BMC Genomics 2004, 5:54 http://www.biomedcentral.com/1471-2164/5/54 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 iso- lated 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 MgCl2, 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 data- base, 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-spe- cific polymorphisms were assayed by allele specific ampli- fication, 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 infor- mation about genotyping methodology. 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 esti- mation; 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 regen- eration 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. Acknowledgements We thank the Axolotl Colony. We thank Greg Chinchar and Betty David- son for providing RNA to make cDNA libraries V3 and V4. We acknowl- edge the support of the National Science Foundation, the National Center for Research Resources at the National Institutes of Health, the Kentucky Spinal Cord and Head Injury Research Trust, and the NSF EPSCOR initia- tive in Functional Genomics at University of Kentucky. References 1. Beetschen J-C: How did urodele embryos come into promi- nence as a model system. Int J Dev Biol 1996, 40:629-636. 2. Gardiner DM, Endo T, Bryant SV: The molecular basis of amphib- ian limb regeneration: integrating the old with the new. Semin Cell Dev Biol 2002, 13:345-352. 3. Echeverri K, Tanaka EM: Ectoderm to mesoderm lineage switching during axolotl tail regeneration. Science 2002, 298:1933-1936. 4. Del Rio-Tsonis K, Jung JC, Chiu IM, Tsonis PA: Conservation of fibroblast growth factor function in lens regeneration. Proc Natl Acad Sci USA 1997, 94:13701-13706. 5. Ikegami Y, Mitsuda S, Araki M: Neural cell differentiation from retinal pigment epithelial cells of the newt: an organ culture model for the urodele retinal regeneration. J Neurobiol 2002, 50:209-20. 6. Chernoff EAG, Stocum DL, Nye HLD, Cameron JA: Urodele spinal cord regeneration and related processes. Dev Dyn 2003, 226:295-307. 7. Ferretti P: Re-examining jaw regeneration in urodeles: what have we learnt? Int J Dev Biol 1996, 40:807-811. 8. Zhang J, Wu SM: Goalpha labels ON bipolar cells in the tiger salamander retina. J Comp Neurol 2003, 461:276-289. 9. Falck P, Hanken J, Olsson L: Cranial neural crest emergence and migration in the Mexican axolotl (Ambystoma mexicanum). Zoology-Jena 2002, 105:195-202. 10. Zhang C, Dube DK, Huang X, Zajdel RW, Bhatia R, Foster D, Leman- ski SL, Lemanski LF: A point mutation in bioactive RNA results in the failure of mutant heart correction in mexican axolotls. Anat Embryol 2003, 206:495-506. 11. Kauer JS: On the scents of smell in the salamander. Nature 2002, 417:336-342. 12. Voss SR, Prudic KL, Oliver JC, Shaffer HB: Candidate gene analysis of metamorphic timing in ambystomatid salamanders. Mol Ecol 2003, 12:1217-1223. 13. Riley SPD, Shaffer HB, Voss SR, Fitzpatrick BM: Hybridization between a rare, native tiger salamander (Ambystoma californiense. Ecol Appl 2003, 13:1263-1275. 14. Borland S, Crawford K, Brand V: Setting the stage: Developmen- tal biology in pre-college classrooms. Int J Dev Biol 2003, 47:85-91. 15. Jancovich JK, Mao J, Chinchar VG, Wyatt C, Case ST, Kumar S, Valente G, Subrmanian S, Davidson EW, Collins JP, Jacobs JB: Genomic sequence of a ranavirus (family Iridoviridae) asso- ciated with salamander mortalities in North America. Virology 2003, 316:90-103. 16. Shaffer HB, McKnight ML: The polytypic species revisited: genetic differentiation and molecular phylogenetics of the Page 16 of 17 (page number not for citation purposes)