From biomedicine to natural history research: EST resources for ambystomatid salamanders

1471-2164-5-54 1471-2164 Research article From biomedicine to natural history research: EST resources for ambystomatid salamanders Putta Srikrishna sputt2@uky.edu Smith J Jeramiah jjsmit3@uky.edu Walker A John jawalk2@uky.edu Rondet Mathieu mrondet@uci.edu Weisrock W David weisrock@uky.edu Monaghan James james.monaghan@uky.edu Samuels K Amy aksamu2@uky.edu Kump Kevin kevinkump@gmail.com King C David dck163@psu.edu Maness J Nicholas njmaness@wisc.edu Habermann Bianca habermann@mpi-cbg.de Tanaka Elly tanaka@mpi-cbg.de Bryant V Susan svbryant@uci.edu Gardiner M David dmgardin@uci.edu Parichy M David dparichy@mail.utexas.edu Voss S Randal srvoss@uky.edu

Department of Biology, University of Kentucky, Lexington, KY 40506, USA

Department of Developmental and Cell Biology and the Developmental Biology Center, University of California, Irvine, CA 92697, USA

The Life Sciences Consortium, 519 Wartik Laboratory, Penn State University, University Park, PA 16802, USA

Department of Zoology, University of Wisconsin-Madison, 250 N. Mills, Madison, WI 53706, USA

Scionics Computer Innovation GmbH, Pfotenhauerstrasse 110, 01307 Dresden, Germany

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany

Section of Integrative Biology and Section of Molecular, Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA

BMC Genomics 1471-2164 2004 5 1 54 http://www.biomedcentral.com/1471-2164/5/54 1531038810.1186/1471-2164-5-54 19 7 2004 13 8 2004 13 8 2004 2004 Putta et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.

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.

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^thcentury. 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 234567. 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 89101112131415. 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 × 10⁹bp)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.

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.

Tissue

cDNAs sequenced

GARD

limb blastema

1029

MATH

limb blastema

16244

tail blastema

1422

brain

3196

liver

792

spleen

337

heart

gill

3039

stage 22 embryo

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

^aIncludes 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

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

MexCluster_7987_Contig3

252

(NM_001402) eukaryotic translation elongation f1

MexCluster_9285_Contig1

240

(NM_001101) beta actin; beta cytoskeletal actin

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

MexCluster_9187_Contig1

105

(NM_007355) heat shock 90 kDa protein 1, beta

A. t. tigrinum

TigCluster_6298_Contig1

654

(NM_000519) delta globin

E^-38

TigCluster_10099_Contig2

193

(NM_001614) actin, gamma 1 propeptide

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

TigCluster_5960_Contig1

(NM_001101) beta actin; beta cytoskeletal actin

TigCluster_7383_Contig1

(NM_001614) actin, gamma 1 propeptide

TigCluster_6645_Contig1

(NM_001063) transferrin

TigCluster_7226_Contig4

(NM_006009) tubulin, alpha 3

E^-160

TigCluster_7191_Contig1

(NM_019016) keratin 24

E^-89

TigCluster_10121_Contig1

(NM_005141) fibrinogen, beta chain preproprotein

TigCluster_6705_Contig1

(NM_000558) alpha 1 globin

E^-39

TigCluster_7854_Contig1

(NM_021870) fibrinogen, gamma chain isoform

E^-121

TigCluster_6139_Contig1

(NM_001404) eukaryotic translation elongation f1

TigCluster_7226_Contig2

(NM_006009) tubulin, alpha 3

TigCluster_10231_Contig1

(NM_003018) surfactant, pulmonary-associated prot.

E^-08

TigCluster_6619_Contig1

(NM_000041) apolipoprotein E

E^-38

TigCluster_7232_Contig2

(NM_003651) cold shock domain protein A

E^-46

TigCluster_5768_Contig1

(NM_003380) vimentin

E^-177

TigCluster_9784_Contig3

|XP_218445.1| similar to RIKEN cDNA 1810065E05

E^-15

Figure 2

Venn diagram of BLAST comparisons among amphibian EST projects

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 http://www.sanger.ac.uk/Projects/X_tropicalis

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^-80or 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)

binding (0005488)

3117

2578

chaparone (0003754)

100

enzyme regulation (003023)

193

223

motor (0003774)

signal transduction (0004871)

344

375

structural protein (0005198)

501

411

transcriptional reg. (0030528)

296

221

translational reg. (0045182)

bone remodeling (0046849)

circulation (0008015)

immune response (000695)

182

263

respiratory ex. (0009605)

254

288

respiratory in. (0009719)

stress (0006950)

263

320

Biological Process (0008150)

Cellular (0009987)

activation (0001775)

aging and death (0008219)

158

148

communication (0007154)

701

696

differentiation (0030154)

extracellular mat. (0043062)

growth and main. (0008151)

1731

1445

migration (0016477)

motility (0006928)

163

154

Developmental (0007275)

aging (0007568)

embryonic (0009790)

growth (0040007)

morphogenesis (0009653)

350

272

pigment (0048066)

post embryonic (0009791)

reproduction (0000003)

Physiological (0007582)

coagulation (0050817)

death and aging (0016265)

159

148

homeostasis (0042592)

metabolism (0008152)

3059

2513

secretion (0046903)

sex differentiation (0007548)

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) 2425; 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

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)

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.

TigCluster_6648_Contig1

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

TigCluster_5768_Contig1

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

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^-80yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of E<10^-20yielded 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