Conserved Vertebrate Chromosome Segments in the Large Salamander Genome

Corresponding author: S. Randal Voss, Department of Biology, Colorado State University, Fort Collins, CO 80523., srvoss{at}lamar.colostate.edu (E-mail)

Communicating editor: N. A. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Urodele amphibians (salamanders) are important models for embryological, physiological, and natural history research and are also a biomedically important group because they are the only vertebrates capable of regenerating entire organ systems. To enhance the utility of salamanders for biomedical research and for understanding genome evolution, genetic linkage analysis was used to identify chromosome segments that are homologous between ambystomatid salamanders and distantly related vertebrate model organisms. A total of 347 loci (AFLPs, RAPDs, and protein-coding loci) were mapped using an interspecific meiotic mapping panel (Ambystoma mexicanum and A. tigrinum tigrinum; family Ambystomatidae). Genome size in Ambystoma was estimated to be 7291 cM, the largest linkage map estimate reported for any organism. However, the relatively large size of the salamander genome did not hinder efforts to map and identify conserved syntenies from a small sample of 24 protein-coding loci. Chromosomal segments that are conserved between fishes and mammals are also conserved in these salamanders. Thus, comparative gene mapping appears to be an efficient strategy for identifying orthologous loci between ambystomatid salamanders and genomically well-characterized vertebrate model organisms.

COMPARATIVE gene mapping among vertebrate species is providing insight into the relative importance and timing of gene duplications, gene losses, gene order rearrangement, and chromosomal exchanges during vertebrate genome evolution (APARICIO et al. 1997 ; EHRLICH et al. 1997 ; O'BRIEN et al. 1997 ; FORCE et al. 1999 ; GROENEN et al. 2000 ; NARUSE et al. 2000 ; POSTLETHWAIT et al. 2000 ; WOODS et al. 2000 ). Nevertheless, relatively little progress has been made in extending genome analysis to representative species of all the major vertebrate groups. Although chicken is becoming a model for one "reptilian" lineage (GROENEN et al. 2000 ), no amphibian genome has been characterized extensively. This is somewhat surprising given the historical importance of amphibians as model organisms and the pivotal phylogenetic position of this lineage for understanding evolutionary transitions from fishes to amniotes.

For more than a century, urodele amphibians (salamanders) have been used as models for embryology, physiology, and natural history research (e.g., ROY et al. 2000 ; VOSS and SHAFFER 2000 ). Moreover, salamanders are the only adult vertebrates that can regenerate organ systems completely and are thus a potentially valuable group for understanding mechanisms of tissue repair and repatterning, as well as for designing clinical therapies to treat human trauma, amputation, and disease (reviewed by DINSMORE 1996 ). To enhance the utility of salamanders for biomedical, genomic, and other research, we initiated a project to develop a comparative gene map that will allow identification of orthologous loci between salamanders and other vertebrates. We chose Mexican axolotl (Ambystoma mexicanum) and tiger salamander (A. tigrinum tigrinum) because these species are arguably the most important given their long histories as research models and their continued use in contemporary research programs. Although strikingly different in morphology and life history, it is possible to cross the axolotl and tiger salamander (as well as other closely and distantly related species; VOSS and SHAFFER 1996 ) to create mapping panels for genetic linkage analysis. In previous work, axolotl/tiger salamander hybrids were backcrossed to axolotl to generate map cross families herein referred to as the AxTg mapping panel. This mapping panel already has been used to map a major quantitative trait locus (QTL) for metamorphic failure (VOSS and SHAFFER 1997 ) and to test candidate genes for metamorphic failure and a mutation affecting pigment cell morphogenesis (PARICHY et al. 1999 ; VOSS et al. 2000 ).

Although previous studies utilized the AxTg mapping panel for genetic linkage analyses (VOSS and SHAFFER 1997 ; PARICHY et al. 1999 ; VOSS et al. 2000 ), there was no investigation of chromosomal segment homologies to other vertebrates. The likelihood of identifying homologous chromosomal segments seems reasonable given that conserved syntenies are being established between fishes and mammals (NARUSE et al. 2000 ; POSTLETHWAIT et al. 2000 ). However, one potentially confounding factor is the relatively large size of salamander genomes when compared to other vertebrates; although ambystomatid salamanders have relatively few chromosomes (N = 14), physical genome size is estimated to be an order of magnitude larger than human (STRAUS 1971 ). Here we report that Ambystoma also has a correspondingly large genetic linkage map, in fact the largest known in any organism. Despite this large genome and genetic map, we show that protein-coding loci can be mapped efficiently in this system and conserved gene orders can be identified between salamanders and distantly related vertebrates with much smaller genomes. Thus, some regions of chromosomal synteny have been maintained between these amphibians and other vertebrate lineages, suggesting that genome cross-referencing between salamanders, humans, and other model vertebrates ultimately will provide new insights into genome evolution and new tools for biomedical research.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Salamander crosses, DNA markers, and polymorphism detection:
A mapping panel (AxTg) was created by crossing two male F₁ A. mexicanum/A. tigrinum tigrinum hybrids to a single A. mexicanum female (VOSS 1995 ). DNA was isolated from 44 backcross offspring and typed for 314 amplified fragment length polymorphisms (AFLPs), nine randomly amplified polymorphic DNA markers (RAPDs), and 24 protein-coding loci. A total of 262 AFLPs were identified previously (VOSS and SHAFFER 1997 ). Additional AFLP marker loci were scored using primer sets for EcoRI/MseI and NotI/HpaII restriction fragments (Table 1 and Table 2; see VOS et al. 1995 and VOSS and SHAFFER 1997 for a description of AFLP methods). RAPD primers are listed in Table 3; the RAPD protocol was described previously (VOSS 1993 ). Table 4 lists protein-coding loci that were mapped, as well as PCR primers and conditions. Protein-coding loci were amplified using 150 ng DNA, 75 ng each primer, 1.5 mM MgCl₂, 0.3 units Taq, and 32 cycles of a three-step profile (45 sec denature, 60 sec anneal, and 30–60 sec extend). Protein-coding loci were verified by sequencing PCR products from the grandparental A. mexicanum and A. t. tigrinum individuals from which the mapping panel was derived. Nucleotide sequence alignments between A. mexicanum and A. t. tigrinum protein-coding loci revealed polymorphisms that were scored using restriction digestion, single-strand conformation polymorphism (SSCP), allele-specific amplification (presence/absence of PCR product), or length variation. SSCP methods followed VOSS et al. 2000 . Length variants were resolved using 1–4% agarose gels.

Genetic linkage analysis and estimation of genome size:
Linkage analysis was performed using Map Manager QTXb07 (MEER et al. 2000 ). To heuristically search for syntenic groups, loci were grouped simultaneously into linkage groups using a search/linkage criteria of P = 0.001. The method suggested by HULBERT et al. 1988 was used to estimate total genome size:

Orthology and establishment of conserved synteny:
Preliminary orthologies for Ambystoma protein-coding loci were established using BLASTX searches (ALTSCHUL et al. 1997 ) and the nonredundant protein sequence database (E value 10^-5; http://www.ncbi.nlm.nih.gov/blast/blast.cgi). Conserved syntenies were identified by comparing ambystomatid linkage groups with two or more protein-coding loci to human, mouse, and zebrafish genome maps. Genome maps from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the Human Genome Project at University of California, Santa Cruz (http://genome.ucsc.edu/index.html) were used to locate positions of presumptive orthologues in human, and the Mouse Genome Database (http://www.informatics.jax.org) and ZFIN (http://zfish.uoregon.edu/) were used to locate presumptive orthologues in mouse and zebrafish.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA markers segregated according to Mendelian expectation:
As a preliminary step toward building a genetic linkage map for these salamander species, we asked whether molecular markers segregated according to Mendelian expectations. We thus compared the proportions of homozygous and heterozygous genotypes for each of the AFLP and RAPD markers as well as protein-coding loci scored in the AxTg mapping panel. This analysis revealed that none of the protein-coding loci and only 5% of the anonymous molecular marker loci (AFLPs and RAPDs) exhibited segregation patterns that deviated significantly from an expected 1:1 ratio of homozygous and heterozygous genotypes. Thus, the vast majority of loci segregated in a Mendelian manner between these closely related species.

A partial genetic linkage map was constructed:
We used the 347 markers scored for segregation in the AxTg mapping panel to generate a framework genetic linkage map in which a total of 56 linkage groups were identified (Fig 1; updated versions of this map also can be found via the world wide web at http://lamar.colostate.edu/~srvoss/SGP/). The majority of these linkage groups (N = 31) consisted of four or fewer linked DNA markers (range, 0–61.7 cM). Nevertheless, several large linkage groups comprising as many as 21 loci were identified, 10 of which were >100 cM (range, 101.1–295.1 cM). A total of 48 loci were found to be unlinked. The entire group of linked markers provided 3475 cM of genome coverage. Given that ambystomatid salamanders have a haploid chromosome number of 14, the observation of many small linkage groups suggests that additional markers will be needed to comprehensively map all chromosomal regions. To estimate the genome size of Ambystoma from these partial linkage data, we determined the total number of marker pairs that were linked at LOD > 3.0 and then applied the method of HULBERT et al. 1988 . A total of 494 significantly linked marker pairs yielded an estimated genome size of 7291 cM.

View larger version (89K): In this window In a new window Download PPT slide   Figure 1. Partial genetic linkage map for Ambystoma using the AxTg panel. For each linkage group, distance (centimorgans) is shown to the left; DNA markers are shown to the right.

Protein-coding loci were mapped efficiently using the AxTg panel, revealing conserved syntenies with other vertebrates:
The generation of a partial genetic linkage map, comprising principally anonymous markers, nevertheless allowed us to ask whether protein loci can be mapped easily using the AxTg mapping panel and whether regions of conserved synteny can be identified in salamanders relative to other vertebrates. To these ends, we targeted 26 protein-coding loci for mapping, which represented a diversity of developmental and physiological protein products. Several of these loci are known to be genetically and physically linked in other vertebrate species. Of the 26 loci targeted, 24 (92%) exhibited nucleotide polymorphism between A. mexicanum and A. t. tigrinum (the exceptions were POMC and TOP1). Table 5 shows map positions for these 24 protein-coding loci relative to presumptive orthologues from human, mouse, and zebrafish. Despite the large genetic and physical size of the salamander genome, we identified five conserved syntenies between salamander and other distantly related vertebrate species. Four of the conserved syntenies include loci of HOX gene complexes. A conserved synteny containing HOXA loci was identified by mapping HOXA4 and HOXA9 to LG 43. A second conserved synteny was identified by mapping HOXB13 and three additional loci (THRA, COL1A1, and DLX3) to LG 23. All four of these loci are located on human chromosome 17 (Hsa 17), and combinations of these genes are syntenic in zebrafish and mouse. A third conserved synteny was identified by mapping HOXC10 and WNT1 to LG 7. On Hsa 12, HOXC complex genes are syntenic with WNT1. A fourth conserved synteny was identified by mapping HOXD8, HOXD11, and AOX1 to LG 9. On Hsa 2, HOXD complex genes are syntenic with AOX1. Finally, a fifth conserved synteny was identified by mapping PDGFRA-KIT to LG 12.

Several syntenies unique to axolotl also were observed: KITLG-UVCP, FOXG1B-CSPG2, and GAPD-d (d = white color mutant; PARICHY et al. 1999 ). UVCP has not been identified in mammalian models and it has yet to be mapped in reptiles and fishes. The linkage of FOXG1B and CSPG2 in Ambystoma is not observed in human, as presumptive orthologues of these genes are found on Hsa 14 and Hsa 5, respectively. Linkage between Gapd-d presumptively localizes the white color mutant to a conserved, vertebrate chromosomal segment containing loci that are syntenic with GAPD.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study represents the first attempt to examine amphibian genome structure by determining the order and location of hundreds of molecular markers. In turn, the partial genetic linkage map we constructed has implications for the feasibility of genetic mapping in this research model organism, the size and structure of ambystomatid genomes, and genome evolution in vertebrates.

Genetic linkage analysis in Ambystoma:
Our crossing design using the two interfertile species, Mexican axolotl and tiger salamander (A. mexicanum and A. t. tigrinum), allowed genetic mapping to be accomplished in a highly efficient manner. Nucleotide variation was found for all but two protein-coding loci between these species and all polymorphic loci were subsequently mapped. Clearly, considerable molecular differences have accrued between these two species since their estimated time of divergence some 10–15 mya (B. SHAFFER, personal communication). However, these differences do not greatly influence the segregation of molecular marker genotypes in hybrid crosses, and, more generally, do not greatly affect the viability and fertility of hybrid offspring (VOSS 1995 ). The extent to which fertile hybrid crosses can be made among salamanders is somewhat unusual for vertebrate species (VOSS and SHAFFER 1996 ). One possible explanation is a conservative rate of chromosomal evolution for ambystomatid salamanders as compared to other vertebrates. For example, giemsa banding patterns of A. mexicanum and A. t. tigrinum are identical for 4 of the 14 chromosomes, and there is no gross evidence for translocations or inversions among the remainder (CUNY and MALACINSKI 1985 ). If chromosomal evolution has occurred at a relatively lower rate in Ambystoma, it may be a particularly useful model for reconstructing the ancestral tetrapod condition that subsequently gave rise to the human genome. Indeed, chromosome number in Ambystoma approximates that expected of the common ancestor of fish and amniotes (POSTLETHWAIT et al. 2000 ).

Estimate of the ambystomatid genetic map:
Our genetic linkage map estimate of 7291 cM is the largest reported for any organism. This is not unexpected because salamanders are known to have physically large genomes (MORESCALCHI 1975 ), and genetic and physical map sizes often are correlated (SYBENGA 1996 ). The C value of A. tigrinum is estimated at 32 x 10⁹ bp (STRAUS 1971 ), an order of magnitude larger than most vertebrates but only moderate in size when compared to other salamanders (GRAUR and LI 2000 ). Nevertheless, it is possible that our estimate is biased upward because we used a small mapping panel. Moreover, this estimate also assumes a random sampling of molecular marker loci, an assumption that may not always be met (BECKER et al. 1995 ; KIEM et al. 1997 ; SALIBA-COLOMBANI et al. 2000 ). For example, a recent analysis of tomato found that >50% of mapped anonymous molecular marker loci (AFLPs and RAPDs) were located in heterochromatic regions near centromeres (SALIBA-COLOMBANI et al. 2000 ). Our goal of mapping 500 additional protein-coding loci, which is underway, ultimately will provide a comprehensive genome map and a more accurate estimate of genome size.

Comparative gene mapping:
Despite the relatively large size of the salamander genome, conserved syntenies were identified among Ambystoma, zebrafish, human, and mouse. Our data indicate that both tightly (e.g., PDGFRA-KIT) and distantly (HOXD8,11-AOX1) linked genes in human are syntenic in Ambystoma. Moreover, our results show that DLX3-COL1A1-HOXB13-THRA are linked in Ambystoma as they are on Hsa 17. This finding provides support for the hypothesis that a large conserved block of Hsa 17 loci has been maintained in zebrafish and human (as well as cat, rat, and mouse) since their divergence from a common ancestor some 450 mya (POSTLETHWAIT et al. 2000 ). Our result suggests that Hsa 17 loci have remained linked for an additional 350 million years of independent evolution within the amphibian clade. Although linkage over this time frame might suggest some type of functional constraint, it is interesting to note that Hsa 17 loci are distributed among multiple chromosomes in chicken, indicating that if such a constraint exists it cannot be absolute (GROENEN et al. 2000 ). Moreover, comparison of syntenic loci among chicken, human, and mouse suggests greater overall conservation between chicken and human than between chicken and mouse, despite the fact that human and mouse shared a more recent common ancestor (BURT et al. 1999 ). Clearly, the problem of reconstructing vertebrate genome evolution is complex and will undoubtedly require multiple representative genomes from each of the major vertebrate groups to distinguish lineage-specific patterns and processes (e.g., genome duplications in fishes, translocations in mouse, chromosomal fissions in chicken) from true conservation and homology. The development of genetic linkage maps for anuran amphibians (e.g., Rana, HOTZ et al. 1997 ; Xenopus, http://www.virginia.edu/~develbio/trop/), will contribute to these efforts and provide an interesting comparison to salamander.

Extent of conserved synteny between salamander and human:
Knowledge of the extent of conserved synteny between two species is informative for estimating rates of chromosome evolution and for identifying orthologous loci (INTERNATIONAL HUMAN GENOME SEQUENCING CONSORTIUM 2001 ). Using the human genome working draft (http://genome.ucsc.edu/index.html), we determined the physical distance between syntenic loci that presumably flank conserved chromosomal segments in salamander. Overall, the genetic linkage distances (centimorgans) of conserved segments in salamander were positively correlated to the physical distances (megabases) observed in human (Fig 2). For example, the linkage distance of PDGFRA-KIT was estimated at 2.60 cM in salamander and 0.36 Mb in human, while the linkage distance of AOX1-HOXD was estimated at 62.3 cM in salamander and 25.69 Mb in human. If large conserved syntenies are common between salamander and human, this would greatly simplify the search for orthologous candidate genes. However, we note that conserved syntenies reported in this article correspond primarily to chromosomal segments containing HOX clusters, which may bias upward estimates of synteny conservation because of functional constraints associated with cis-regulatory elements (e.g., VAN DER HOEVEN et al. 1996 ; KMITA et al. 2000 ).

View larger version (17K): In this window In a new window Download PPT slide   Figure 2. Genetic linkage distance (cM) of conserved segments identified in salamander relative to physical distance (Mb) in human.

Conclusions:
Our study shows that genetic linkage analysis is an efficient method for identifying homologous chromosomal segments between ambystomatid salamanders and genomically well-characterized vertebrate models. Given the importance of Ambystoma as a research model (http://www.indiana.edu/~axolotl/) and its pivotal phylogenetic position for understanding evolutionary transitions from fishes to amniotes, additional protein-coding loci are being mapped to allow candidate gene identification within the ambystomatid system and to provide an amphibian perspective to vertebrate genome evolution.

	ACKNOWLEDGMENTS

We thank J. Ma and K. Mason for providing unpublished sequences, and D. King for web site support. This work was supported by National Science Foundation grants BSR-9101128, IBN-9509802, IBN-0080112, IBN-0093251, and institutional funds from Colorado State University to S.R.V., and National Institutes of Health grant RO1 HD40165-01 to D.M.P.

Manuscript received January 18, 2001; Accepted for publication March 20, 2001.

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