|203336| catechol-O-methyltransferase E -47 TigSingletonClusters_Salamander_17_N04_ab1 gi|203467| voltage-gated K+ channel protein (RK5) E -08 MexSingletonClusters_v1_p8_c16_triplex5ld_ gi|203583| cytosolic retinol-binding protein (CRBP) E -77 TigCluster_6321_Contig1 E -18 MexCluster_5399_Contig1 gi|204647| heme oxygenase gene E -67 TigCluster_2577_Contig1 E -67 MexCluster_4647_Contig1 gi|204664| heat shock protein 27 (Hsp27) E -83 TigSingletonClusters_Salamander_12_M05_ab1 E -51 MexSingletonClusters_BL285C_F02 gi|205404| metabotropic glutamate receptor 3 E -41 TigSingletonClusters_Salamander_2_B24_ab1 gi|205508| myelin/oligodendrocyte glycoprotein E -26 TigCluster_5740_V2_p10_M20_TriplEx5ld_ gi|205531| metallothionein-2 and metallothionein 1 E -08 TigSingletonClusters_V2_p5_A2_TriplEx5ld_ gi|205537| microtubule-associated protein 1A E -59 MexCluster_1645_Contig1 gi|205633| Na, K-ATPase alpha-2 subunit E -149 TigSingletonClusters_Contig328 0 TigSingletonClusters_Contig45 gi|205683| smallest neurofilament protein (NF-L) E -63 MexSingletonClusters_NT016A_A09 gi|205693| nerve growth factor-induced (NGFI-A) E -95 TigSingletonClusters_I09_Ag2_p9_K24_M13R E -24 MexSingletonClusters_NT007A_E07 gi|205754| neuronal protein (NP25) E -64 TigCluster_7148_Contig1 E -57 MexCluster_9504_Contig1 gi|206161| peripheral-type benzodiazepine receptor E -73 MexSingletonClusters_BL016B_B02 gi|206166| protein kinase C type III E -36 TigCluster_981_Contig1 E -27 MexSingletonClusters_nm_19_k3_t3_ gi|206170| brain type II Ca2+/calmodulin-dependent E -117 MexSingletonClusters_v11_p42_j20_t3_049_ab1 gi|207138| norvegicus syntaxin B 1e -079 MexSingletonClusters_nm_14_h19_t3_ gi|207473| neural receptor protein-tyrosine kinase E -40 TigSingletonClusters_Contig336 E -34 TigSingletonClusters_E10_Ag2_p18_O19_M13 gi|2116627| SNAP-25A E -123 MexCluster_211_Contig1 gi|220713| calcineurin A alpha E -63 TigSingletonClusters_Salamander_7_K14_ab1 E -87 MexSingletonClusters_NT014A_G03 gi|220839| platelet-derived growth factor A chain E -21 TigSingletonClusters_Salamander_9_M15_ab1 E -56 TigSingletonClusters_Salamander_19_M06_ab1 gi|2501807| brain digoxin carrier protein E -55 MexSingletonClusters_Contig100 gi|2746069| MAP-kinase phosphatase (cpg21) E -108 TigSingletonClusters_Salamander_11_A16_ab1 E -70 MexCluster_8345_Contig1 gi|2832312| survival motor neuron (smn) E -40 TigCluster_8032_Contig1 E -49 MexCluster_3580_Contig1 gi|294567| heat shock protein 70 (HSP70) 0 TigCluster_8592_Contig2 E -161 TigSingletonClusters_Salamander_17_N08_ab1 gi|2961528| carboxyl-terminal PDZ E -10 MexSingletonClusters_BL286C_D09 gi|298325| sodium-dependent neurotransmitter tran. E -12 TigSingletonClusters_Contig95 E -22 MexSingletonClusters_Contig461 gi|2996031| brain finger protein (BFP) E -08 TigSingletonClusters_Salamander_11_O19_ab1 E -23 TigSingletonClusters_E16_Ag2_p8_O20_M13R gi|3135196| Ca2+/calmodulin-dependent E -33 MexSingletonClusters_Contig188 gi|3252500| CC chemokine receptor protein E -15 MexCluster_6961_Contig1 gi|3319323| suppressor of cytokine signaling-3 E -08 MexSingletonClusters_nm_14_p15_t3_ gi|349552| P-selectin E -16 TigCluster_218_Contig2 E -99 MexSingletonClusters_Contig506 gi|3707306| Normalized rat embryo, cDNA clone E -14 TigSingletonClusters_I16_Ag2_p5_N7_M13R gi|3711670| Normalized rat muscle, cDNA clone E -35 MexSingletonClusters_V1_p1_a10_Triplex5Ld gi|3727094| Normalized rat ovary, cDNA clone E -15 TigSingletonClusters_v2_p1_D20_triplex5ld E -16 MexSingletonClusters_NT005B_F02 gi|3811504| Normalized rat brain, cDNA clone E -35 TigSingletonClusters_Salamander_22_I04_ab1 E -34 TigSingletonClusters_Ag2_p34_N23_M13R gi|405556| adenylyl cyclase-activated serotonin E -17 TigSingletonClusters_Salamander_1_H02_ab1 gi|4103371| putative potassium channel TWIK E -22 MexCluster_4589_Contig1 gi|4135567| Normalized rat embryo, cDNA clone E -32 TigSingletonClusters_Contig220 E -09 TigCluster_4093_Contig1 gi|4228395| cDNA clone UI-R-A0-bc-h-02-0-UI E -104 MexSingletonClusters_nm_21_2_m7_t3_ gi|425471| nuclear factor kappa B p105 subunit E -22 TigCluster_8535_Contig1 E -11 MexSingletonClusters_v6_p1_j6_triplex5_1ld_ gi|430718| Sprague Dawley inducible nitric oxide E -13 TigSingletonClusters_Salamander_15_D22_ab1 E -41 MexCluster_3498_Contig1 gi|436934| Sprague Dawley protein kinase C rec. 0 TigCluster_6648_Contig1 0 MexSingletonClusters_BL279A_B12 gi|464196| phosphodiesterase I E -49 TigSingletonClusters_Salamander_25_P03_ab1 E -75 MexCluster_8708_Contig1 gi|466438| 40kDa ribosomal protein E -168 TigCluster_5877_Contig1 E -168 MexSingletonClusters_nm_14_a9_t3_ gi|493208| stress activated protein kinase alpha II E -51 TigSingletonClusters_Salamander_11_A13_ab1 gi|517393| tau microtubule-associated protein E -44 TigSingletonClusters_Salamander_12_J14_ab1 gi|55933| c-fos E -26 MexSingletonClusters_nm_21_2_l13_t3_ gi|56822| major synaptic vesicel protein p38 E -39 TigCluster_2065_Contig1 E -50 MexCluster_10965_Contig1 gi|56828| nuclear oncoprotein p53 E -75 TigCluster_5315_Contig1 E -66 MexCluster_4245_Contig1 gi|56909| pJunB gene E -50 TigSingletonClusters_G05_Ag2_p9_G8_M13R E -09 MexSingletonClusters_NT013D_C12 gi|56919| region fragment for protein kinase C E -33 TigSingletonClusters_Salamander_21_H19_ab1 E -24 MexCluster_9585_Contig1 gi|57007| ras-related mRNA rab3 E -61 TigCluster_4885_Contig1 E -63 TigSingletonClusters_Salamander_1_M03_ab1 gi|57238| silencer factor B E -13 MexSingletonClusters_NT008B_D05 gi|57341| transforming growth factor-beta 1 E -13 TigSingletonClusters_Salamander_24_I16_ab1 E -20 MexCluster_9533_Contig1 gi|57479| vimentin 0 TigCluster_5768_Contig1 0 MexSingletonClusters_BL283B_A11 gi|596053| immediate early gene transcription E -12 TigSingletonClusters_Salamander_13_J19_ab1 E -16 MexSingletonClusters_v6_p4_j2_triplex5_1ld_ gi|790632| macrophage inflammatory protein-1alpha E -22 TigCluster_2146_Contig1 gi|951175| limbic system-associated membrane prot. E -11 MexSingletonClusters_v11_p54_o4_t3_ gi|971274| neurodegeneration associated protein 1 E -09 TigSingletonClusters_Salamander_2_J12_ab1 E -11 Similar gene expression programs may underlie regeneration of vertebrate appendages such as fish fins and tetrapod limbs. Regeneration could depend on reiterative expression of genes that function in patterning, morphogenesis, and metabolism during normal development and homeostasis. Or, regeneration could depend in part on novel genes that function exclusively in this process. We investigated these alternatives by searching  A. mexicanum  limb regeneration ESTs against UNIGENE zebrafish fin regeneration ESTs (Figure  3 ). This search identified 1357 significant BLAST hits (TBLASTX;  E <10 -7 ) that corresponded to 1058 unique zebrafish ESTs. We then asked whether any of these potential regeneration homologues were represented uniquely in limb and fin regeneration databases (and not in databases derived from other zebrafish tissues). A search of the 1058 zebrafish ESTs against > 400,000 zebrafish ESTs that were sampled from non-regenerating tissues revealed 43 that were unique to the zebrafish regeneration database (Table  7 ). Conceivably, these 43 ESTs may represent transcripts important to appendage regeneration. For example, our search identified several genes (e.g.  hspc128 , pre- B-cell colony enhancing factor 1 ,  galectin 4 ,  galectin 8 ) that may be expressed in progenitor cells that proliferate and differentiate during appendage regeneration. Overall, our results suggest that regeneration is achieved largely through the reiterative expression of genes having additional functions in other developmental contexts, however a small number of genes may be expressed uniquely during appendage regeneration. Figure 3 Results of BLASTN and TBLASTX searches to identify 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. Table 7 Ambystoma  limb regeneration contigs that show sequence similarity to zebrafish fin regeneration ESTs Mex. Contigs Human ID E-value Zfish ID E-value Contig94 gi|10835079| 1e -63 gnl|UG|Dr#S12319632 1e -58 nm_30_a11_t3_ gi|32306539| 1e -58 gnl|UG|Dr#S12312602 1e -35 Contig615 gi|4502693| 1e -70 gnl|UG|Dr#S12313407 1e -34 nm_23_l13_t3_ No Human Hit gnl|UG|Dr#S12320916 1e -31 nm_9_e22_t3_ gi|4758788| 1e -98 gnl|UG|Dr#S12309914 1e -29 nm_8_l17_t3_ gi|21361310| 1e -16 gnl|UG|Dr#S12313396 1e -27 Contig531 gi|13775198| 1e -27 gnl|UG|Dr#S12309680 1e -26 Contig152 gi|5453712| 1e -32 gnl|UG|Dr#S12239884 1e -26 nm_32h_j20_t3_ gi|39777601| 1e -79 gnl|UG|Dr#S12136499 1e -25 Contig1011 gi|39752675| 1e -65 gnl|UG|Dr#S12136499 1e -24 v11_p50_b24_t3_ gi|41208832| 1e -36 gnl|UG|Dr#S12319219 1e -23 Contig589 gi|4506505| 1e -56 gnl|UG|Dr#S12312662 1e -22 Contig785 gi|33695095| 1e -61 gnl|UG|Dr#S12264765 1e -22 Contig157 gi|21361122| 1e -138 gnl|UG|Dr#S12313094 1e -21 v11_p42_j20_t3_049_ab1 gi|47591841| 1e -100 gnl|UG|Dr#S12137806 1e -21 Contig610 gi|10801345| 1e -114 gnl|UG|Dr#S12310326 1e -20 nm_27_o1_t3_ gi|7706429| 1e -72 gnl|UG|Dr#S12310422 1e -19 Contig439 gi|4504799| 1e -25 gnl|UG|Dr#S12309233 1e -19 nm_31_d5_t3_ gi|8923956| 1e -50 gnl|UG|Dr#S12264745 1e -17 v11_p41_h12_t3_026_ab1 No Human Hit gnl|UG|Dr#S12320916 1e -17 Contig129 gi|34932414| 1e -103 gnl|UG|Dr#S12313534 1e -17 nm_14_j21_t3_ gi|4505325| 1e -42 gnl|UG|Dr#S12136571 1e -17 Contig1321 gi|4501857| 1e -80 gnl|UG|Dr#S12309233 1e -17 nm_19_k3_t3_ gi|26051212| 1e -106 gnl|UG|Dr#S12137637 1e -17 Contig488 gi|4557525| 1e -105 gnl|UG|Dr#S12311975 1e -15 nm_35h_k19_t3_ gi|16950607| 1e -43 gnl|UG|Dr#S12196214 1e -15 Contig195 gi|4557231| 1e -99 gnl|UG|Dr#S12309233 1e -14 nm_14_h19_t3_ gi|4503787| 1e -86 gnl|UG|Dr#S12310912 1e -13 v11_p51_d20_t3_ gi|30520322| 1e -19 gnl|UG|Dr#S12321150 1e -13 g3-n14 gi|13654278| 1e -23 gnl|UG|Dr#S12318856 1e -13 nm_29_f2_t3_ gi|4506517| 1e -65 gnl|UG|Dr#S12312662 1e -13 g4-h23 gi|24111250| 1e -33 gnl|UG|Dr#S12312651 1e -13 Math_p2_A2_T3_ No human Hit gnl|UG|Dr#S12078998 1e -13 nm_35h_f4_t3_ gi|41148476| 1e -67 gnl|UG|Dr#S12319663 1e -13 Contig952 gi|21264558| 1e -61 gnl|UG|Dr#S12318843 1e -12 g4-g21 gi|11995474| 1e -65 gnl|UG|Dr#S12192716 1e -12 Contig854 gi|8922789| 1e -117 gnl|UG|Dr#S12313534 1e -11 Contig1105 gi|6912638|| 1e -83 gnl|UG|Dr#S12079967 1e -11 nm_26_f7_t3_ gi|30181238| 1e -83 gnl|UG|Dr#S12319880 1e -11 Contig949 gi|21284385| 1e -68 gnl|UG|Dr#S12290856 1e -11 g3-n3 gi|18490991| 1e -64 gnl|UG|Dr#S12320832 1e -10 v11_p41_m16_t3_007_ab1 gi|4885661| 1e -33 gnl|UG|Dr#S12310912 1e -10 Contig653 gi|4505047| 1e -124 gnl|UG|Dr#S12239868 1e -09 Contig1349 gi|9665259| 1e -46 gnl|UG|Dr#S12320840 1e -09 6h12 gi|31317231| 1e -43 gnl|UG|Dr#S12321311 1e -09 v11_p43h_i14_t3_070_ab1 No Human Hit gnl|UG|Dr#S12320916 1e -09 nm_35h_d11_t3_ gi|7661790| 1e -35 gnl|UG|Dr#S12196146 1e -09 nm_35h_k22_t3_ gi|5031977| 1e -124 gnl|UG|Dr#S12242267 1e -09 v11_p48_g2_t3_087_ab1 gi|11496277| 1e -60 gnl|UG|Dr#S12312396 1e -09 nm_30_e11_t3_ gi|32483357| 1e -56 gnl|UG|Dr#S12309103 1e -08 nm_28_f23_t3_ gi|42544191| 1e -25 gnl|UG|Dr#S12239884 1e -08 nm_12_p16_t3_ gi|21361553| 1e -21 gnl|UG|Dr#S12310912 1e -08 nm_32h_a8_t3_ gi|11386179| 1e -22 gnl|UG|Dr#S12312152 1e -08 Human RefSeq sequence ID's are provided to allow cross-referencing. DNA sequence polymorphisms within and between  A. mexicanum  and  A. t. tigrinum The identification of single nucleotide polymorphisms (SNPs) within and between orthologous sequences of  A. mexicanum  and  A. t. tigrinum  is needed to develop DNA markers for genome mapping [ 32 ], quantitative genetic analysis [ 33 ], and population genetics [ 34 ]. We estimated within species polymorphism for both species by calculating the frequency of SNPs among ESTs within the 20 largest contigs (Table  4 ). These analyses considered a total of 30,638 base positions for  A. mexicanum  and 18,765 base positions for  A. t. tigrinum . Two classes of polymorphism were considered in this analysis: those occurring at moderate (identified in 10–30% of the EST sequences) and high frequencies (identified in at least 30% of the EST sequences). Within the  A. mexicanum  contigs, 0.49% and 0.06% of positions were polymorphic at moderate and high frequency, while higher levels of polymorphism were observed for  A. t. tigrinum  (1.41% and 0.20%). Higher levels of polymorphism are expected for  A. t. tigrinum  because they exist in larger, out-bred populations in nature. To identify SNPs between species, we had to first identify presumptive, interspecific orthologues. We did this by performing BLASTN searches between the  A. mexicanum  and  A. t. tigrinum  assemblies, and the resulting alignments 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 disproportionately small effect on the overall total length of BLAST alignments. A threshold of  E< 10 -80 yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of  E< 10 -20  yielded 2820 alignments 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. mexicanum  and  A. t. tigrinum  transcripts are estimated to be 97% identical at the nucleotide level, including both protein 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 natural 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 perspective, 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 facilitate 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 informative 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. mexicanum and A. t. tigrinum  EST contigs were aligned to identify orthologous genes with species-specific sequence variations (SNPs or Insertion/Deletions = INDELs). Primer pairs corresponding 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. mexicanum  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 individual 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 amplify the corresponding sequence in a related  A. tigrinum  complex species, and for small DNA fragments in the range of 150–500 bp, approximately half are expected to have informative polymorphisms. Table 8 EST loci used in a population-level PCR amplification screen in  A. ordinarium Locus ID Forward Primer 5' to 3' Reverse Primer 5' to 3' 1F8 AAGAAGGTCGGGATTGTGGGTAA CAGCCTTCCTCTTCATCTTTGTCTTG 1H3 GGCAAATGCTGGTCCCAACACAAA GGACAACACTGCCAAATACCACAT 2C8 GCAAGCACCAGCCACATAAAG GGCCACCATAACCACTCTGCT 3B10 TCAAAACGAATAAGGGAAGAGCGACTG TTGCCCCCATAATAAGCCATCCATC 5E7 ACGCTTCGCTGGGGTTGACAT CGGTAGGATTTCTGGTAGCGAGCAC 5F4 CCGAGATGAGATTTATAGAAGGAC TAGGGGAAGTTAAACATAGATAGAA 6A3 GTTTATGAAGGCGAGAGGGCTATGACCA ATCTTGTTCTCCTCGCCAGTGCTCTTGT 6B1 TGATGCTGGCGAGTACAAACCCCCTTCT TTTACCATTCCTTCCCTTCGGCAGCACA 6B3 ACCACGTGCTGTCTTCCCATCCAT ACGAAGCTCATTGTAGAAGGTGTG 6B4 CCCACGATGAATTGGAATTGGACAT CTGCCTGCCAGACCTACAGACTATCGT 6C4 ATGGCGCCAAAGTGATGAGTA GGGCCAGGCACACGACCACAAT 6D2 ATCAAGGCTGGCATGGTGGTCA GGGGGTCGTTCTTGCTGTCA 6H8 GAAGAAGACAGAAACGCAGGAGAAAAAC CGGGCGGGGGCGGGTCACAGTAAAAC BL005B_A01.5.1 GACAGGTCATGAACTTTTGAAAATAA AAAGTATATGTACCAAATGGGAGAGC BL006A_G07.5.1 GATGTCCTCTCCACTATACAAGTGTG GTTTGACTTGTCACCACTTTATCAAC BL012D_F02.5.1 ACAGCCAGAAATAGAAACTTTGAACT TGAAAGTATGTATTGTTTTCACAGGG BL013C_E01.5.1 AGGATGAAATAATATGCTGTGCTTC ACCGTGATAAACTCCATCCCTT BL014D_B11.5.1 AGCAAAACTCCTCTATGAATCTCG ATTGCACACTAAATAGGTGAATACGA BL279A_G10.5.1 ATGGCAGGATGAAGAAAGACAT ATGCACTTTGGACCCACTGAG Et.fasta.Contig1023.5.1 TGTGGTTATTGGACTACTTCACTCTC AAACGTCCATTTGACACTGTATTTTA Et.fasta.Contig1166.5.1 GAATGAAGAGAAAATGTTTTGAAGGT GCACAGTATTGGCTATGAGCAC Et.fasta.Contig1311.5.1 AGAAAACTGTGTCAAGCTTATTTTCC CAACTTAGTGTTCACATTTCTGAGGT Et.fasta.Contig1335.5.1 CCACTTATGGTAGTTCCCACTTTTAT GCTAAAGAATACCAAGAACCTTTGAC Et.fasta.Contig1381.5.1 GTCACAGGTATAACATTGAAAGGATG TAAATGAATCAAACATTGAAGAGAGC Et.fasta.Contig1459.5.1 ATAACAAGGACATGTTCTGCTGG CTAGCAGAACCCTGTATAGCCTG Et.fasta.Contig1506.5.1 AGGATATCCGCTCAGAAATATGAAG CTGACCACTTGCAAAACTTACTACCT Et.fasta.Contig1578.5.1 CCTAGAACATTACCAAAACAGACTCA AATGAAGAAGTATTGCATGTGAGAAC Et.fasta.Contig1647.5.1 GTACAACGTCAGGCAAAGCTATTCT ATCTCCAACACCGTGGCTAAT Et.fasta.Contig1717.5.1 GAACTTGTTGGCAGGTTTCTCTT CTAGTGATAGGTTGGACATACCAGAG Et.fasta.Contig1796.5.1 TGTGGGTATGTATATGGCTAACTTGT AGATTTTATGTGCTACTGCATTTACG Et.fasta.Contig1908.5.1 CTCATGACTTAATTGCTGTTCTTCG ATAACCATTCTGAGGTTTTGAGTTG Et.fasta.Contig1941.5.1 ATCTCCTGCTTCATCTCTTGATTTAT TAACAGATTTAATAAACGTCCCCTTC Et.fasta.Contig1943.5.1 AGTACGATGAATCTGGTCCTTCAAT CCACAATACTGACATACTCTGGTCTT Et.fasta.Contig325.5.1 GTGAAGTCAGTGAGTAAAGTCCATGT CTAGGATACCAGTGGGAGAGTGTAAT Et.fasta.Contig330.5.1 GTCATCACCTCCACTACTTCACAAG TTTTGGCACTGTAAGATTCTATGAAC Et.fasta.Contig536.5.1 CCTTAGGTAGAACAGACTGAAGCAG GAAACATGAAACTGGACTTGTTTTAG Et.fasta.Contig917.5.1 GGATGCAGATTCTTCCTATTTTACTC CTGGTCACTTTACTTGTTTTCAGTGT Et.fasta.Contig926.5.1 TTCATCACATTCTACTTCACAAATCA CTAGGCAAGCAAGCTTTCTAATAGTT Et.fasta.Contig93.5.1 GAATAAAAGCAACAATTGCAGAGTTA CTCGACTCCTTCTACGATCTCTACTC Et.fasta.Contig990.5.1 GTTTAGGTTAGTATGAAGGATCCCAA TGCCAGTACTCACCAATTAGTAAAAG G1-C12 CCCAAATCCAGGAGTTCAAA TGGGACCTGGGGCTTCATT G1-C13 TTGCCCGAGAAAAGGAAGGACATA CAAGGGTGGGTGAGGGACATC G1-C5 F-CACTGTTGACTTGGGTTATGTTATT CTGCTCCTAGGGTTTGTGAAG G1-C7 CCCGTGTGGCTGGCTTGTGC TCGGCTACTTTGGTGTTTTTCTCCCTCAT G1-C9 TGGTCCGGCAACAGCATCAGA GCTTTTCGGTATTCAACGGCAGAGTG G1-C9 TGGTCCGGCAACAGCATCAGA GCTTTTCGGTATTCAACGGCAGAGTG G1-D5 AGACCCTTGCTGTGTAACTGCT GACTGGGACTGACTTCTATGACG G1-D6 CAGCGTGCCCACCCGATAGAA TCCCAAAAAGTAAAATGTGCAAAGAAAA G1-D7 CAGCGGTGGAAATGACAAACAGG CCAAGACGACGAGGAACGGTATT G1-E12 CAACCATGAGAGGAGGCCAGAGAAC AAAACAGCACTACCTACAAAACCCTATT G1-F1 TTAGTTTGGGTGCAGACAGGA GGTGCTCAACAACAAATCAACT G1-F20 TCCCCAACAACTCCAGCAGAT GGAAACCACCTAGACGAAAAATG G1-I18 CATGTTTGTGGGTGTGGTGAA AAAAGCGGCATCTGGTAAGG G1-I19 ACCCAGACCTGTCCACCTCA GAACAGCTCTCCAATCCACAAG G1-I21 CCAAGCGAAGGAGGCGTGTG CATGTGGCTCTTTGTTTCTGGA G1-I5 TAATCGTGTTTGGTGGCATCCTTGAGTC AGCAGCAGTTCCATTTTCCCACACCA G1-I8 ACCTGCAGTGGGCTAAGACC ATGGAAATAATAAAATAAAATGTT G1-J10 CGTTCGCTTTGCCTGCCACA GGCTCTTCCCCGGTCGTCCAC G1-J17 AGCGCCTTCTACACGGACAC TATGCCCCAATTACTCTTCTGC G1-J2 TACAGTAACTATGCCAAGATGAAATG CAATATGGATAATGGCTGTAGACC G1-J20 ATCCTCCAAGCTCACTACAACA CCAGCCCCTTCCCAAACAG G1-J9 CTGTCATTGCCTGCATCGGGGAGAAG TGTTGAGGGGAAGCAGTTTTG G1-K2 GCTTTCGCCTTTGACACCTC GGCCGGACCATTGCTGAAGAAG G1-L11 AAAGTGACCATCCAGTGCCCAAACCT CCGGCCGAAACTGACGAGATACATTAG G1-L13 TCAGCTGCACTAGGTTTGTC CATTTTGATTTGCTCCATAA G1-L19 GACAACCTTGAATCCTTTATG AGATGTTGGTTGGTGACTTAT G1-L20 TGGGCATAGATGGCAAGGAAAAA CCCCCAGCATCTCGCATACAC G1-L7 GTGCTACAGGAAGGAATGGATG TAGCACAGGAACAGCCGACAATAA G1-M14 CCGCTTGGACATGAGGAGAT TGGCAAAGAAACAGAACACAACTA G1-M19 GAGAAGTAGTGTCCCGGCAGAAAC ATGGGTGAAAACTTAGGTGAAATG G1-N9 GCGGGGCAATACATGACGTTCCACAG GACCCCCATCTCCGTTTCCCATTCC G1-O1 GGGGTAGAGCACAGTCCAGTT TTGCAAGGCCGAAAAGGTG G1-O12 GGAATTCCGGGGCACTACT TCGCGAGGACGGGGAAGAG G1-O24 CGGCCTTCCTGCAGTACAACCATC TCGGCAACGTGAAGACCATA G2-A11 GCCCCTGGAAGCTGTTGTGA GGGGTCCATCCGAGTCC G2-A7 TTACCCCACAGACAAAATCAACACC GGCGGCCCCTCATAGCAC G2-B1 GGGCCTAGTCCTGCTGGTC CAAAGAGTGCGGAGAAATGG G2-B8 CAACATGCGACCACTATAGCCACTTCCT CGCCACCGCCACCACCACA G2-C2 TTTGCAGGAAGAGTCATAACACAG GTCAACAACACCCTTTTCCCTTCCT G2-D1 GCAGGTCGGCAAGAAGCTAAAGAAGGAA AGGGTTGGTTTGAAAGGATGTGCTGGTAA G2-E17 GGAGCACCAAATTCAAGTCAG CGTCCCCGGTCAATCTCCAC G2-E19 CCAGTTTGAGCCCCAGGAG TCGCGGCAGTCAAGAGGTC G2-F17 TATCCTCTTATTGCTGCATTCTCCTCAC AGTACGGCCGTTCACCATCTCTG G2-F2 CACACCACAGACGCATTGAC TCCCCAGCCTGTGTAGAAC G2-G13 GGGAGGGGAGAAGGCTACCA ATACACGGCTTCCATGCTTCTTCTT G2-G15 CCACGGCCCCACATCCAGC TCCCGCAGAATTTCCGTATCCAT G2-G21 TCCAAGAGGGTGTGAGGTGAAC AAAGCCATGCGAAGCGGAAGAC G2-G23 GGTTTGGTACTTCAGCGGATGT CCAAAGCCTGTACTATGCGAAAAG G2-G5 CGGTCCCTACTGTGGTCTATGGTTTTCA GGCTCTGCATATCCTCGGTCACACTTCC G2-G6 CCCATGGCTGCAAGGATTACG CAGGGGTTGTTGGGAGGCAGTGT G2-H18 TTGTCAAATGGGCGAGTTCA TGTTTTGCACCCAGTTTTTG G2-I18 GATCTCCTCAGGTCTCTTTCA GATTATGGGCCGGTGTCTCT G2-I23 TGACTTTCCCAATGTGAGCAGAC CAGAGGTGGTGTTACAGCAGCAGTTT G2-J12 CCTCTTGTCCCAGTGCCAGTG TCCAGGGATCCGAAACAAAG G2-J21 CCGCCTCAGCCTGTTTCTCTACTTTT CTTTGAATTTCTGCTTTTGGTGCTCTGC G2-K12 ACATTAGTCCTGGTTACGAGAGC AAAGGGCAGTCCAGCATTGA G2-K2 CTGCCCAAGAAGACCGAGAGCCACAAG AGCGCCCCCTGCACCAAAATCA G2-L16 CCAAGGGTAGGAGAACAAGACA ATGGCATGCTGGGAAATCA G2-L21 GAATCTAGGTCCAAGCAGTCCCATCT GACCATCACACCACTACCCACACTCA G2-L3 TGAAAGAGGCCAGAAACAAGTAG TTCCCAAGGTCTCCATAACAAT G2-L4 TGGCCAAGAAGATGAAACAGGAAGAGGAG TGGCAAAGGACACGACGCAGAG G2-M14 CGGCCTCCTCGACGCATACG CCAGGCCGGCCCATTGTTC G2-M24 ACGGAGCACGGTCAGATTTCACG CCCGGCTGGCTCTTCTTGCTCTT G2-M3 CGATCCGCATTGAACGAGT TGTGGCAGGAAGGAGAAGG G2-N2 CGTGTTTTCCTCCTATGTCGACTTCTTTG ACGTGCTCTGCCTTTCTTGATCTTGTGTT G3-D7 AGGATTTCTTGGCCGGTGGAGTGG GAAGTTGAGGGCCTGGGTGGGGAAGTA NT001D_E08.5.1 AGAAGTTCCTAGATGAGTTGGAGGAG AATTAATTTCCTAAACCAGGTGACAG NT010B_E09.5.1 GAAGAGGTCCTAAAATATCAAGATGC ATGATAGACTTCGTCCTTGTCATAGA NT014D_E01.5.1 AAAGAAGTCCCGCATCTAACCT ATTAAATATGAGAAGATGTGTGCAGG V2_p1_b8 AGTCACTGTGTTACATTATCACCCAC ATAATTATACACTGCGGTCTGCATCT V2_p1_c5 AGTACCTGTTCGACAAGCACAC TGAGAACATAGACAAGTTAACATACACC V2_p1_d10 GAGATAGAAAGGCTGCATAAAGAAAT TATGTTTCAACAATGTACAGGAAACC V2_p1_d4 CACCAGAACAAGCTGTATTTTTATGT TGGTTTGCATCATATATTAAAGGGTA V2_p1_g7 GACTTCAAGCACATTGGGAAAC ATTGTAAACTTGATAGGCTGGTGAG V2_p2_g6 AGAATTCCCAATAGCACCTGAAAT CACTTGGTAAATACATACACACAGCA V2_p2_h2 CTTTTTGGCCTGGTCTTTTTG AGATTCTTCAGACTCGTCCTTCTTT V2_p3_a5 TTTACACAGAAACCTTGTTTATTTGGC TTTAAGGATGCTTAGAGGCAAAGTATT V2_p3_b1 AGTCACTGTGTTACATTATCACCCAC TATACACTGCGGTCTGCATCTACT V2_p5_b3 AATGGGATGAAGAGCGAGAAT CTGCCCCATTGACATTTACCTA V2_p5_h3 CCTTCAGACGAAAACAGCACTAAG TACAGTGTATGAGAGCCCAATATTTC V2_p6_a4 AGAAATACATCAAATATCGGGTGG AAAAAGGACAATGTTCAGCTCTCT V3_p1_a21 ACCAAGTTCTTGGAAAGTGGTG CTTAGTGTCTCCTGGGTTTGAATAG V3_p1_b13 GTCTTGGTACTCAATGAAGGAGATG TCAATCTGATGAAGAGTTTACATGTCT Comparative gene mapping Salamanders occupy a pivotal phylogenetic position for reconstructing the ancestral tetrapod genome structure and for providing perspective on the extremely derived anuran  Xenopus  (37) that is currently providing the bulk of amphibian genome information. Here we show the utility of ambystomatid ESTs for identifying chromosomal regions that are conserved between salamanders and other vertebrates. A region of conserved synteny that corresponds to human chromosome ( Hsa ) 17q has been identified in several non-mammalian taxa including reptiles (38) and fishes (39). In a previous study Voss et al. (40) identified a region of conserved synteny between  Ambystoma  and  Hsa  17q that included collagen type 1 alpha 1 ( Col1a1 ), thyroid hormone receptor alpha ( Thra ), homeo box b13 ( Hoxb13) , and distal-less 3 ( Dlx3)  (Figure  4 ). To evaluate both the technical feasibility of mapping ESTs and the likelihood that presumptive orthologues map to the same synteny group, we searched our assemblies for presumptive  Hsa  17 orthologues and then developed a subset of these loci for genetic linkage mapping. Using a joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs, 97 Hsa 17 presumptive orthologues were identified. We chose 15 genes from this list and designed PCR primers to amplify a short DNA fragment containing 1 or more presumptive SNPs that were identified in the joint assembly (Table  9 ). All but two of these genes were mapped, indicating a high probability of mapping success using markers developed from the joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs. All 6 ESTs that exhibited 'best hits' to loci within the previously defined human- Ambystoma  synteny group did map to this region ( Hspc009 ,  Sui1 ,  Krt17 ,  Krt24 ,  Flj13855,  and  Rpl19 ). Our results show that BLAST-based definitions of orthology are informative between salamanders and human. All other presumptive  Hsa  17 loci mapped to  Ambystoma  chromosomal regions outside of the previously defined synteny group. It is interesting to note that two of these loci mapped to the same ambystomatid linkage group ( Cgi-125 ,  Flj20345 ), but in human the presumptive orthologues are 50 Mb apart and distantly flank the syntenic loci in Figure  4 . Assuming orthology has been assigned correctly for these loci, this suggests a dynamic history for some  Hsa  17 orthologues during vertebrate evolution. Figure 4 Comparison of gene order between  Ambystoma  linkage group 1 and an 11 Mb region of Hsa17 (37.7 Mb to 48.7 Mb) . Lines connect the positions of putatively orthologous genes. Table 9 Presumptive human chromosome 17 loci that were mapped in  Ambystoma Marker ID Primers a Diagnosis b LG c Symbol d RefSeq ID e E-value f Pl_6_E/F_6 F-GAAAACCTGCTCAGCATTAGTGT ASA ul PFN1 NP_005013 E -34 R-TCTATTACCATAGCATTAATTGGCAG Pl_5_G/H_5 F-CTATTTCATCTGAGTACCGTTGAATG PE (A) 23 CGI-125 NP_057144 E -56 R-TAATGTAGAACTAAATGGCATCCTTC E-CCATGGTGCAGGAAGAGAGCCTATAT Pl_0.4_A/B_1 F-GTCTCATTATCCGCAAACCTGT SP 1 RPL19 NP_000972 E -67 R-ATTCTCATCCTCCTCATCCACGAC Pl_4_B_7/8 F-CCTAGAACATTACCAAAACAGACTCA RD (Dpn II) 1 KRT10 NP_061889 E -17 R-AATGAAGAAGTATTGCATGTGAGAAC Pl_4_B_9/10 F-GAACTTGTTGGCAGGTTTCTCTT RD (AciI) 1 KRT17 NP_000413 E -146 R-CTAGTGATAGGTTGGACATACCAGAG Pl_10_C/D_4 F-CTCCACTATTTAAAGGACATGCTACA PE (A) 1 SUI1 NP_005792 E -48 R-TTAATATAGCACAACATTGCCTCATT E-TGCTACATTAATGTAATAAACGGCATCATC Pl_6_E/F_11 F-AAGAGAAGTTCCTAGATGAGTTGGAG PE (A) 1 HSPC009 NP_054738 E -26 R-TGAAGAGAGAACTCAAAGTGTCTGAT E-TCATGTTTTGCTCTGCTGTGCAGT Pl_9_A/B_10 F-TGATAGTTTCTGGATTAAGACGAGTG PE (T) 1 FLJ13855 NP_075567 E -15 R-CTTAGAGCCATTGTTACAAGATGTTC E-GTGATCTAGTGGGATCAAACCCTAAAGACC Pl_10_C/D_9 F-AAAGTGCCAAGAAGGAGATTAACTT PE (T) 9 NME1 NP_000260 E -71 R-GAGCTCAGAAAACAAGGCAGTAAC E-AAATGGATCTACGAGTAGACCTTGACCC Pl_9_C/D_9 F-GAGTCTCCTTTAGGATTGACGTATCT PE (T) 23 FLJ20345 NP_060247 E -17 R-GCTATGTGAGCAGAGATAAAAGTCAG E-GTTACAGCATCAGTGGGATGTGGTATGT Pl_8_C/D_9 F-AGGATACCAACCTCTGTGCTATACAT PE (C) 15 H3F3B NP_005315 E -66 R-TAAATGTATTTACAAACCGAAAGCAA E-CGTGGCGAGCGTGCCTAGT Pl_9_C/D_4 F-GTGGTTATTTGTAACATTTCGTTGAC PE (A) 8 SFRS2 NP_003007 E -40 R-AATTACATTTGGGCTTCTCAATTTAC E-TTTTTAAACGCGTAAAAATGTTAACAGA Pl_6_C/D_5 F-CCGTAAATGTTTCTAAATGACAGTTG PE (G) 2 ACTG1 NP_001605 0 R-GGAAAGAAAGTACAATCAAGTCCTTC E-GATTGAAAACTGGAACCGAAAGAAGATAAA a Sequences are 5' amplification primers, 3' amplification primers, or primer extension probes, and are preceded by F-, R-, and E- respectively.  b Genotyping methods are abbreviated: allele specific amplification (ASA), size polymorphism (SP), restriction digestion (RD), primer extension (PE). Diagnostic restriction enzymes and diagnostic extension bases are provided in parentheses.  c Ambystoma linkage group ID. "ul" designates markers that are unlinked.  d Official gene symbols as defined by the Human Genome Organization Gene Nomenclature Committee  .  e Best BLASTX hit (highest e-value) from the human RefSeq database using the contig from which each marker was designed as a query sequence.  f Highest E-value statistic obtained by searching contigs, from which EST markers were designed, against the human RefSeq database. Future directions Ambystomatid salamanders are classic model organisms that continue to inform biological research in a variety of areas. Their future importance in regenerative biology and metamorphosis will almost certainly escalate as genome resources and other molecular and cellular approaches become widely available. Among the genomic resources currently under development (see [ 41 ]) are a comparative genome map, which will allow mapping of candidate genes, QTL, and comparative anchors for cross-referencing the salamander genome to fully sequenced vertebrate models. In closing, we reiterate a second benefit to resource development in  Ambystoma . Genome resources in  Ambystoma  can be extended to multiple, closely related species to explore the molecular basis of natural, phenotypic variation. Such extensions can better inform our understanding of ambystomatid biodiversity in nature and draw attention to the need for conserving such naturalistic systems. Several paedomorphic species, including  A. mexicanum , are on the brink of extinction. We can think of no better investment than one that simultaneously enhances research in all areas of biology and draws attention to the conservation needs of model organisms in their natural habitats.
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 primarily from regenerating larval tissues in  A. mexicanum  and several non-regenerating larval tissues in  A. t. tigrinum . Table 1 Tissues selected to make cDNA libraries. ID Tissue cDNAs sequenced GARD limb blastema 1029 MATH limb blastema 16244 V1 tail blastema 1422 V2 brain 3196 V3 liver 792 V4 spleen 337 V5 heart 38 V6 gill 3039 V7 stage 22 embryo 96 AG liver, gonad, lung, kidney, heart, gill 19871 Further information is found in Methods and Materials.
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 primarily from regenerating larval tissues in  A. mexicanum  and several non-regenerating larval tissues in  A. t. tigrinum .A. mexicanumA. t. tigrinumTissues selected to make cDNA libraries.IDTissuecDNAs sequencedFurther information is found in Methods and Materials.EST sequencing and clustering A total of 46,064 cDNA clones were sequenced, yielding 39,982 high quality sequences for  A. mexicanum  and  A. t. tigrinum  (Table  2 ). Of these, 3,745 corresponded to mtDNA and were removed from the dataset; complete mtDNA genome data for these and other ambystomatid species will be reported elsewhere. The remaining nuclear ESTs for each species were clustered and assembled separately. We included in our  A. mexicanum  assembly an additional 16,030 high quality ESTs that were generated recently for regenerating tail and neurula stage embryos [ 20 ]. Thus, a total of 32,891 and 19,376 ESTs were clustered for  A. mexicanum  and  A. t. tigrinum , respectively. Using PaCE clustering and CAP3 assembly, a similar number of EST clusters and contigs were identified for each species (Table  2 ). Overall contig totals were 11,190 and 9,901 for  A. mexicanum  and  A. t. tigrinum  respectively. Thus, although 13,515 more  A. mexicanum  ESTs were assembled, a roughly equivalent number of contigs were obtained for both species. This indicates that EST development was more efficient for  A. t. tigrinum,  presumably because ESTs were obtained primarily from the normalized AG library; indeed, there were approximately twice as many ESTs on average per  A. mexicanum  contig (Table  2 ). Thus, our EST project yielded an approximately equivalent number of contigs for  A. mexicanum  and  A. t. tigrinum , and overall we identified > 21,000 different contigs. Assuming that 20% of the contigs correspond to redundant loci, which has been found generally in large EST projects [ 21 ], we identified transcripts for approximately 17,000 different ambystomatid loci. If ambystomatid salamanders have approximately the same number of loci as other vertebrates (e.g. [ 22 ]), we have isolated roughly half the expected number of genes in the genome. Table 2 EST summary and assembly results. A. mex A. t. tig cDNA clones sequenced 21830 24234 high-quality sequences 19383 20599 mt DNA sequence 2522 1223 seqs submitted to NCBI 16861 19376 sequences assembled 32891 a 19376  PaCE clusters 11381 10226  ESTs in contigs 25457 12676  contigs 3756 3201  singlets 7434 6700  putative transcripts 11190 9901 a Includes 16,030 ESTs from [20].
EST sequencing and clustering
A total of 46,064 cDNA clones were sequenced, yielding 39,982 high quality sequences for  A. mexicanum  and  A. t. tigrinum  (Table  2 ). Of these, 3,745 corresponded to mtDNA and were removed from the dataset; complete mtDNA genome data for these and other ambystomatid species will be reported elsewhere. The remaining nuclear ESTs for each species were clustered and assembled separately. We included in our  A. mexicanum  assembly an additional 16,030 high quality ESTs that were generated recently for regenerating tail and neurula stage embryos [ 20 ]. Thus, a total of 32,891 and 19,376 ESTs were clustered for  A. mexicanum  and  A. t. tigrinum , respectively. Using PaCE clustering and CAP3 assembly, a similar number of EST clusters and contigs were identified for each species (Table  2 ). Overall contig totals were 11,190 and 9,901 for  A. mexicanum  and  A. t. tigrinum  respectively. Thus, although 13,515 more  A. mexicanum  ESTs were assembled, a roughly equivalent number of contigs were obtained for both species. This indicates that EST development was more efficient for  A. t. tigrinum,  presumably because ESTs were obtained primarily from the normalized AG library; indeed, there were approximately twice as many ESTs on average per  A. mexicanum  contig (Table  2 ). Thus, our EST project yielded an approximately equivalent number of contigs for  A. mexicanum  and  A. t. tigrinum , and overall we identified > 21,000 different contigs. Assuming that 20% of the contigs correspond to redundant loci, which has been found generally in large EST projects [ 21 ], we identified transcripts for approximately 17,000 different ambystomatid loci. If ambystomatid salamanders have approximately the same number of loci as other vertebrates (e.g. [ 22 ]), we have isolated roughly half the expected number of genes in the genome.A. mexicanumA. t. tigrinumA. mexicanumA. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinum,A. mexicanumA. mexicanumA. t. tigrinumEST summary and assembly results.A. mexA. mexA. t. tigA. t. tiga Includes 16,030 ESTs from [20].Identification of vertebrate sequences similar to  Ambystoma  contigs We searched all contigs against several vertebrate databases to identify sequences that exhibited significant sequence similarity. As our objective was to reliably annotate as many contigs as possible, we first searched against 19,804 sequences in the NCBI human RefSeq database (Figure  1 ), which is actively reviewed and curated by biologists. This search revealed 5619 and 4973 "best hit" matches for the  A. mexicanum  and  A. t. tigrinum  EST datasets at a BLASTX threshold of  E  = 10 -7 . The majority of contigs were supported at more stringent E-value thresholds (Table  3 ). Non-matching contigs were subsequently searched against the Non-Redundant (nr) Protein database and  Xenopus tropicalus  and  X. laevis  UNIGENE ESTs (Figure  1 ). These later two searches yielded a few hundred more 'best hit' matches, however a relatively large number of ESTs from both ambystomatid species were not similar to any sequences from the databases above. Presumably, these non-matching sequences were obtained from the non-coding regions of transcripts or they contain protein-coding sequences that are novel to salamander. Although the majority are probably of the former type, we did identify 3,273 sequences from the non-matching set that had open reading frames (ORFs) of at least 200 bp, and 911 of these were greater than 300 bp. Figure 1 Results of BLASTX and TBLASTX searches to identify best BLAST hits for  Ambystoma  contigs searched against NCBI human RefSeq, nr, and  Xenopus  Unigene databases . Table 3 Ambystoma  contig search of NCBI human RefSeq, nr, and  Xenopus  Unigene databases. A. mex A. t. tig # BLASTX Best Matches 6283 5545 < E -100 630 870 < E -50  > E -100 2015 1990 < E -20  > E -50 2153 1595 < E -10  > E -20 967 745 < E -7  > E -10 518 345 The distribution of ESTs among contigs can provide perspective on gene expression when clones are randomly sequenced from non-normalized cDNA libraries. In general, frequently sampled transcripts may be expressed at higher levels. We identified the 20 contigs from  A. mexicanum  and  A. t. tigrinum  that contained the most assembled ESTs (Table  4 ). The largest  A. t. tigrinum  contigs contained fewer ESTs than the largest  A. mexicanum  contigs, probably because fewer overall  A. t. tigrinum  clones were sequenced, with the majority selected from a normalized library. However, we note that the contig with the most ESTs was identified for  A. t. tigrinum:  delta globin. In both species, transcripts corresponding to globin genes were sampled more frequently than all other loci. This may reflect the fact that amphibians, unlike mammals, have nucleated red blood cells that are transcriptionally active. In addition to globin transcripts, a few other house-keeping genes were identified in common from both species, however the majority of the contigs were unique to each list. Overall, the strategy of sequencing cDNAs from a diverse collection of tissues (from normalized and non-normalized libraries) yielded different sets of highly redundant contigs. Only 25% and 28% of the  A. mexicanum  and  A. t. tigrinum  contigs, respectively, were identified in common (Figure  2 ). We also note that several hundred contigs were identified in common between Xenopus and  Ambystoma ; this will help facilitate comparative studies among these amphibian models. Table 4 Top 20 contigs with the most assembled ESTs. Contig ID # ESTs Best Human Match E-value MexCluster_4615_Contig1 415 (NM_000519) delta globin E -39 MexCluster_600_Contig1 354 (NM_182985) ring finger protein 36 isoform a E -110 MexCluster_6279_Contig1 337 (NM_000559) A-gamma globin E -32 MexCluster_10867_Contig1 320 (NM_000558) alpha 1 globin E -38 MexCluster_5357_Contig1 307 (NM_000558) alpha 1 globin E -37 MexCluster_9285_Contig3 285 (NM_001614) actin, gamma 1 propeptide 0 MexCluster_7987_Contig3 252 (NM_001402) eukaryotic translation elongation f1 0 MexCluster_9285_Contig1 240 (NM_001101) beta actin; beta cytoskeletal actin 0 MexCluster_9279_Contig3 218 (NM_000223) keratin 12 E -113 MexCluster_11203_Contig1 181 (NM_002032) ferritin, heavy polypeptide 1 E -70 MexCluster_8737_Contig2 152 (NM_058242) keratin 6C E -131 MexCluster_3193_Contig1 145 (NM_004499) heterogeneous nuclear ribonucleoprotein E -90 MexCluster_8737_Contig7 134 (NM_058242) keratin 6C E -131 MexCluster_5005_Contig3 132 (NM_031263) heterogeneous nuclear ribonucleoprotein E -124 MexCluster_6225_Contig1 125 (NM_001152) solute carrier family 25, member 5 E -151 MexCluster_1066_Contig1 122 [31015660] IMAGE:6953586 E -16 MexCluster_8737_Contig4 114 (NM_058242) keratin 6C; keratin, epidermal type II E -132 MexCluster_8187_Contig2 113 (NM_005507) cofilin 1 (non-muscle) E -65 MexCluster_2761_Contig1 109 (NM_001961) eukaryotic translation elongation factor2 0 MexCluster_9187_Contig1 105 (NM_007355) heat shock 90 kDa protein 1, beta 0 A. t. tigrinum TigCluster_6298_Contig1 654 (NM_000519) delta globin E -38 TigCluster_10099_Contig2 193 (NM_001614) actin, gamma 1 propeptide 0 TigCluster_6470_Contig1 167 (NM_000558) alpha 1 globin E -39 TigCluster_9728_Contig2 142 (NM_000477) albumin precursor E -140 TigCluster_6594_Contig1 117 (NM_001402) eukaryotic translation elongation f1 0 TigCluster_5960_Contig1 91 (NM_001101) beta actin; beta cytoskeletal actin 0 TigCluster_7383_Contig1 77 (NM_001614) actin, gamma 1 propeptide 0 TigCluster_6645_Contig1 76 (NM_001063) transferrin 0 TigCluster_7226_Contig4 74 (NM_006009) tubulin, alpha 3 E -160 TigCluster_7191_Contig1 67 (NM_019016) keratin 24 E -89 TigCluster_10121_Contig1 64 (NM_005141) fibrinogen, beta chain preproprotein 0 TigCluster_6705_Contig1 63 (NM_000558) alpha 1 globin E -39 TigCluster_7854_Contig1 62 (NM_021870) fibrinogen, gamma chain isoform E -121 TigCluster_6139_Contig1 52 (NM_001404) eukaryotic translation elongation f1 0 TigCluster_7226_Contig2 51 (NM_006009) tubulin, alpha 3 0 TigCluster_10231_Contig1 44 (NM_003018) surfactant, pulmonary-associated prot. E -08 TigCluster_6619_Contig1 36 (NM_000041) apolipoprotein E E -38 TigCluster_7232_Contig2 35 (NM_003651) cold shock domain protein A E -46 TigCluster_5768_Contig1 34 (NM_003380) vimentin E -177 TigCluster_9784_Contig3 32 |XP_218445.1| similar to RIKEN cDNA 1810065E05 E -15 Figure 2 Venn diagram of BLAST comparisons among amphibian EST projects.  Values provided are numbers of reciprocal best BLAST hits ( E< 10 -20 ) among quality masked  A. mexicanum  and  A. t. tigrinum  assemblies and a publicly available  X. tropicalis  EST assembly
Identification of vertebrate sequences similar to  Ambystoma  contigs
AmbystomaWe searched all contigs against several vertebrate databases to identify sequences that exhibited significant sequence similarity. As our objective was to reliably annotate as many contigs as possible, we first searched against 19,804 sequences in the NCBI human RefSeq database (Figure  1 ), which is actively reviewed and curated by biologists. This search revealed 5619 and 4973 "best hit" matches for the  A. mexicanum  and  A. t. tigrinum  EST datasets at a BLASTX threshold of  E  = 10 -7 . The majority of contigs were supported at more stringent E-value thresholds (Table  3 ). Non-matching contigs were subsequently searched against the Non-Redundant (nr) Protein database and  Xenopus tropicalus  and  X. laevis  UNIGENE ESTs (Figure  1 ). These later two searches yielded a few hundred more 'best hit' matches, however a relatively large number of ESTs from both ambystomatid species were not similar to any sequences from the databases above. Presumably, these non-matching sequences were obtained from the non-coding regions of transcripts or they contain protein-coding sequences that are novel to salamander. Although the majority are probably of the former type, we did identify 3,273 sequences from the non-matching set that had open reading frames (ORFs) of at least 200 bp, and 911 of these were greater than 300 bp.A. mexicanumA. t. tigrinumEXenopus tropicalusX. laevisAmbystoma  contig search of NCBI human RefSeq, nr, and  Xenopus  Unigene databases.AmbystomaXenopusA. mexA. mexA. t. tigA. t. tigThe distribution of ESTs among contigs can provide perspective on gene expression when clones are randomly sequenced from non-normalized cDNA libraries. In general, frequently sampled transcripts may be expressed at higher levels. We identified the 20 contigs from  A. mexicanum  and  A. t. tigrinum  that contained the most assembled ESTs (Table  4 ). The largest  A. t. tigrinum  contigs contained fewer ESTs than the largest  A. mexicanum  contigs, probably because fewer overall  A. t. tigrinum  clones were sequenced, with the majority selected from a normalized library. However, we note that the contig with the most ESTs was identified for  A. t. tigrinum:  delta globin. In both species, transcripts corresponding to globin genes were sampled more frequently than all other loci. This may reflect the fact that amphibians, unlike mammals, have nucleated red blood cells that are transcriptionally active. In addition to globin transcripts, a few other house-keeping genes were identified in common from both species, however the majority of the contigs were unique to each list. Overall, the strategy of sequencing cDNAs from a diverse collection of tissues (from normalized and non-normalized libraries) yielded different sets of highly redundant contigs. Only 25% and 28% of the  A. mexicanum  and  A. t. tigrinum  contigs, respectively, were identified in common (Figure  2 ). We also note that several hundred contigs were identified in common between Xenopus and  Ambystoma ; this will help facilitate comparative studies among these amphibian models.A. mexicanumA. t. tigrinumA. t. tigrinumA. mexicanumA. t. tigrinumA. t. tigrinum:A. mexicanumA. t. tigrinumAmbystomaTop 20 contigs with the most assembled ESTs.Contig ID# ESTsBest Human MatchE-valueA. t. tigrinumA. t. tigrinumFunctional annotation For the 10,592 contigs that showed significant similarity to sequences from the human RefSeq database, we obtained Gene Ontology (23) information to describe ESTs in functional terms. Although there are hundreds of possible annotations, we chose a list of descriptors for molecular and biological processes that we believe are of interest for research programs currently utilizing salamanders as model organisms (Table  5 ). In all searches, we counted each match between a contig and a RefSeq sequence as identifying a different ambystomatid gene, even when different contigs matched the same RefSeq reference. In almost all cases, approximately the same number of matches was found per functional descriptor for both species. This was not simply because the same loci were being identified for both species, as only 20% of the total number of searched contigs shared sufficient identity (BLASTN;  E< 10 -80  or  E< 10 -20 ) to be potential homologues. In this sense, the sequencing effort between these two species was complementary in yielding a more diverse collection of ESTs that were highly similar to human gene sequences. Table 5 Functional annotation of contigs A. mex A. t. tig Molecular Function (0016209)  antioxidant (0016209) 25 29  binding (0005488) 3117 2578  chaparone (0003754) 100 85  enzyme regulation (003023) 193 223  motor (0003774) 73 75  signal transduction (0004871) 344 375  structural protein (0005198) 501 411  transcriptional reg. (0030528) 296 221  translational reg. (0045182) 94 59  bone remodeling (0046849) 8 8  circulation (0008015) 23 78  immune response (000695) 182 263  respiratory ex. (0009605) 254 288  respiratory in. (0009719) 72 58  stress (0006950) 263 320 Biological Process (0008150) Cellular (0009987)  activation (0001775) 4 6  aging and death (0008219) 158 148  communication (0007154) 701 696  differentiation (0030154) 31 20  extracellular mat. (0043062) 4 4  growth and main. (0008151) 1731 1445  migration (0016477) 8 14  motility (0006928) 163 154 Developmental (0007275)  aging (0007568) 32 21  embryonic (0009790) 6 1  growth (0040007) 2 2  morphogenesis (0009653) 350 272  pigment (0048066) 13 26  post embryonic (0009791) 8 13  reproduction (0000003) 42 27 Physiological (0007582)  coagulation (0050817) 22 73  death and aging (0016265) 159 148  homeostasis (0042592) 22 27  metabolism (0008152) 3059 2513  secretion (0046903) 9 16  sex differentiation (0007548) 3 2 Numbers in parentheses reference GO numbers [23].
Functional annotation
For the 10,592 contigs that showed significant similarity to sequences from the human RefSeq database, we obtained Gene Ontology (23) information to describe ESTs in functional terms. Although there are hundreds of possible annotations, we chose a list of descriptors for molecular and biological processes that we believe are of interest for research programs currently utilizing salamanders as model organisms (Table  5 ). In all searches, we counted each match between a contig and a RefSeq sequence as identifying a different ambystomatid gene, even when different contigs matched the same RefSeq reference. In almost all cases, approximately the same number of matches was found per functional descriptor for both species. This was not simply because the same loci were being identified for both species, as only 20% of the total number of searched contigs shared sufficient identity (BLASTN;  E< 10 -80  or  E< 10 -20 ) to be potential homologues. In this sense, the sequencing effort between these two species was complementary in yielding a more diverse collection of ESTs that were highly similar to human gene sequences.E<E<Functional annotation of contigsA. mexA. mexA. t. tigA. t. tigMolecular Function (0016209)Biological Process (0008150)Numbers in parentheses reference GO numbers [23].Informatic searches for regeneration probes The value of a salamander model to regeneration research will ultimately rest on the ease in which data and results can be cross-referenced to other vertebrate models. For example, differences in the ability of mammals and salamanders to regenerate spinal cord may reflect differences in the way cells of the ependymal layer respond to injury. As is observed in salamanders, ependymal cells in adult mammals also proliferate and differentiate after spinal cord injury (SCI) [ 24 , 25 ]; immediately after contusion injury in adult rat, ependymal cell numbers increase and proliferation continues for at least 4 days [[ 26 ]; but see [ 27 ]]. Rat ependymal cells share some of the same gene expression and protein properties of embryonic stem cells [ 28 ], however no new neurons have been observed to derive from these cells  in vivo  after SCI [ 29 ]. Thus, although endogenous neural progenitors of the ependymal layer may have latent regenerative potential in adult mammals, this potential is not realized. Several recently completed microarray analyses of spinal cord injury in rat now make it possible to cross-reference information between amphibians and mammals. For example, we searched the complete list of significantly up and down regulated genes from Carmel et al. [ 30 ] and Song et al. [ 31 ] against all  Ambystoma  ESTs. Based upon amino acid sequence similarity of translated ESTs (TBLASTX;  E <10 -7 ), we identified DNA sequences corresponding to 69 of these 164 SCI rat genes (Table  6 ). It is likely that we have sequence corresponding to other presumptive orthologues from this list as many of our ESTs only contain a portion of the coding sequence or the untranslated regions (UTR), and in many cases our searches identified closely related gene family members. Thus, many of the genes that show interesting expression patterns after SCI in rat can now be examined in salamander. Table 6 Ambystoma  contigs that show sequence similarity to rat spinal cord injury genes. Ambystoma Contig ID RAT cDNA clone E-value MexCluster_7440_Contig1 gi|1150557|c-myc, exon 2 E -29 MexCluster_4624_Contig1 gi|1468968| brain acyl-CoA synthtase II E -09 TigCluster_4083_Contig1 E -09 TigSingletonClusters_Salamander_4_G20_ab1 gi|1552375| SKR6 gene, a CB1 cannabinoid recept. E -08 MexSingletonClusters_NT009B_B04 gi|17352488| cyclin ania-6a E -46 TigCluster_3719_Contig1 E -114 TigCluster_8423_Contig1 gi|1778068| binding zyginI E -102 TigCluster_7064_Contig1 gi|1836160| Ca2+/calmodulin-dependent E -20 MexCluster_3225_Contig1 gi|1906612| Rattus norvegicus CXC chemokine E -68 TigSingletonClusters_Salamander_13_F03_ab1 E -38 MexSingletonClusters_BL285B_A06 gi|203042| (Na+, K+)-ATPase-beta-2 subunit E -63 TigCluster_6994_Contig1 E -65 MexSingletonClusters_BL014B_F12 gi|203048| plasma membrane Ca2+ ATPase-isoform 2 E -112 TigSingletonClusters_Salamander_5_F07_ab1 E -92 MexCluster_1251_Contig1 gi|203167| GTP-binding protein (G-alpha-i1) E -110 TigSingletonClusters_Salamander_3_P14_ab1 E -152 TigSingletonClusters_Salamander_22_B01_ab1 gi|203336| catechol-O-methyltransferase E -47 TigSingletonClusters_Salamander_17_N04_ab1 gi|203467| voltage-gated K+ channel protein (RK5) E -08 MexSingletonClusters_v1_p8_c16_triplex5ld_ gi|203583| cytosolic retinol-binding protein (CRBP) E -77 TigCluster_6321_Contig1 E -18 MexCluster_5399_Contig1 gi|204647| heme oxygenase gene E -67 TigCluster_2577_Contig1 E -67 MexCluster_4647_Contig1 gi|204664| heat shock protein 27 (Hsp27) E -83 TigSingletonClusters_Salamander_12_M05_ab1 E -51 MexSingletonClusters_BL285C_F02 gi|205404| metabotropic glutamate receptor 3 E -41 TigSingletonClusters_Salamander_2_B24_ab1 gi|205508| myelin/oligodendrocyte glycoprotein E -26 TigCluster_5740_V2_p10_M20_TriplEx5ld_ gi|205531| metallothionein-2 and metallothionein 1 E -08 TigSingletonClusters_V2_p5_A2_TriplEx5ld_ gi|205537| microtubule-associated protein 1A E -59 MexCluster_1645_Contig1 gi|205633| Na, K-ATPase alpha-2 subunit E -149 TigSingletonClusters_Contig328 0 TigSingletonClusters_Contig45 gi|205683| smallest neurofilament protein (NF-L) E -63 MexSingletonClusters_NT016A_A09 gi|205693| nerve growth factor-induced (NGFI-A) E -95 TigSingletonClusters_I09_Ag2_p9_K24_M13R E -24 MexSingletonClusters_NT007A_E07 gi|205754| neuronal protein (NP25) E -64 TigCluster_7148_Contig1 E -57 MexCluster_9504_Contig1 gi|206161| peripheral-type benzodiazepine receptor E -73 MexSingletonClusters_BL016B_B02 gi|206166| protein kinase C type III E -36 TigCluster_981_Contig1 E -27 MexSingletonClusters_nm_19_k3_t3_ gi|206170| brain type II Ca2+/calmodulin-dependent E -117 MexSingletonClusters_v11_p42_j20_t3_049_ab1 gi|207138| norvegicus syntaxin B 1e -079 MexSingletonClusters_nm_14_h19_t3_ gi|207473| neural receptor protein-tyrosine kinase E -40 TigSingletonClusters_Contig336 E -34 TigSingletonClusters_E10_Ag2_p18_O19_M13 gi|2116627| SNAP-25A E -123 MexCluster_211_Contig1 gi|220713| calcineurin A alpha E -63 TigSingletonClusters_Salamander_7_K14_ab1 E -87 MexSingletonClusters_NT014A_G03 gi|220839| platelet-derived growth factor A chain E -21 TigSingletonClusters_Salamander_9_M15_ab1 E -56 TigSingletonClusters_Salamander_19_M06_ab1 gi|2501807| brain digoxin carrier protein E -55 MexSingletonClusters_Contig100 gi|2746069| MAP-kinase phosphatase (cpg21) E -108 TigSingletonClusters_Salamander_11_A16_ab1 E -70 MexCluster_8345_Contig1 gi|2832312| survival motor neuron (smn) E -40 TigCluster_8032_Contig1 E -49 MexCluster_3580_Contig1 gi|294567| heat shock protein 70 (HSP70) 0 TigCluster_8592_Contig2 E -161 TigSingletonClusters_Salamander_17_N08_ab1 gi|2961528| carboxyl-terminal PDZ E -10 MexSingletonClusters_BL286C_D09 gi|298325| sodium-dependent neurotransmitter tran. E -12 TigSingletonClusters_Contig95 E -22 MexSingletonClusters_Contig461 gi|2996031| brain finger protein (BFP) E -08 TigSingletonClusters_Salamander_11_O19_ab1 E -23 TigSingletonClusters_E16_Ag2_p8_O20_M13R gi|3135196| Ca2+/calmodulin-dependent E -33 MexSingletonClusters_Contig188 gi|3252500| CC chemokine receptor protein E -15 MexCluster_6961_Contig1 gi|3319323| suppressor of cytokine signaling-3 E -08 MexSingletonClusters_nm_14_p15_t3_ gi|349552| P-selectin E -16 TigCluster_218_Contig2 E -99 MexSingletonClusters_Contig506 gi|3707306| Normalized rat embryo, cDNA clone E -14 TigSingletonClusters_I16_Ag2_p5_N7_M13R gi|3711670| Normalized rat muscle, cDNA clone E -35 MexSingletonClusters_V1_p1_a10_Triplex5Ld gi|3727094| Normalized rat ovary, cDNA clone E -15 TigSingletonClusters_v2_p1_D20_triplex5ld E -16 MexSingletonClusters_NT005B_F02 gi|3811504| Normalized rat brain, cDNA clone E -35 TigSingletonClusters_Salamander_22_I04_ab1 E -34 TigSingletonClusters_Ag2_p34_N23_M13R gi|405556| adenylyl cyclase-activated serotonin E -17 TigSingletonClusters_Salamander_1_H02_ab1 gi|4103371| putative potassium channel TWIK E -22 MexCluster_4589_Contig1 gi|4135567| Normalized rat embryo, cDNA clone E -32 TigSingletonClusters_Contig220 E -09 TigCluster_4093_Contig1 gi|4228395| cDNA clone UI-R-A0-bc-h-02-0-UI E -104 MexSingletonClusters_nm_21_2_m7_t3_ gi|425471| nuclear factor kappa B p105 subunit E -22 TigCluster_8535_Contig1 E -11 MexSingletonClusters_v6_p1_j6_triplex5_1ld_ gi|430718| Sprague Dawley inducible nitric oxide E -13 TigSingletonClusters_Salamander_15_D22_ab1 E -41 MexCluster_3498_Contig1 gi|436934| Sprague Dawley protein kinase C rec. 0 TigCluster_6648_Contig1 0 MexSingletonClusters_BL279A_B12 gi|464196| phosphodiesterase I E -49 TigSingletonClusters_Salamander_25_P03_ab1 E -75 MexCluster_8708_Contig1 gi|466438| 40kDa ribosomal protein E -168 TigCluster_5877_Contig1 E -168 MexSingletonClusters_nm_14_a9_t3_ gi|493208| stress activated protein kinase alpha II E -51 TigSingletonClusters_Salamander_11_A13_ab1 gi|517393| tau microtubule-associated protein E -44 TigSingletonClusters_Salamander_12_J14_ab1 gi|55933| c-fos E -26 MexSingletonClusters_nm_21_2_l13_t3_ gi|56822| major synaptic vesicel protein p38 E -39 TigCluster_2065_Contig1 E -50 MexCluster_10965_Contig1 gi|56828| nuclear oncoprotein p53 E -75 TigCluster_5315_Contig1 E -66 MexCluster_4245_Contig1 gi|56909| pJunB gene E -50 TigSingletonClusters_G05_Ag2_p9_G8_M13R E -09 MexSingletonClusters_NT013D_C12 gi|56919| region fragment for protein kinase C E -33 TigSingletonClusters_Salamander_21_H19_ab1 E -24 MexCluster_9585_Contig1 gi|57007| ras-related mRNA rab3 E -61 TigCluster_4885_Contig1 E -63 TigSingletonClusters_Salamander_1_M03_ab1 gi|57238| silencer factor B E -13 MexSingletonClusters_NT008B_D05 gi|57341| transforming growth factor-beta 1 E -13 TigSingletonClusters_Salamander_24_I16_ab1 E -20 MexCluster_9533_Contig1 gi|57479| vimentin 0 TigCluster_5768_Contig1 0 MexSingletonClusters_BL283B_A11 gi|596053| immediate early gene transcription E -12 TigSingletonClusters_Salamander_13_J19_ab1 E -16 MexSingletonClusters_v6_p4_j2_triplex5_1ld_ gi|790632| macrophage inflammatory protein-1alpha E -22 TigCluster_2146_Contig1 gi|951175| limbic system-associated membrane prot. E -11 MexSingletonClusters_v11_p54_o4_t3_ gi|971274| neurodegeneration associated protein 1 E -09 TigSingletonClusters_Salamander_2_J12_ab1 E -11 Similar gene expression programs may underlie regeneration of vertebrate appendages such as fish fins and tetrapod limbs. Regeneration could depend on reiterative expression of genes that function in patterning, morphogenesis, and metabolism during normal development and homeostasis. Or, regeneration could depend in part on novel genes that function exclusively in this process. We investigated these alternatives by searching  A. mexicanum  limb regeneration ESTs against UNIGENE zebrafish fin regeneration ESTs (Figure  3 ). This search identified 1357 significant BLAST hits (TBLASTX;  E <10 -7 ) that corresponded to 1058 unique zebrafish ESTs. We then asked whether any of these potential regeneration homologues were represented uniquely in limb and fin regeneration databases (and not in databases derived from other zebrafish tissues). A search of the 1058 zebrafish ESTs against > 400,000 zebrafish ESTs that were sampled from non-regenerating tissues revealed 43 that were unique to the zebrafish regeneration database (Table  7 ). Conceivably, these 43 ESTs may represent transcripts important to appendage regeneration. For example, our search identified several genes (e.g.  hspc128 , pre- B-cell colony enhancing factor 1 ,  galectin 4 ,  galectin 8 ) that may be expressed in progenitor cells that proliferate and differentiate during appendage regeneration. Overall, our results suggest that regeneration is achieved largely through the reiterative expression of genes having additional functions in other developmental contexts, however a small number of genes may be expressed uniquely during appendage regeneration. Figure 3 Results of BLASTN and TBLASTX searches to identify 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. Table 7 Ambystoma  limb regeneration contigs that show sequence similarity to zebrafish fin regeneration ESTs Mex. Contigs Human ID E-value Zfish ID E-value Contig94 gi|10835079| 1e -63 gnl|UG|Dr#S12319632 1e -58 nm_30_a11_t3_ gi|32306539| 1e -58 gnl|UG|Dr#S12312602 1e -35 Contig615 gi|4502693| 1e -70 gnl|UG|Dr#S12313407 1e -34 nm_23_l13_t3_ No Human Hit gnl|UG|Dr#S12320916 1e -31 nm_9_e22_t3_ gi|4758788| 1e -98 gnl|UG|Dr#S12309914 1e -29 nm_8_l17_t3_ gi|21361310| 1e -16 gnl|UG|Dr#S12313396 1e -27 Contig531 gi|13775198| 1e -27 gnl|UG|Dr#S12309680 1e -26 Contig152 gi|5453712| 1e -32 gnl|UG|Dr#S12239884 1e -26 nm_32h_j20_t3_ gi|39777601| 1e -79 gnl|UG|Dr#S12136499 1e -25 Contig1011 gi|39752675| 1e -65 gnl|UG|Dr#S12136499 1e -24 v11_p50_b24_t3_ gi|41208832| 1e -36 gnl|UG|Dr#S12319219 1e -23 Contig589 gi|4506505| 1e -56 gnl|UG|Dr#S12312662 1e -22 Contig785 gi|33695095| 1e -61 gnl|UG|Dr#S12264765 1e -22 Contig157 gi|21361122| 1e -138 gnl|UG|Dr#S12313094 1e -21 v11_p42_j20_t3_049_ab1 gi|47591841| 1e -100 gnl|UG|Dr#S12137806 1e -21 Contig610 gi|10801345| 1e -114 gnl|UG|Dr#S12310326 1e -20 nm_27_o1_t3_ gi|7706429| 1e -72 gnl|UG|Dr#S12310422 1e -19 Contig439 gi|4504799| 1e -25 gnl|UG|Dr#S12309233 1e -19 nm_31_d5_t3_ gi|8923956| 1e -50 gnl|UG|Dr#S12264745 1e -17 v11_p41_h12_t3_026_ab1 No Human Hit gnl|UG|Dr#S12320916 1e -17 Contig129 gi|34932414| 1e -103 gnl|UG|Dr#S12313534 1e -17 nm_14_j21_t3_ gi|4505325| 1e -42 gnl|UG|Dr#S12136571 1e -17 Contig1321 gi|4501857| 1e -80 gnl|UG|Dr#S12309233 1e -17 nm_19_k3_t3_ gi|26051212| 1e -106 gnl|UG|Dr#S12137637 1e -17 Contig488 gi|4557525| 1e -105 gnl|UG|Dr#S12311975 1e -15 nm_35h_k19_t3_ gi|16950607| 1e -43 gnl|UG|Dr#S12196214 1e -15 Contig195 gi|4557231| 1e -99 gnl|UG|Dr#S12309233 1e -14 nm_14_h19_t3_ gi|4503787| 1e -86 gnl|UG|Dr#S12310912 1e -13 v11_p51_d20_t3_ gi|30520322| 1e -19 gnl|UG|Dr#S12321150 1e -13 g3-n14 gi|13654278| 1e -23 gnl|UG|Dr#S12318856 1e -13 nm_29_f2_t3_ gi|4506517| 1e -65 gnl|UG|Dr#S12312662 1e -13 g4-h23 gi|24111250| 1e -33 gnl|UG|Dr#S12312651 1e -13 Math_p2_A2_T3_ No human Hit gnl|UG|Dr#S12078998 1e -13 nm_35h_f4_t3_ gi|41148476| 1e -67 gnl|UG|Dr#S12319663 1e -13 Contig952 gi|21264558| 1e -61 gnl|UG|Dr#S12318843 1e -12 g4-g21 gi|11995474| 1e -65 gnl|UG|Dr#S12192716 1e -12 Contig854 gi|8922789| 1e -117 gnl|UG|Dr#S12313534 1e -11 Contig1105 gi|6912638|| 1e -83 gnl|UG|Dr#S12079967 1e -11 nm_26_f7_t3_ gi|30181238| 1e -83 gnl|UG|Dr#S12319880 1e -11 Contig949 gi|21284385| 1e -68 gnl|UG|Dr#S12290856 1e -11 g3-n3 gi|18490991| 1e -64 gnl|UG|Dr#S12320832 1e -10 v11_p41_m16_t3_007_ab1 gi|4885661| 1e -33 gnl|UG|Dr#S12310912 1e -10 Contig653 gi|4505047| 1e -124 gnl|UG|Dr#S12239868 1e -09 Contig1349 gi|9665259| 1e -46 gnl|UG|Dr#S12320840 1e -09 6h12 gi|31317231| 1e -43 gnl|UG|Dr#S12321311 1e -09 v11_p43h_i14_t3_070_ab1 No Human Hit gnl|UG|Dr#S12320916 1e -09 nm_35h_d11_t3_ gi|7661790| 1e -35 gnl|UG|Dr#S12196146 1e -09 nm_35h_k22_t3_ gi|5031977| 1e -124 gnl|UG|Dr#S12242267 1e -09 v11_p48_g2_t3_087_ab1 gi|11496277| 1e -60 gnl|UG|Dr#S12312396 1e -09 nm_30_e11_t3_ gi|32483357| 1e -56 gnl|UG|Dr#S12309103 1e -08 nm_28_f23_t3_ gi|42544191| 1e -25 gnl|UG|Dr#S12239884 1e -08 nm_12_p16_t3_ gi|21361553| 1e -21 gnl|UG|Dr#S12310912 1e -08 nm_32h_a8_t3_ gi|11386179| 1e -22 gnl|UG|Dr#S12312152 1e -08 Human RefSeq sequence ID's are provided to allow cross-referencing.
Informatic searches for regeneration probes
The value of a salamander model to regeneration research will ultimately rest on the ease in which data and results can be cross-referenced to other vertebrate models. For example, differences in the ability of mammals and salamanders to regenerate spinal cord may reflect differences in the way cells of the ependymal layer respond to injury. As is observed in salamanders, ependymal cells in adult mammals also proliferate and differentiate after spinal cord injury (SCI) [ 24 , 25 ]; immediately after contusion injury in adult rat, ependymal cell numbers increase and proliferation continues for at least 4 days [[ 26 ]; but see [ 27 ]]. Rat ependymal cells share some of the same gene expression and protein properties of embryonic stem cells [ 28 ], however no new neurons have been observed to derive from these cells  in vivo  after SCI [ 29 ]. Thus, although endogenous neural progenitors of the ependymal layer may have latent regenerative potential in adult mammals, this potential is not realized. Several recently completed microarray analyses of spinal cord injury in rat now make it possible to cross-reference information between amphibians and mammals. For example, we searched the complete list of significantly up and down regulated genes from Carmel et al. [ 30 ] and Song et al. [ 31 ] against all  Ambystoma  ESTs. Based upon amino acid sequence similarity of translated ESTs (TBLASTX;  E <10 -7 ), we identified DNA sequences corresponding to 69 of these 164 SCI rat genes (Table  6 ). It is likely that we have sequence corresponding to other presumptive orthologues from this list as many of our ESTs only contain a portion of the coding sequence or the untranslated regions (UTR), and in many cases our searches identified closely related gene family members. Thus, many of the genes that show interesting expression patterns after SCI in rat can now be examined in salamander.in vivoAmbystomaEAmbystoma  contigs that show sequence similarity to rat spinal cord injury genes.AmbystomaAmbystoma Contig IDRAT cDNA cloneE-valueSimilar gene expression programs may underlie regeneration of vertebrate appendages such as fish fins and tetrapod limbs. Regeneration could depend on reiterative expression of genes that function in patterning, morphogenesis, and metabolism during normal development and homeostasis. Or, regeneration could depend in part on novel genes that function exclusively in this process. We investigated these alternatives by searching  A. mexicanum  limb regeneration ESTs against UNIGENE zebrafish fin regeneration ESTs (Figure  3 ). This search identified 1357 significant BLAST hits (TBLASTX;  E <10 -7 ) that corresponded to 1058 unique zebrafish ESTs. We then asked whether any of these potential regeneration homologues were represented uniquely in limb and fin regeneration databases (and not in databases derived from other zebrafish tissues). A search of the 1058 zebrafish ESTs against > 400,000 zebrafish ESTs that were sampled from non-regenerating tissues revealed 43 that were unique to the zebrafish regeneration database (Table  7 ). Conceivably, these 43 ESTs may represent transcripts important to appendage regeneration. For example, our search identified several genes (e.g.  hspc128 , pre- B-cell colony enhancing factor 1 ,  galectin 4 ,  galectin 8 ) that may be expressed in progenitor cells that proliferate and differentiate during appendage regeneration. Overall, our results suggest that regeneration is achieved largely through the reiterative expression of genes having additional functions in other developmental contexts, however a small number of genes may be expressed uniquely during appendage regeneration.A. mexicanumEhspc128B-cell colony enhancing factor 1galectin 4galectin 8Ambystoma  limb regeneration contigs that show sequence similarity to zebrafish fin regeneration ESTsAmbystomaMex. ContigsHuman IDE-valueZfish IDE-valueHuman RefSeq sequence ID's are provided to allow cross-referencing.DNA sequence polymorphisms within and between  A. mexicanum  and  A. t. tigrinum The identification of single nucleotide polymorphisms (SNPs) within and between orthologous sequences of  A. mexicanum  and  A. t. tigrinum  is needed to develop DNA markers for genome mapping [ 32 ], quantitative genetic analysis [ 33 ], and population genetics [ 34 ]. We estimated within species polymorphism for both species by calculating the frequency of SNPs among ESTs within the 20 largest contigs (Table  4 ). These analyses considered a total of 30,638 base positions for  A. mexicanum  and 18,765 base positions for  A. t. tigrinum . Two classes of polymorphism were considered in this analysis: those occurring at moderate (identified in 10–30% of the EST sequences) and high frequencies (identified in at least 30% of the EST sequences). Within the  A. mexicanum  contigs, 0.49% and 0.06% of positions were polymorphic at moderate and high frequency, while higher levels of polymorphism were observed for  A. t. tigrinum  (1.41% and 0.20%). Higher levels of polymorphism are expected for  A. t. tigrinum  because they exist in larger, out-bred populations in nature. To identify SNPs between species, we had to first identify presumptive, interspecific orthologues. We did this by performing BLASTN searches between the  A. mexicanum  and  A. t. tigrinum  assemblies, and the resulting alignments 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 disproportionately small effect on the overall total length of BLAST alignments. A threshold of  E< 10 -80 yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of  E< 10 -20  yielded 2820 alignments 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. mexicanum  and  A. t. tigrinum  transcripts are estimated to be 97% identical at the nucleotide level, including both protein 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 natural 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 perspective, 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.
DNA sequence polymorphisms within and between  A. mexicanum  and  A. t. tigrinum
A. mexicanumA. t. tigrinumThe identification of single nucleotide polymorphisms (SNPs) within and between orthologous sequences of  A. mexicanum  and  A. t. tigrinum  is needed to develop DNA markers for genome mapping [ 32 ], quantitative genetic analysis [ 33 ], and population genetics [ 34 ]. We estimated within species polymorphism for both species by calculating the frequency of SNPs among ESTs within the 20 largest contigs (Table  4 ). These analyses considered a total of 30,638 base positions for  A. mexicanum  and 18,765 base positions for  A. t. tigrinum . Two classes of polymorphism were considered in this analysis: those occurring at moderate (identified in 10–30% of the EST sequences) and high frequencies (identified in at least 30% of the EST sequences). Within the  A. mexicanum  contigs, 0.49% and 0.06% of positions were polymorphic at moderate and high frequency, while higher levels of polymorphism were observed for  A. t. tigrinum  (1.41% and 0.20%). Higher levels of polymorphism are expected for  A. t. tigrinum  because they exist in larger, out-bred populations in nature.A. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumA. t. tigrinumTo identify SNPs between species, we had to first identify presumptive, interspecific orthologues. We did this by performing BLASTN searches between the  A. mexicanum  and  A. t. tigrinum  assemblies, and the resulting alignments 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 disproportionately small effect on the overall total length of BLAST alignments. A threshold of  E< 10 -80 yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of  E< 10 -20  yielded 2820 alignments 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. mexicanum  and  A. t. tigrinum  transcripts are estimated to be 97% identical at the nucleotide level, including both protein 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 natural 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 perspective, 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.A. mexicanumA. t. tigrinumEEE<E<EE<A. mexicanumA. t. tigrinumA. tigrinumExtending 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 facilitate 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 informative 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. mexicanum and A. t. tigrinum  EST contigs were aligned to identify orthologous genes with species-specific sequence variations (SNPs or Insertion/Deletions = INDELs). Primer pairs corresponding 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. mexicanum  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 individual 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 amplify the corresponding sequence in a related  A. tigrinum  complex species, and for small DNA fragments in the range of 150–500 bp, approximately half are expected to have informative polymorphisms. Table 8 EST loci used in a population-level PCR amplification screen in  A. ordinarium Locus ID Forward Primer 5' to 3' Reverse Primer 5' to 3' 1F8 AAGAAGGTCGGGATTGTGGGTAA CAGCCTTCCTCTTCATCTTTGTCTTG 1H3 GGCAAATGCTGGTCCCAACACAAA GGACAACACTGCCAAATACCACAT 2C8 GCAAGCACCAGCCACATAAAG GGCCACCATAACCACTCTGCT 3B10 TCAAAACGAATAAGGGAAGAGCGACTG TTGCCCCCATAATAAGCCATCCATC 5E7 ACGCTTCGCTGGGGTTGACAT CGGTAGGATTTCTGGTAGCGAGCAC 5F4 CCGAGATGAGATTTATAGAAGGAC TAGGGGAAGTTAAACATAGATAGAA 6A3 GTTTATGAAGGCGAGAGGGCTATGACCA ATCTTGTTCTCCTCGCCAGTGCTCTTGT 6B1 TGATGCTGGCGAGTACAAACCCCCTTCT TTTACCATTCCTTCCCTTCGGCAGCACA 6B3 ACCACGTGCTGTCTTCCCATCCAT ACGAAGCTCATTGTAGAAGGTGTG 6B4 CCCACGATGAATTGGAATTGGACAT CTGCCTGCCAGACCTACAGACTATCGT 6C4 ATGGCGCCAAAGTGATGAGTA GGGCCAGGCACACGACCACAAT 6D2 ATCAAGGCTGGCATGGTGGTCA GGGGGTCGTTCTTGCTGTCA 6H8 GAAGAAGACAGAAACGCAGGAGAAAAAC CGGGCGGGGGCGGGTCACAGTAAAAC BL005B_A01.5.1 GACAGGTCATGAACTTTTGAAAATAA AAAGTATATGTACCAAATGGGAGAGC BL006A_G07.5.1 GATGTCCTCTCCACTATACAAGTGTG GTTTGACTTGTCACCACTTTATCAAC BL012D_F02.5.1 ACAGCCAGAAATAGAAACTTTGAACT TGAAAGTATGTATTGTTTTCACAGGG BL013C_E01.5.1 AGGATGAAATAATATGCTGTGCTTC ACCGTGATAAACTCCATCCCTT BL014D_B11.5.1 AGCAAAACTCCTCTATGAATCTCG ATTGCACACTAAATAGGTGAATACGA BL279A_G10.5.1 ATGGCAGGATGAAGAAAGACAT ATGCACTTTGGACCCACTGAG Et.fasta.Contig1023.5.1 TGTGGTTATTGGACTACTTCACTCTC AAACGTCCATTTGACACTGTATTTTA Et.fasta.Contig1166.5.1 GAATGAAGAGAAAATGTTTTGAAGGT GCACAGTATTGGCTATGAGCAC Et.fasta.Contig1311.5.1 AGAAAACTGTGTCAAGCTTATTTTCC CAACTTAGTGTTCACATTTCTGAGGT Et.fasta.Contig1335.5.1 CCACTTATGGTAGTTCCCACTTTTAT GCTAAAGAATACCAAGAACCTTTGAC Et.fasta.Contig1381.5.1 GTCACAGGTATAACATTGAAAGGATG TAAATGAATCAAACATTGAAGAGAGC Et.fasta.Contig1459.5.1 ATAACAAGGACATGTTCTGCTGG CTAGCAGAACCCTGTATAGCCTG Et.fasta.Contig1506.5.1 AGGATATCCGCTCAGAAATATGAAG CTGACCACTTGCAAAACTTACTACCT Et.fasta.Contig1578.5.1 CCTAGAACATTACCAAAACAGACTCA AATGAAGAAGTATTGCATGTGAGAAC Et.fasta.Contig1647.5.1 GTACAACGTCAGGCAAAGCTATTCT ATCTCCAACACCGTGGCTAAT Et.fasta.Contig1717.5.1 GAACTTGTTGGCAGGTTTCTCTT CTAGTGATAGGTTGGACATACCAGAG Et.fasta.Contig1796.5.1 TGTGGGTATGTATATGGCTAACTTGT AGATTTTATGTGCTACTGCATTTACG Et.fasta.Contig1908.5.1 CTCATGACTTAATTGCTGTTCTTCG ATAACCATTCTGAGGTTTTGAGTTG Et.fasta.Contig1941.5.1 ATCTCCTGCTTCATCTCTTGATTTAT TAACAGATTTAATAAACGTCCCCTTC Et.fasta.Contig1943.5.1 AGTACGATGAATCTGGTCCTTCAAT CCACAATACTGACATACTCTGGTCTT Et.fasta.Contig325.5.1 GTGAAGTCAGTGAGTAAAGTCCATGT CTAGGATACCAGTGGGAGAGTGTAAT Et.fasta.Contig330.5.1 GTCATCACCTCCACTACTTCACAAG TTTTGGCACTGTAAGATTCTATGAAC Et.fasta.Contig536.5.1 CCTTAGGTAGAACAGACTGAAGCAG GAAACATGAAACTGGACTTGTTTTAG Et.fasta.Contig917.5.1 GGATGCAGATTCTTCCTATTTTACTC CTGGTCACTTTACTTGTTTTCAGTGT Et.fasta.Contig926.5.1 TTCATCACATTCTACTTCACAAATCA CTAGGCAAGCAAGCTTTCTAATAGTT Et.fasta.Contig93.5.1 GAATAAAAGCAACAATTGCAGAGTTA CTCGACTCCTTCTACGATCTCTACTC Et.fasta.Contig990.5.1 GTTTAGGTTAGTATGAAGGATCCCAA TGCCAGTACTCACCAATTAGTAAAAG G1-C12 CCCAAATCCAGGAGTTCAAA TGGGACCTGGGGCTTCATT G1-C13 TTGCCCGAGAAAAGGAAGGACATA CAAGGGTGGGTGAGGGACATC G1-C5 F-CACTGTTGACTTGGGTTATGTTATT CTGCTCCTAGGGTTTGTGAAG G1-C7 CCCGTGTGGCTGGCTTGTGC TCGGCTACTTTGGTGTTTTTCTCCCTCAT G1-C9 TGGTCCGGCAACAGCATCAGA GCTTTTCGGTATTCAACGGCAGAGTG G1-C9 TGGTCCGGCAACAGCATCAGA GCTTTTCGGTATTCAACGGCAGAGTG G1-D5 AGACCCTTGCTGTGTAACTGCT GACTGGGACTGACTTCTATGACG G1-D6 CAGCGTGCCCACCCGATAGAA TCCCAAAAAGTAAAATGTGCAAAGAAAA G1-D7 CAGCGGTGGAAATGACAAACAGG CCAAGACGACGAGGAACGGTATT G1-E12 CAACCATGAGAGGAGGCCAGAGAAC AAAACAGCACTACCTACAAAACCCTATT G1-F1 TTAGTTTGGGTGCAGACAGGA GGTGCTCAACAACAAATCAACT G1-F20 TCCCCAACAACTCCAGCAGAT GGAAACCACCTAGACGAAAAATG G1-I18 CATGTTTGTGGGTGTGGTGAA AAAAGCGGCATCTGGTAAGG G1-I19 ACCCAGACCTGTCCACCTCA GAACAGCTCTCCAATCCACAAG G1-I21 CCAAGCGAAGGAGGCGTGTG CATGTGGCTCTTTGTTTCTGGA G1-I5 TAATCGTGTTTGGTGGCATCCTTGAGTC AGCAGCAGTTCCATTTTCCCACACCA G1-I8 ACCTGCAGTGGGCTAAGACC ATGGAAATAATAAAATAAAATGTT G1-J10 CGTTCGCTTTGCCTGCCACA GGCTCTTCCCCGGTCGTCCAC G1-J17 AGCGCCTTCTACACGGACAC TATGCCCCAATTACTCTTCTGC G1-J2 TACAGTAACTATGCCAAGATGAAATG CAATATGGATAATGGCTGTAGACC G1-J20 ATCCTCCAAGCTCACTACAACA CCAGCCCCTTCCCAAACAG G1-J9 CTGTCATTGCCTGCATCGGGGAGAAG TGTTGAGGGGAAGCAGTTTTG G1-K2 GCTTTCGCCTTTGACACCTC GGCCGGACCATTGCTGAAGAAG G1-L11 AAAGTGACCATCCAGTGCCCAAACCT CCGGCCGAAACTGACGAGATACATTAG G1-L13 TCAGCTGCACTAGGTTTGTC CATTTTGATTTGCTCCATAA G1-L19 GACAACCTTGAATCCTTTATG AGATGTTGGTTGGTGACTTAT G1-L20 TGGGCATAGATGGCAAGGAAAAA CCCCCAGCATCTCGCATACAC G1-L7 GTGCTACAGGAAGGAATGGATG TAGCACAGGAACAGCCGACAATAA G1-M14 CCGCTTGGACATGAGGAGAT TGGCAAAGAAACAGAACACAACTA G1-M19 GAGAAGTAGTGTCCCGGCAGAAAC ATGGGTGAAAACTTAGGTGAAATG G1-N9 GCGGGGCAATACATGACGTTCCACAG GACCCCCATCTCCGTTTCCCATTCC G1-O1 GGGGTAGAGCACAGTCCAGTT TTGCAAGGCCGAAAAGGTG G1-O12 GGAATTCCGGGGCACTACT TCGCGAGGACGGGGAAGAG G1-O24 CGGCCTTCCTGCAGTACAACCATC TCGGCAACGTGAAGACCATA G2-A11 GCCCCTGGAAGCTGTTGTGA GGGGTCCATCCGAGTCC G2-A7 TTACCCCACAGACAAAATCAACACC GGCGGCCCCTCATAGCAC G2-B1 GGGCCTAGTCCTGCTGGTC CAAAGAGTGCGGAGAAATGG G2-B8 CAACATGCGACCACTATAGCCACTTCCT CGCCACCGCCACCACCACA G2-C2 TTTGCAGGAAGAGTCATAACACAG GTCAACAACACCCTTTTCCCTTCCT G2-D1 GCAGGTCGGCAAGAAGCTAAAGAAGGAA AGGGTTGGTTTGAAAGGATGTGCTGGTAA G2-E17 GGAGCACCAAATTCAAGTCAG CGTCCCCGGTCAATCTCCAC G2-E19 CCAGTTTGAGCCCCAGGAG TCGCGGCAGTCAAGAGGTC G2-F17 TATCCTCTTATTGCTGCATTCTCCTCAC AGTACGGCCGTTCACCATCTCTG G2-F2 CACACCACAGACGCATTGAC TCCCCAGCCTGTGTAGAAC G2-G13 GGGAGGGGAGAAGGCTACCA ATACACGGCTTCCATGCTTCTTCTT G2-G15 CCACGGCCCCACATCCAGC TCCCGCAGAATTTCCGTATCCAT G2-G21 TCCAAGAGGGTGTGAGGTGAAC AAAGCCATGCGAAGCGGAAGAC G2-G23 GGTTTGGTACTTCAGCGGATGT CCAAAGCCTGTACTATGCGAAAAG G2-G5 CGGTCCCTACTGTGGTCTATGGTTTTCA GGCTCTGCATATCCTCGGTCACACTTCC G2-G6 CCCATGGCTGCAAGGATTACG CAGGGGTTGTTGGGAGGCAGTGT G2-H18 TTGTCAAATGGGCGAGTTCA TGTTTTGCACCCAGTTTTTG G2-I18 GATCTCCTCAGGTCTCTTTCA GATTATGGGCCGGTGTCTCT G2-I23 TGACTTTCCCAATGTGAGCAGAC CAGAGGTGGTGTTACAGCAGCAGTTT G2-J12 CCTCTTGTCCCAGTGCCAGTG TCCAGGGATCCGAAACAAAG G2-J21 CCGCCTCAGCCTGTTTCTCTACTTTT CTTTGAATTTCTGCTTTTGGTGCTCTGC G2-K12 ACATTAGTCCTGGTTACGAGAGC AAAGGGCAGTCCAGCATTGA G2-K2 CTGCCCAAGAAGACCGAGAGCCACAAG AGCGCCCCCTGCACCAAAATCA G2-L16 CCAAGGGTAGGAGAACAAGACA ATGGCATGCTGGGAAATCA G2-L21 GAATCTAGGTCCAAGCAGTCCCATCT GACCATCACACCACTACCCACACTCA G2-L3 TGAAAGAGGCCAGAAACAAGTAG TTCCCAAGGTCTCCATAACAAT G2-L4 TGGCCAAGAAGATGAAACAGGAAGAGGAG TGGCAAAGGACACGACGCAGAG G2-M14 CGGCCTCCTCGACGCATACG CCAGGCCGGCCCATTGTTC G2-M24 ACGGAGCACGGTCAGATTTCACG CCCGGCTGGCTCTTCTTGCTCTT G2-M3 CGATCCGCATTGAACGAGT TGTGGCAGGAAGGAGAAGG G2-N2 CGTGTTTTCCTCCTATGTCGACTTCTTTG ACGTGCTCTGCCTTTCTTGATCTTGTGTT G3-D7 AGGATTTCTTGGCCGGTGGAGTGG GAAGTTGAGGGCCTGGGTGGGGAAGTA NT001D_E08.5.1 AGAAGTTCCTAGATGAGTTGGAGGAG AATTAATTTCCTAAACCAGGTGACAG NT010B_E09.5.1 GAAGAGGTCCTAAAATATCAAGATGC ATGATAGACTTCGTCCTTGTCATAGA NT014D_E01.5.1 AAAGAAGTCCCGCATCTAACCT ATTAAATATGAGAAGATGTGTGCAGG V2_p1_b8 AGTCACTGTGTTACATTATCACCCAC ATAATTATACACTGCGGTCTGCATCT V2_p1_c5 AGTACCTGTTCGACAAGCACAC TGAGAACATAGACAAGTTAACATACACC V2_p1_d10 GAGATAGAAAGGCTGCATAAAGAAAT TATGTTTCAACAATGTACAGGAAACC V2_p1_d4 CACCAGAACAAGCTGTATTTTTATGT TGGTTTGCATCATATATTAAAGGGTA V2_p1_g7 GACTTCAAGCACATTGGGAAAC ATTGTAAACTTGATAGGCTGGTGAG V2_p2_g6 AGAATTCCCAATAGCACCTGAAAT CACTTGGTAAATACATACACACAGCA V2_p2_h2 CTTTTTGGCCTGGTCTTTTTG AGATTCTTCAGACTCGTCCTTCTTT V2_p3_a5 TTTACACAGAAACCTTGTTTATTTGGC TTTAAGGATGCTTAGAGGCAAAGTATT V2_p3_b1 AGTCACTGTGTTACATTATCACCCAC TATACACTGCGGTCTGCATCTACT V2_p5_b3 AATGGGATGAAGAGCGAGAAT CTGCCCCATTGACATTTACCTA V2_p5_h3 CCTTCAGACGAAAACAGCACTAAG TACAGTGTATGAGAGCCCAATATTTC V2_p6_a4 AGAAATACATCAAATATCGGGTGG AAAAAGGACAATGTTCAGCTCTCT V3_p1_a21 ACCAAGTTCTTGGAAAGTGGTG CTTAGTGTCTCCTGGGTTTGAATAG V3_p1_b13 GTCTTGGTACTCAATGAAGGAGATG TCAATCTGATGAAGAGTTTACATGTCT
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 facilitate 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 informative 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 ].A. mexicanumA. t. tigrinumA. ordinariumAmbystoma ordinariumAmbystoma ordinariumA. mexicanumA. t. tigrinumTo identify informative markers for  A. ordinarium ,  A. mexicanum and A. t. tigrinum  EST contigs were aligned to identify orthologous genes with species-specific sequence variations (SNPs or Insertion/Deletions = INDELs). Primer pairs corresponding 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. mexicanum  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 individual 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 amplify the corresponding sequence in a related  A. tigrinum  complex species, and for small DNA fragments in the range of 150–500 bp, approximately half are expected to have informative polymorphisms.A. ordinariumA. mexicanum and A. t. tigrinumA. ordinariumA. mexicanumA. t. tigrinumA. ordinariumA. mexicanumA. t. tigrinumA. tigrinumEST loci used in a population-level PCR amplification screen in  A. ordinariumA. ordinariumLocus IDForward Primer 5' to 3'Reverse Primer 5' to 3'Comparative gene mapping Salamanders occupy a pivotal phylogenetic position for reconstructing the ancestral tetrapod genome structure and for providing perspective on the extremely derived anuran  Xenopus  (37) that is currently providing the bulk of amphibian genome information. Here we show the utility of ambystomatid ESTs for identifying chromosomal regions that are conserved between salamanders and other vertebrates. A region of conserved synteny that corresponds to human chromosome ( Hsa ) 17q has been identified in several non-mammalian taxa including reptiles (38) and fishes (39). In a previous study Voss et al. (40) identified a region of conserved synteny between  Ambystoma  and  Hsa  17q that included collagen type 1 alpha 1 ( Col1a1 ), thyroid hormone receptor alpha ( Thra ), homeo box b13 ( Hoxb13) , and distal-less 3 ( Dlx3)  (Figure  4 ). To evaluate both the technical feasibility of mapping ESTs and the likelihood that presumptive orthologues map to the same synteny group, we searched our assemblies for presumptive  Hsa  17 orthologues and then developed a subset of these loci for genetic linkage mapping. Using a joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs, 97 Hsa 17 presumptive orthologues were identified. We chose 15 genes from this list and designed PCR primers to amplify a short DNA fragment containing 1 or more presumptive SNPs that were identified in the joint assembly (Table  9 ). All but two of these genes were mapped, indicating a high probability of mapping success using markers developed from the joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs. All 6 ESTs that exhibited 'best hits' to loci within the previously defined human- Ambystoma  synteny group did map to this region ( Hspc009 ,  Sui1 ,  Krt17 ,  Krt24 ,  Flj13855,  and  Rpl19 ). Our results show that BLAST-based definitions of orthology are informative between salamanders and human. All other presumptive  Hsa  17 loci mapped to  Ambystoma  chromosomal regions outside of the previously defined synteny group. It is interesting to note that two of these loci mapped to the same ambystomatid linkage group ( Cgi-125 ,  Flj20345 ), but in human the presumptive orthologues are 50 Mb apart and distantly flank the syntenic loci in Figure  4 . Assuming orthology has been assigned correctly for these loci, this suggests a dynamic history for some  Hsa  17 orthologues during vertebrate evolution. Figure 4 Comparison of gene order between  Ambystoma  linkage group 1 and an 11 Mb region of Hsa17 (37.7 Mb to 48.7 Mb) . Lines connect the positions of putatively orthologous genes. Table 9 Presumptive human chromosome 17 loci that were mapped in  Ambystoma Marker ID Primers a Diagnosis b LG c Symbol d RefSeq ID e E-value f Pl_6_E/F_6 F-GAAAACCTGCTCAGCATTAGTGT ASA ul PFN1 NP_005013 E -34 R-TCTATTACCATAGCATTAATTGGCAG Pl_5_G/H_5 F-CTATTTCATCTGAGTACCGTTGAATG PE (A) 23 CGI-125 NP_057144 E -56 R-TAATGTAGAACTAAATGGCATCCTTC E-CCATGGTGCAGGAAGAGAGCCTATAT Pl_0.4_A/B_1 F-GTCTCATTATCCGCAAACCTGT SP 1 RPL19 NP_000972 E -67 R-ATTCTCATCCTCCTCATCCACGAC Pl_4_B_7/8 F-CCTAGAACATTACCAAAACAGACTCA RD (Dpn II) 1 KRT10 NP_061889 E -17 R-AATGAAGAAGTATTGCATGTGAGAAC Pl_4_B_9/10 F-GAACTTGTTGGCAGGTTTCTCTT RD (AciI) 1 KRT17 NP_000413 E -146 R-CTAGTGATAGGTTGGACATACCAGAG Pl_10_C/D_4 F-CTCCACTATTTAAAGGACATGCTACA PE (A) 1 SUI1 NP_005792 E -48 R-TTAATATAGCACAACATTGCCTCATT E-TGCTACATTAATGTAATAAACGGCATCATC Pl_6_E/F_11 F-AAGAGAAGTTCCTAGATGAGTTGGAG PE (A) 1 HSPC009 NP_054738 E -26 R-TGAAGAGAGAACTCAAAGTGTCTGAT E-TCATGTTTTGCTCTGCTGTGCAGT Pl_9_A/B_10 F-TGATAGTTTCTGGATTAAGACGAGTG PE (T) 1 FLJ13855 NP_075567 E -15 R-CTTAGAGCCATTGTTACAAGATGTTC E-GTGATCTAGTGGGATCAAACCCTAAAGACC Pl_10_C/D_9 F-AAAGTGCCAAGAAGGAGATTAACTT PE (T) 9 NME1 NP_000260 E -71 R-GAGCTCAGAAAACAAGGCAGTAAC E-AAATGGATCTACGAGTAGACCTTGACCC Pl_9_C/D_9 F-GAGTCTCCTTTAGGATTGACGTATCT PE (T) 23 FLJ20345 NP_060247 E -17 R-GCTATGTGAGCAGAGATAAAAGTCAG E-GTTACAGCATCAGTGGGATGTGGTATGT Pl_8_C/D_9 F-AGGATACCAACCTCTGTGCTATACAT PE (C) 15 H3F3B NP_005315 E -66 R-TAAATGTATTTACAAACCGAAAGCAA E-CGTGGCGAGCGTGCCTAGT Pl_9_C/D_4 F-GTGGTTATTTGTAACATTTCGTTGAC PE (A) 8 SFRS2 NP_003007 E -40 R-AATTACATTTGGGCTTCTCAATTTAC E-TTTTTAAACGCGTAAAAATGTTAACAGA Pl_6_C/D_5 F-CCGTAAATGTTTCTAAATGACAGTTG PE (G) 2 ACTG1 NP_001605 0 R-GGAAAGAAAGTACAATCAAGTCCTTC E-GATTGAAAACTGGAACCGAAAGAAGATAAA a Sequences are 5' amplification primers, 3' amplification primers, or primer extension probes, and are preceded by F-, R-, and E- respectively.  b Genotyping methods are abbreviated: allele specific amplification (ASA), size polymorphism (SP), restriction digestion (RD), primer extension (PE). Diagnostic restriction enzymes and diagnostic extension bases are provided in parentheses.  c Ambystoma linkage group ID. "ul" designates markers that are unlinked.  d Official gene symbols as defined by the Human Genome Organization Gene Nomenclature Committee  .  e Best BLASTX hit (highest e-value) from the human RefSeq database using the contig from which each marker was designed as a query sequence.  f Highest E-value statistic obtained by searching contigs, from which EST markers were designed, against the human RefSeq database.
Comparative gene mapping
Salamanders occupy a pivotal phylogenetic position for reconstructing the ancestral tetrapod genome structure and for providing perspective on the extremely derived anuran  Xenopus  (37) that is currently providing the bulk of amphibian genome information. Here we show the utility of ambystomatid ESTs for identifying chromosomal regions that are conserved between salamanders and other vertebrates. A region of conserved synteny that corresponds to human chromosome ( Hsa ) 17q has been identified in several non-mammalian taxa including reptiles (38) and fishes (39). In a previous study Voss et al. (40) identified a region of conserved synteny between  Ambystoma  and  Hsa  17q that included collagen type 1 alpha 1 ( Col1a1 ), thyroid hormone receptor alpha ( Thra ), homeo box b13 ( Hoxb13) , and distal-less 3 ( Dlx3)  (Figure  4 ). To evaluate both the technical feasibility of mapping ESTs and the likelihood that presumptive orthologues map to the same synteny group, we searched our assemblies for presumptive  Hsa  17 orthologues and then developed a subset of these loci for genetic linkage mapping. Using a joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs, 97 Hsa 17 presumptive orthologues were identified. We chose 15 genes from this list and designed PCR primers to amplify a short DNA fragment containing 1 or more presumptive SNPs that were identified in the joint assembly (Table  9 ). All but two of these genes were mapped, indicating a high probability of mapping success using markers developed from the joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs. All 6 ESTs that exhibited 'best hits' to loci within the previously defined human- Ambystoma  synteny group did map to this region ( Hspc009 ,  Sui1 ,  Krt17 ,  Krt24 ,  Flj13855,  and  Rpl19 ). Our results show that BLAST-based definitions of orthology are informative between salamanders and human. All other presumptive  Hsa  17 loci mapped to  Ambystoma  chromosomal regions outside of the previously defined synteny group. It is interesting to note that two of these loci mapped to the same ambystomatid linkage group ( Cgi-125 ,  Flj20345 ), but in human the presumptive orthologues are 50 Mb apart and distantly flank the syntenic loci in Figure  4 . Assuming orthology has been assigned correctly for these loci, this suggests a dynamic history for some  Hsa  17 orthologues during vertebrate evolution.XenopusHsaAmbystomaHsaCol1a1ThraHoxb13)Dlx3)HsaA. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumAmbystomaHspc009Sui1Krt17Krt24Flj13855,Rpl19HsaAmbystomaCgi-125Flj20345HsaPresumptive human chromosome 17 loci that were mapped in  AmbystomaAmbystomaMarker IDPrimers aDiagnosis bLG cSymbol dRefSeq ID eE-value fa Sequences are 5' amplification primers, 3' amplification primers, or primer extension probes, and are preceded by F-, R-, and E- respectively.  b Genotyping methods are abbreviated: allele specific amplification (ASA), size polymorphism (SP), restriction digestion (RD), primer extension (PE). Diagnostic restriction enzymes and diagnostic extension bases are provided in parentheses.  c Ambystoma linkage group ID. "ul" designates markers that are unlinked.  d Official gene symbols as defined by the Human Genome Organization Gene Nomenclature Committee  .  e Best BLASTX hit (highest e-value) from the human RefSeq database using the contig from which each marker was designed as a query sequence.  f Highest E-value statistic obtained by searching contigs, from which EST markers were designed, against the human RefSeq database.Future directions Ambystomatid salamanders are classic model organisms that continue to inform biological research in a variety of areas. Their future importance in regenerative biology and metamorphosis will almost certainly escalate as genome resources and other molecular and cellular approaches become widely available. Among the genomic resources currently under development (see [ 41 ]) are a comparative genome map, which will allow mapping of candidate genes, QTL, and comparative anchors for cross-referencing the salamander genome to fully sequenced vertebrate models. In closing, we reiterate a second benefit to resource development in  Ambystoma . Genome resources in  Ambystoma  can be extended to multiple, closely related species to explore the molecular basis of natural, phenotypic variation. Such extensions can better inform our understanding of ambystomatid biodiversity in nature and draw attention to the need for conserving such naturalistic systems. Several paedomorphic species, including  A. mexicanum , are on the brink of extinction. We can think of no better investment than one that simultaneously enhances research in all areas of biology and draws attention to the conservation needs of model organisms in their natural habitats.
Future directions
Ambystomatid salamanders are classic model organisms that continue to inform biological research in a variety of areas. Their future importance in regenerative biology and metamorphosis will almost certainly escalate as genome resources and other molecular and cellular approaches become widely available. Among the genomic resources currently under development (see [ 41 ]) are a comparative genome map, which will allow mapping of candidate genes, QTL, and comparative anchors for cross-referencing the salamander genome to fully sequenced vertebrate models. In closing, we reiterate a second benefit to resource development in  Ambystoma . Genome resources in  Ambystoma  can be extended to multiple, closely related species to explore the molecular basis of natural, phenotypic variation. Such extensions can better inform our understanding of ambystomatid biodiversity in nature and draw attention to the need for conserving such naturalistic systems. Several paedomorphic species, including  A. mexicanum , are on the brink of extinction. We can think of no better investment than one that simultaneously enhances research in all areas of biology and draws attention to the conservation needs of model organisms in their natural habitats.AmbystomaAmbystomaA. mexicanumConclusions Approximately 40,000 cDNA sequences were isolated from a variety of tissues to develop expressed sequence tags for two model salamander species ( A. mexicanum  and  A. t. tigrinum ). An approximately equivalent number of contigs were identified for each species, with 21,091 unique contigs identified overall. The strategy to sequence cDNAs from a diverse collection of tissues from normalized and non-normalized libraries yielded different sets of highly redundant contigs. Only 25% and 28% of the  A. mexicanum  and  A. t. tigrinum  contigs, respectively, were identified in common. To demonstrate the utility of these EST resources, we searched databases to identify new probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human/ Ambystoma  synteny group with marker loci, and extended PCR primer sets designed for  A. mexicanum  /  A. t. tigrinum  orthologues to a related tiger salamander species. Over 100 new probes were identified for regeneration research using informatic approaches. With respect to comparative mapping, 13 of 15 EST markers were mapped successfully, and 6 EST markers were mapped to a previously defined synteny group in  Ambystoma . These results indicate a high probability of mapping success using EST markers developed from the joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs. Finally, we found that primer sets designed for  A. mexicanum  /  A. t. tigrinum  EST orthologues can be used to amplify the corresponding sequence in a related  A. tigrinum  complex species. Overall, the EST resources reported here will enable a diversity of new research areas using ambystomatid salamanders.
Conclusions
Approximately 40,000 cDNA sequences were isolated from a variety of tissues to develop expressed sequence tags for two model salamander species ( A. mexicanum  and  A. t. tigrinum ). An approximately equivalent number of contigs were identified for each species, with 21,091 unique contigs identified overall. The strategy to sequence cDNAs from a diverse collection of tissues from normalized and non-normalized libraries yielded different sets of highly redundant contigs. Only 25% and 28% of the  A. mexicanum  and  A. t. tigrinum  contigs, respectively, were identified in common. To demonstrate the utility of these EST resources, we searched databases to identify new probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human/ Ambystoma  synteny group with marker loci, and extended PCR primer sets designed for  A. mexicanum  /  A. t. tigrinum  orthologues to a related tiger salamander species. Over 100 new probes were identified for regeneration research using informatic approaches. With respect to comparative mapping, 13 of 15 EST markers were mapped successfully, and 6 EST markers were mapped to a previously defined synteny group in  Ambystoma . These results indicate a high probability of mapping success using EST markers developed from the joint assembly of  A. mexicanum  and  A. t. tigrinum  contigs. Finally, we found that primer sets designed for  A. mexicanum  /  A. t. tigrinum  EST orthologues can be used to amplify the corresponding sequence in a related  A. tigrinum  complex species. Overall, the EST resources reported here will enable a diversity of new research areas using ambystomatid salamanders.A. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumAmbystomaA. mexicanumA. t. tigrinumAmbystomaA. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumA. tigrinumMethods cDNA library construction Ten cDNA libraries were constructed for the project using various larval tissues of  A. mexicanum  and  A. t. tigrinum  (Table  1 ). Larval  A. mexicanum  were obtained from adult animals whose ancestry traces back to the Axolotl Colony [ 17 ]. Larval  A. t. tigrinum  were obtained from Charles Sullivan Corp. The GARD and MATH A. mexicanum limb regeneration libraries were constructed using regenerating forelimb mesenchyme. Total RNAs were collected from anterior and posterior limbs amputated at the mid-stylopod level on 15 cm animals, and from the resulting regenerates at 12 h, 2 days, 5 days and early bud stages. One hundred μg fractions of each were pooled together and polyA-selected to yield 5 μg that was utilized for directional library construction (Lambda Zap, Stratagene). The V1 ( A. mex ), V2 ( A. tig ), V4-5 ( A. tig ), and V6-7 ( A. mex ) libraries were made from an assortment of larval tissues (see Table  1 ) using the SMART cDNA cloning kits (Clontech). Total RNAs were isolated and reverse transcribed to yield cDNAs that were amplified by long distance PCR and subsequently cloned into pTriplEX. The V3 and AG libraries were constructed by commercial companies (BioS&T and Agencourt, respectively). cDNA template preparation and sequencing cDNA inserts were mass excised as phagemids, picked into microtitre plates, grown overnight in LB broth, and then diluted (1/20) to spike PCR reactions: (94°C for 2 min; then 30 cycles at 94°C for 45 sec, 58°C for 45°sec, and 72°C for 7 min). All successful amplifications with inserts larger than ~500 bp were sequenced (ABI Big Dye or Amersham Dye terminator chemistry and 5' universal primer). Sequencing and clean-up reactions was carried out according to manufacturers' protocols. ESTs were deposited into NCBI database under accession numbers BI817205-BI818091 and CN033008-CN045937 and CN045944-CN069430. EST sequence processing and assembly The PHRED base-calling program [ 42 ] was used to generate sequence and quality scores from trace files. PHRED files were then quality clipped and vector/contaminant screened. An in-house program called QUALSCREEN was used to quality clip the ends of sequence traces. Starting at the ends of sequence traces, this program uses a 20 bp sliding window to identify a continuous run of bases that has an average PHRED quality score of 15. Mitochondrial DNA sequences were identified by searching all ESTs against the complete mtDNA genome sequence of  A. mexicanum  (AJ584639). Finally, all sequences less than 100 bp were removed. The average length of the resulting ESTs was 629 bp. The resulting high quality ESTs were clustered initially using PaCE [ 43 ] on the U.K. HP Superdome computer. Multi-sequence clusters were used as input sequence sets for assembly using CAP3 [ 44 ] with an 85% sequence similarity threshold. Clusters comprising single ESTs were assembled again using CAP3 with an 80% sequence similarity threshold to identify multi-EST contigs that were missed during the initial analysis. This procedure identified 550 additional contigs comprising 1150 ESTs. Functional annotation All contigs and singletons were searched against the human RefSeq database (Oct. 2003 release) using BLASTX. The subset of sequences that yielded no BLAST hit was searched against the non-redundant protein sequence database (Feb. 2004) using BLASTX. The remaining subset of sequences that yielded no BLAST hit was searched against  Xenopus laevis  and  X. tropicalis  UNIGENE ESTs (Mar. 2004) using TBLASTX. Zebrafish ESTs were downloaded from UNIGENE ESTs (May 2004). BLAST searches were done with an E-value threshold of E <10-7 unless specified. Sequence comparison of  A. mexicanum  and  A. t. tigrinum  assemblies All low quality base calls within contigs were masked using a PHRED base quality threshold of 16. To identify polymorphisms for linkage mapping, contigs from  A. mexicanum  and  A. t. tigrinum  assemblies were joined into a single assembly using CAP3 and the following criteria: an assembly threshold of 12 bp to identify initial matches, a minimum 100 bp match length, and 85% sequence identity. To identify putatively orthologous genes from  A. mexicanum  and  A. t. tigrinum  assemblies, and generate an estimate of gene sequence divergence, assemblies were compared using BLASTN with a threshold of  E  <10 -20 . Following BLAST, alignments were filtered to obtain reciprocal best BLAST hits. Extending  A. mexicanum  /  A. t. tigrinum  sequence information to  A. ordinarium Polymorphic DNA marker loci were identified by locating single nucleotide polymorphisms (SNPs) in the joint  A. mexicanum  and  A. t. tigrinum  assembly. Polymerase chain reaction (PCR) primers were designed using  Primer 3  [ 45 ] to amplify 100 – 500 bp SNP-containing fragments from 123 different protein-coding loci (Table  8 ). DNA was isolated from salamander tail clips using SDS, RNAse and proteinase K treatment, followed by phenol-chloroform extraction. Fragments were amplified using 150 ng DNA, 75 ng each primer, 1.5 mM MgCl 2 , 0.25 U Taq, and a 3-step profile (94°C for 4 min; 33 cycles of 94°C for 45 s, 60°C for 45 s, 72°C for 30 s; and 72°C for 7 min). DNA fragments were purified and sequenced using ABI Big Dye or Amersham Dye terminator chemistry. Single nucleotide polymorphisms were identified by eye from sequence alignments. Linkage mapping of human chromosome 17 orthologous genes Putative salamander orthologues of genes on human chromosome 17 ( Hsa  17) were identified by comparing the joint  A. mexicanum  and  A. t. tigrinum  assembly to sequences from the human RefSeq (NCBI) protein database, using BLASTX at threshold  E< 10 -7 . Linkage distance and arrangement among markers was estimated using MapManager QTXb19 software [ 46 ] and the Kosambi mapping function at a threshold of p = 0.001. All markers were mapped using DNA from a previously described meiotic mapping panel [ 40 ]. All PCR primers and primer extension probes were designed using  Primer 3  [ 45 ] and  Array Designer2  (Premier Biosoft) software. Species-specific polymorphisms were assayed by allele specific amplification, restriction digestion, or primer extension, using the reagent and PCR conditions described above. Primer extension markers were genotyped using the AcycloPrime-FP SNP detection assay (Perkin Elmer). See Table  9  for amplification and extension primer sequences, and information about genotyping methodology.
Methods
cDNA library construction Ten cDNA libraries were constructed for the project using various larval tissues of  A. mexicanum  and  A. t. tigrinum  (Table  1 ). Larval  A. mexicanum  were obtained from adult animals whose ancestry traces back to the Axolotl Colony [ 17 ]. Larval  A. t. tigrinum  were obtained from Charles Sullivan Corp. The GARD and MATH A. mexicanum limb regeneration libraries were constructed using regenerating forelimb mesenchyme. Total RNAs were collected from anterior and posterior limbs amputated at the mid-stylopod level on 15 cm animals, and from the resulting regenerates at 12 h, 2 days, 5 days and early bud stages. One hundred μg fractions of each were pooled together and polyA-selected to yield 5 μg that was utilized for directional library construction (Lambda Zap, Stratagene). The V1 ( A. mex ), V2 ( A. tig ), V4-5 ( A. tig ), and V6-7 ( A. mex ) libraries were made from an assortment of larval tissues (see Table  1 ) using the SMART cDNA cloning kits (Clontech). Total RNAs were isolated and reverse transcribed to yield cDNAs that were amplified by long distance PCR and subsequently cloned into pTriplEX. The V3 and AG libraries were constructed by commercial companies (BioS&T and Agencourt, respectively).
cDNA library construction
Ten cDNA libraries were constructed for the project using various larval tissues of  A. mexicanum  and  A. t. tigrinum  (Table  1 ). Larval  A. mexicanum  were obtained from adult animals whose ancestry traces back to the Axolotl Colony [ 17 ]. Larval  A. t. tigrinum  were obtained from Charles Sullivan Corp. The GARD and MATH A. mexicanum limb regeneration libraries were constructed using regenerating forelimb mesenchyme. Total RNAs were collected from anterior and posterior limbs amputated at the mid-stylopod level on 15 cm animals, and from the resulting regenerates at 12 h, 2 days, 5 days and early bud stages. One hundred μg fractions of each were pooled together and polyA-selected to yield 5 μg that was utilized for directional library construction (Lambda Zap, Stratagene). The V1 ( A. mex ), V2 ( A. tig ), V4-5 ( A. tig ), and V6-7 ( A. mex ) libraries were made from an assortment of larval tissues (see Table  1 ) using the SMART cDNA cloning kits (Clontech). Total RNAs were isolated and reverse transcribed to yield cDNAs that were amplified by long distance PCR and subsequently cloned into pTriplEX. The V3 and AG libraries were constructed by commercial companies (BioS&T and Agencourt, respectively).A. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumA. mexA. tigA. tigA. mexcDNA template preparation and sequencing cDNA inserts were mass excised as phagemids, picked into microtitre plates, grown overnight in LB broth, and then diluted (1/20) to spike PCR reactions: (94°C for 2 min; then 30 cycles at 94°C for 45 sec, 58°C for 45°sec, and 72°C for 7 min). All successful amplifications with inserts larger than ~500 bp were sequenced (ABI Big Dye or Amersham Dye terminator chemistry and 5' universal primer). Sequencing and clean-up reactions was carried out according to manufacturers' protocols. ESTs were deposited into NCBI database under accession numbers BI817205-BI818091 and CN033008-CN045937 and CN045944-CN069430.
cDNA template preparation and sequencing
cDNA inserts were mass excised as phagemids, picked into microtitre plates, grown overnight in LB broth, and then diluted (1/20) to spike PCR reactions: (94°C for 2 min; then 30 cycles at 94°C for 45 sec, 58°C for 45°sec, and 72°C for 7 min). All successful amplifications with inserts larger than ~500 bp were sequenced (ABI Big Dye or Amersham Dye terminator chemistry and 5' universal primer). Sequencing and clean-up reactions was carried out according to manufacturers' protocols. ESTs were deposited into NCBI database under accession numbers BI817205-BI818091 and CN033008-CN045937 and CN045944-CN069430.EST sequence processing and assembly The PHRED base-calling program [ 42 ] was used to generate sequence and quality scores from trace files. PHRED files were then quality clipped and vector/contaminant screened. An in-house program called QUALSCREEN was used to quality clip the ends of sequence traces. Starting at the ends of sequence traces, this program uses a 20 bp sliding window to identify a continuous run of bases that has an average PHRED quality score of 15. Mitochondrial DNA sequences were identified by searching all ESTs against the complete mtDNA genome sequence of  A. mexicanum  (AJ584639). Finally, all sequences less than 100 bp were removed. The average length of the resulting ESTs was 629 bp. The resulting high quality ESTs were clustered initially using PaCE [ 43 ] on the U.K. HP Superdome computer. Multi-sequence clusters were used as input sequence sets for assembly using CAP3 [ 44 ] with an 85% sequence similarity threshold. Clusters comprising single ESTs were assembled again using CAP3 with an 80% sequence similarity threshold to identify multi-EST contigs that were missed during the initial analysis. This procedure identified 550 additional contigs comprising 1150 ESTs.
EST sequence processing and assembly
The PHRED base-calling program [ 42 ] was used to generate sequence and quality scores from trace files. PHRED files were then quality clipped and vector/contaminant screened. An in-house program called QUALSCREEN was used to quality clip the ends of sequence traces. Starting at the ends of sequence traces, this program uses a 20 bp sliding window to identify a continuous run of bases that has an average PHRED quality score of 15. Mitochondrial DNA sequences were identified by searching all ESTs against the complete mtDNA genome sequence of  A. mexicanum  (AJ584639). Finally, all sequences less than 100 bp were removed. The average length of the resulting ESTs was 629 bp. The resulting high quality ESTs were clustered initially using PaCE [ 43 ] on the U.K. HP Superdome computer. Multi-sequence clusters were used as input sequence sets for assembly using CAP3 [ 44 ] with an 85% sequence similarity threshold. Clusters comprising single ESTs were assembled again using CAP3 with an 80% sequence similarity threshold to identify multi-EST contigs that were missed during the initial analysis. This procedure identified 550 additional contigs comprising 1150 ESTs.A. mexicanumFunctional annotation All contigs and singletons were searched against the human RefSeq database (Oct. 2003 release) using BLASTX. The subset of sequences that yielded no BLAST hit was searched against the non-redundant protein sequence database (Feb. 2004) using BLASTX. The remaining subset of sequences that yielded no BLAST hit was searched against  Xenopus laevis  and  X. tropicalis  UNIGENE ESTs (Mar. 2004) using TBLASTX. Zebrafish ESTs were downloaded from UNIGENE ESTs (May 2004). BLAST searches were done with an E-value threshold of E <10-7 unless specified.
Functional annotation
All contigs and singletons were searched against the human RefSeq database (Oct. 2003 release) using BLASTX. The subset of sequences that yielded no BLAST hit was searched against the non-redundant protein sequence database (Feb. 2004) using BLASTX. The remaining subset of sequences that yielded no BLAST hit was searched against  Xenopus laevis  and  X. tropicalis  UNIGENE ESTs (Mar. 2004) using TBLASTX. Zebrafish ESTs were downloaded from UNIGENE ESTs (May 2004). BLAST searches were done with an E-value threshold of E <10-7 unless specified.Xenopus laevisX. tropicalisSequence comparison of  A. mexicanum  and  A. t. tigrinum  assemblies All low quality base calls within contigs were masked using a PHRED base quality threshold of 16. To identify polymorphisms for linkage mapping, contigs from  A. mexicanum  and  A. t. tigrinum  assemblies were joined into a single assembly using CAP3 and the following criteria: an assembly threshold of 12 bp to identify initial matches, a minimum 100 bp match length, and 85% sequence identity. To identify putatively orthologous genes from  A. mexicanum  and  A. t. tigrinum  assemblies, and generate an estimate of gene sequence divergence, assemblies were compared using BLASTN with a threshold of  E  <10 -20 . Following BLAST, alignments were filtered to obtain reciprocal best BLAST hits.
Sequence comparison of  A. mexicanum  and  A. t. tigrinum  assemblies
A. mexicanumA. t. tigrinumAll low quality base calls within contigs were masked using a PHRED base quality threshold of 16. To identify polymorphisms for linkage mapping, contigs from  A. mexicanum  and  A. t. tigrinum  assemblies were joined into a single assembly using CAP3 and the following criteria: an assembly threshold of 12 bp to identify initial matches, a minimum 100 bp match length, and 85% sequence identity. To identify putatively orthologous genes from  A. mexicanum  and  A. t. tigrinum  assemblies, and generate an estimate of gene sequence divergence, assemblies were compared using BLASTN with a threshold of  E  <10 -20 . Following BLAST, alignments were filtered to obtain reciprocal best BLAST hits.A. mexicanumA. t. tigrinumA. mexicanumA. t. tigrinumEExtending  A. mexicanum  /  A. t. tigrinum  sequence information to  A. ordinarium Polymorphic DNA marker loci were identified by locating single nucleotide polymorphisms (SNPs) in the joint  A. mexicanum  and  A. t. tigrinum  assembly. Polymerase chain reaction (PCR) primers were designed using  Primer 3  [ 45 ] to amplify 100 – 500 bp SNP-containing fragments from 123 different protein-coding loci (Table  8 ). DNA was isolated from salamander tail clips using SDS, RNAse and proteinase K treatment, followed by phenol-chloroform extraction. Fragments were amplified using 150 ng DNA, 75 ng each primer, 1.5 mM MgCl 2 , 0.25 U Taq, and a 3-step profile (94°C for 4 min; 33 cycles of 94°C for 45 s, 60°C for 45 s, 72°C for 30 s; and 72°C for 7 min). DNA fragments were purified and sequenced using ABI Big Dye or Amersham Dye terminator chemistry. Single nucleotide polymorphisms were identified by eye from sequence alignments.
Extending  A. mexicanum  /  A. t. tigrinum  sequence information to  A. ordinarium
A. mexicanumA. t. tigrinumA. ordinariumPolymorphic DNA marker loci were identified by locating single nucleotide polymorphisms (SNPs) in the joint  A. mexicanum  and  A. t. tigrinum  assembly. Polymerase chain reaction (PCR) primers were designed using  Primer 3  [ 45 ] to amplify 100 – 500 bp SNP-containing fragments from 123 different protein-coding loci (Table  8 ). DNA was isolated from salamander tail clips using SDS, RNAse and proteinase K treatment, followed by phenol-chloroform extraction. Fragments were amplified using 150 ng DNA, 75 ng each primer, 1.5 mM MgCl 2 , 0.25 U Taq, and a 3-step profile (94°C for 4 min; 33 cycles of 94°C for 45 s, 60°C for 45 s, 72°C for 30 s; and 72°C for 7 min). DNA fragments were purified and sequenced using ABI Big Dye or Amersham Dye terminator chemistry. Single nucleotide polymorphisms were identified by eye from sequence alignments.A. mexicanumA. t. tigrinumPrimer 3Linkage mapping of human chromosome 17 orthologous genes Putative salamander orthologues of genes on human chromosome 17 ( Hsa  17) were identified by comparing the joint  A. mexicanum  and  A. t. tigrinum  assembly to sequences from the human RefSeq (NCBI) protein database, using BLASTX at threshold  E< 10 -7 . Linkage distance and arrangement among markers was estimated using MapManager QTXb19 software [ 46 ] and the Kosambi mapping function at a threshold of p = 0.001. All markers were mapped using DNA from a previously described meiotic mapping panel [ 40 ]. All PCR primers and primer extension probes were designed using  Primer 3  [ 45 ] and  Array Designer2  (Premier Biosoft) software. Species-specific polymorphisms were assayed by allele specific amplification, restriction digestion, or primer extension, using the reagent and PCR conditions described above. Primer extension markers were genotyped using the AcycloPrime-FP SNP detection assay (Perkin Elmer). See Table  9  for amplification and extension primer sequences, and information about genotyping methodology.
Linkage mapping of human chromosome 17 orthologous genes
Putative salamander orthologues of genes on human chromosome 17 ( Hsa  17) were identified by comparing the joint  A. mexicanum  and  A. t. tigrinum  assembly to sequences from the human RefSeq (NCBI) protein database, using BLASTX at threshold  E< 10 -7 . Linkage distance and arrangement among markers was estimated using MapManager QTXb19 software [ 46 ] and the Kosambi mapping function at a threshold of p = 0.001. All markers were mapped using DNA from a previously described meiotic mapping panel [ 40 ]. All PCR primers and primer extension probes were designed using  Primer 3  [ 45 ] and  Array Designer2  (Premier Biosoft) software. Species-specific polymorphisms were assayed by allele specific amplification, restriction digestion, or primer extension, using the reagent and PCR conditions described above. Primer extension markers were genotyped using the AcycloPrime-FP SNP detection assay (Perkin Elmer). See Table  9  for amplification and extension primer sequences, and information about genotyping methodology.HsaA. mexicanumA. t. tigrinumE<Primer 3Array Designer2Author's contributions SP and DK: bioinformatics; JW: clone management and sequencing in support of  A. mexicanum  and  A. t. tigrinum  ESTs; JS: comparative mapping and polymorphism estimation; DW: extending ESTs to  A. ordinarium ; JM, KK, AS, NM: PCR and gel electrophoresis; BH and ET: cDNA library construction and sequencing for spinal cord regeneration ESTs; MR, SB, DG: cDNA library construction and clone management for limb regeneration ESTs; DP and SV conceived of the project and participated in its design and coordination. All authors read and approved the final manuscript.
Author's contributions
SP and DK: bioinformatics; JW: clone management and sequencing in support of  A. mexicanum  and  A. t. tigrinum  ESTs; JS: comparative mapping and polymorphism estimation; DW: extending ESTs to  A. ordinarium ; JM, KK, AS, NM: PCR and gel electrophoresis; BH and ET: cDNA library construction and sequencing for spinal cord regeneration ESTs; MR, SB, DG: cDNA library construction and clone management for limb regeneration ESTs; DP and SV conceived of the project and participated in its design and coordination. All authors read and approved the final manuscript.A. mexicanumA. t. tigrinumA. ordinarium