BACKGROUND
the trinidadian guppy, poecilia reticulata peters, is well known for the highly polymorphic male color patterns, which have been the subject of genetic analysis for almost a century  <cit> .
the vast literature on the ecology and evolution of the guppy and the extensive phenotypic variation in wild populations make the guppy a particularly attractive choice for understanding the molecular basis of adaptation to varying natural conditions.
despite the wealth of field studies, molecular genetic information about the guppy is still scarce.
therefore, a genetic map would be a first step towards identifying quantitative adaptive traits of the guppy, including male ornamentation and predator-driven adaptations found in different river habitats in trinidad, underlying heritable differences in life history traits  <cit> .
the genome size of the guppy is estimated to be around  <dig> mbp, with a diploid set of  <dig> chromosomes, including genetically defined x and y sex chromosomes  <cit> .
a first map of the sex chromosomes, based on classical genetic analysis of male colour patterns, has been sketched out by winge and co-workers  <cit> .
more recently, phang and co-workers used ornamental guppies from singapore to generate a genetic map based on  <dig> rapd markers, and a cross between two laboratory strains of different body shape and colour was mapped using a combination of  <dig> aflp and microsatellite markers  <cit> .
conservation of microsatellites between closely related species and synteny with respect to  <dig> microsatellite markers were suggested by an intergeneric cross between xiphophorus maculatus  <cit>  and p. reticulata  <cit> .
in all of these studies, the number of linkage groups fell short of the chromosome number, indicating that a higher marker density is required for complete coverage of the genetic map.
unfortunately, rapd and aflp markers cannot be easily reused for studying crosses between outbred strains of wild guppies.
we have identified hundreds of expressed sequence tag -linked single nucleotide polymorphism  markers, suitable for genetic mapping of wild guppies.
the fact that these markers are linked to expressed genes will help to exploit syntenic information from fully sequenced genomes of other fish species  <cit> .
this will in turn also facilitate future identification of candidate genes when mapping qualitative morphological as well as quantitative life history traits of the guppy.
RESULTS
a p. reticulata est database
several guppy cdna libraries were constructed using smart technology, as detailed in materials and methods.
as sources of mrna we used whole embryos, newborn fish, adult liver, testis, brain, retina, and skin, in order to obtain a broad spectrum of different expressed sequences.
several feral and laboratory strains were used, including the quare <dig> strain from east trinidad and the tranquille strain from west trinidad  <cit> .
this allows for direct sequence comparison of abundant transcripts between different strains.
between  <dig> and  <dig>  clones were picked at random from each library, depending on its complexity.
the inserts were first sequenced from the 5' end and sequences were compared to embl vertebrate databases  using ncbi blastn and blastx algorithm  <cit>  to assign a possible function.
blast results were parsed and automatically entered into a mysql est database  <cit> .
sequences lacking sufficiently good support by blast hits with an e-value higher than 10- <dig> were not entered into the database, but were set aside for periodically repeated subsequent blast searches.
our est database http://guppy.weigelworld.org currently comprises about  <dig>  entries, which represent about  <dig>  different gene products.
the quare and tranquille strains are represented by about 40% of the entries each, with the remaining 20% derived from other strains, including the laboratory strains blue and istanbul wild  <cit>  .
about  <dig>  ests with a minimum of  <dig> bp sequence available have been deposited to genbank.
the accession numbers are included in table  <dig>
*clones for which sequence information was less than  <dig> nucleotides have not yet been submitted to genbank.
the accession numbers of the submitted clones are: es370951–es387146
the results of the blastx searches were parsed for annotation according to go criteria  <cit> , describing molecular function or biological processes, provided that the e-value of the best hit was lower than 10- <dig> .
most of the proteins encoded by non-redundant annotated guppy ests have binding  or catalytic  activity or are structural proteins .
this distribution resembles that described for the fundulus heteroclitus est database  <cit> , although the guppy database contains relatively more ests derived from retina, testis, and muscle.
among enzymes and structural proteins, muscle proteins are the most abundant, because somite tissue prevails in advanced embryos, the predominant source of our est libraries .
sequences without significant best blast hits or with hits to genes that lacked informative annotation, e.g.
from genomic surveys or non-annotated est products, are listed as unknown in table  <dig>  about 25% of the guppy cdnas in our database is annotated as unknown.
the est database can be searched by clone name, by accession number of the best blast search hit, and by possible biological function of the deduced protein product of each est.
furthermore, the blast hits are saved in a table, in which a full text search for annotations as well as accession numbers can be performed.
a subset of est clones was also sequenced from the 3' end, and the longest orf was extracted from the assembly with its 5' sequence.
information on the coding regions was obtained by database searches using blastx.
contigs representing multiple hits of the same gene product were aligned in order to analyse these for polymorphisms.
we provide a download function for the extraction of the 5' and 3' end cdna sequences, as well as assemblies in the fasta format  <cit> .
it is also possible to perform blast searches against all ests.
development of single nucleotide polymorphism  markers in guppies
for identification of polymorphisms, we used ests to design pcr primers for amplification from genomic dna of different strains.
we primarily amplified 3' utr sequences, because they are less conserved than coding sequences and not interrupted by introns.
the 3' utrs are typically shorter than in mammals, as has been reported for other lower vertebrates  <cit> .
this somewhat reduces the usefulness of 3' utrs for identification of snps by resequencing of genomic dna from different strains.
several est libraries were constructed  and some consisted of size-selected sub fractions .
in the different libraries, between  <dig> and  <dig> % of the cdna sequences had 3' utrs longer than  <dig> bp and those were given priority.
the 3' end of the coding sequence was included where required to produce fragments about  <dig> to  <dig> bp in length.
in many instances, pcr amplification and sequencing revealed that the 3' ends of coding regions contained short  introns.
a comparison of ests from different strains reveals that, as expected, coding sequences are less polymorphic than the 3' utrs.
some primers that were designed to flank an intron were also used, yet their efficient design required prediction of the most likely exon-intron boundaries by alignment of the ests to genomic sequences from other fish species, and the length of the resulting pcr products was unpredictable.
aside from snps, 3' utrs and introns also contained short insertions and deletions  at lower frequency .
of these indels, about  <dig> % were found as parts of either short tandem repeat polymorphisms or of homopolymer stretches .
a second mysql database was established for the management of the strain-specific markers and was linked to the est database.
from the marker database information can be retrieved on the reference clone, the primer pairs used, type , and position of polymorphisms between genomic sequences of the different guppy strains.
the marker database can also accommodate information on available assays for these polymorphisms.
so far, these are mainly maldi tof assays developed for high throughput detection of snp markers  <cit> .
all multiple alignments of genomic and cdna sequences that had been generated for polymorphism detection  <cit>  were loaded into the multiple snp query tool , a database used for snp assay development, especially for design of strain-specific assays .
from  <dig> unique ests that were successfully re-sequenced in  <dig> strains of guppy, we could identify  <dig>  est linked polymorphic loci.
about 75% of the polymorphisms were identified by comparison of only four populations, originally collected in the quare , tranquille and upper aripo rivers , and in central cumaná .
the geographically most distant among these are the quare and the cumaná guppies and we found these to be most genetically divergent.
as shown in table  <dig>  the evaluation of  <dig> polymorphic ests using the msqt, allowed for the selection of  <dig> assays for markers that are each linked to a different gene product and that distinguish between quare and cumaná populations.
a similar or slightly smaller number of assays were predicted to distinguish other pairs of ecotypes .
for analysis of genetic crosses between different feral guppy strains, these est-linked snp markers can be supplemented by about  <dig> microsatellite markers previously described in guppies  <cit> .
numbers above the diagonal refer to the total number of polymorphisms in the dataset, numbers in bold refer to polymorphisms representing one distinct est each.
blast searches with the guppy ests selected for marker development were performed against the tetraodon nigroviridis genome.
the number of hits for each chromosome was approximately proportionate to the length of each t. nigroviridis chromosome and indicated that at least two markers corresponding to each tetraodon chromosome had been identified.
another 38% of these sequences aligned to the 40% of the tetraodon sequences that were not yet assigned to one of its  <dig> chromosomes .
similarly, guppy ests were aligned to genomic sequences of zebrafish , fugu  and medaka , to facilitate prediction of potential coding sequences in regions of interest for mapping, genomic walking, and for prediction of exon-intron boundaries .
since a certain degree of synteny between different fish species has been previously described  <cit> , a higher marker density in regions of interest for genetic mapping may be achieved by ortholog cloning based on synteny predictions on genes in the guppy.
however, even between close relatives, this approach will not work perfectly, given the fact that chromosome numbers are variable and chromosomal rearrangements occur frequently.
molecular phylogeny of the guppy
previous molecular phylogenetic studies on guppies were primarily based on mitochondrial sequences  <cit> .
the molecular resources we developed enable studies on molecular evolution of nuclear genes, e.g.
the identification of genes that have rapidly diversified and are potentially under positive selection, as exemplified by a study comparing genes encoding the long wave length sensitive opsins to other opsins  <cit> .
additional applications include studies on molecular phylogeny of the guppy.
coding sequences of orthologous expressed nuclear genes from nine different fish species, including the guppy, were identified by reciprocal blast.
for phylogenetic analysis, these sequences were concatenated and a bionj tree was reconstructed  <cit> , using splitstree <dig> software, version  <dig>   <cit>  .
the selected genes include highly conserved ribosomal proteins as well as metabolic enzymes and transcription factors .
the number of teleost species in this analysis was limited by available sequence data from so-called non-model species, since the intersection of orthologous genes got smaller the more non-model species were included.
the resulting topology was essentially the same when only four coding sequences were concatenated.
by convergence tests using increasing numbers of nuclear genes we showed that the pairwise distance statistics and the bootstrap values further improved upon addition of more sequences.
this showed that a stable tree topology requires seven or more genes .
furthermore, the topology of the phylogenetic tree was the same when maximum likelihood or maximum parsimony estimation was used instead of bionj .
this is in agreement with previously published comparisons of reconstruction methods, showing that bionj is not inferior to maximum likelihood  <cit> .
this tree confirms previous phylogenetic studies on poeciliid fishes, based on morphological criteria  <cit>  and on genomic sequences  <cit> .
in the future, intraspecific polymorphisms found in nuclear genes of feral guppy populations of different origins will be compared to previous phylogenies of different guppy strains  <cit>  .
this will enable investigation of population structure and mapping of quantitative adaptive traits.
CONCLUSIONS
we established a non-normalized est library of the guppy from embryos and six different adult tissues, containing  <dig>  entries with three-fold redundancy on average.
we show that re-sequencing of 3' utrs from genomic dna of different guppy strains is a powerful strategy to identify polymorphisms existing between feral guppies of geographically different origins.
sequence information of  <dig> nuclear coding genes is sufficient to reconstruct a robust teleost phylogeny including the guppy poecilia reticulata.

 the trinidadian guppy, poecilia reticulata peters, is well known for the highly polymorphic male color patterns, which have been the subject of genetic analysis for almost a century. the vast literature on the ecology and evolution of the guppy and the extensive phenotypic variation in wild populations make the guppy a particularly attractive choice for understanding the molecular basis of adaptation to varying natural conditions. despite the wealth of field studies, molecular genetic information about the guppy is still scarce. therefore, a genetic map would be a first step towards identifying quantitative adaptive traits of the guppy, including male ornamentation and predator-driven adaptations found in different river habitats in trinidad, underlying heritable differences in life history traits. the genome size of the guppy is estimated to be around mbp, with a diploid set of chromosomes, including genetically defined x and y sex chromosomes. a first map of the sex chromosomes, based on classical genetic analysis of male colour patterns, has been sketched out by winge and co-workers. more recently, phang and co-workers used ornamental guppies from singapore to generate a genetic map based on rapd markers, and a cross between two laboratory strains of different body shape and colour was mapped using a combination of aflp and microsatellite markers. conservation of microsatellites between closely related species and synteny with respect to microsatellite markers were suggested by an intergeneric cross between xiphophorus maculatus and p. reticulata. in all of these studies, the number of linkage groups fell short of the chromosome number, indicating that a higher marker density is required for complete coverage of the genetic map. unfortunately, rapd and aflp markers cannot be easily reused for studying crosses between outbred strains of wild guppies. we have identified hundreds of expressed sequence tag -linked single nucleotide polymorphism markers, suitable for genetic mapping of wild guppies. the fact that these markers are linked to expressed genes will help to exploit syntenic information from fully sequenced genomes of other fish species. this will in turn also facilitate future identification of candidate genes when mapping qualitative morphological as well as quantitative life history traits of the guppy.  a p. reticulata est database several guppy cdna libraries were constructed using smart technology, as detailed in materials and methods. as sources of mrna we used whole embryos, newborn fish, adult liver, testis, brain, retina, and skin, in order to obtain a broad spectrum of different expressed sequences. several feral and laboratory strains were used, including the quare strain from east trinidad and the tranquille strain from west trinidad. this allows for direct sequence comparison of abundant transcripts between different strains. between and clones were picked at random from each library, depending on its complexity. the inserts were first sequenced from the 5' end and sequences were compared to embl vertebrate databases using ncbi blastn and blastx algorithm to assign a possible function. blast results were parsed and automatically entered into a mysql est database. sequences lacking sufficiently good support by blast hits with an e-value higher than 10- were not entered into the database, but were set aside for periodically repeated subsequent blast searches. our est database http://guppy.weigelworld.org currently comprises about entries, which represent about different gene products. the quare and tranquille strains are represented by about 40% of the entries each, with the remaining 20% derived from other strains, including the laboratory strains blue and istanbul wild. about ests with a minimum of bp sequence available have been deposited to genbank. the accession numbers are included in table *clones for which sequence information was less than nucleotides have not yet been submitted to genbank. the accession numbers of the submitted clones are: es370951–es387146 the results of the blastx searches were parsed for annotation according to go criteria, describing molecular function or biological processes, provided that the e-value of the best hit was lower than 10-. most of the proteins encoded by non-redundant annotated guppy ests have binding or catalytic activity or are structural proteins. this distribution resembles that described for the fundulus heteroclitus est database, although the guppy database contains relatively more ests derived from retina, testis, and muscle. among enzymes and structural proteins, muscle proteins are the most abundant, because somite tissue prevails in advanced embryos, the predominant source of our est libraries. sequences without significant best blast hits or with hits to genes that lacked informative annotation, e.g. from genomic surveys or non-annotated est products, are listed as unknown in table about 25% of the guppy cdnas in our database is annotated as unknown. the est database can be searched by clone name, by accession number of the best blast search hit, and by possible biological function of the deduced protein product of each est. furthermore, the blast hits are saved in a table, in which a full text search for annotations as well as accession numbers can be performed. a subset of est clones was also sequenced from the 3' end, and the longest orf was extracted from the assembly with its 5' sequence. information on the coding regions was obtained by database searches using blastx. contigs representing multiple hits of the same gene product were aligned in order to analyse these for polymorphisms. we provide a download function for the extraction of the 5' and 3' end cdna sequences, as well as assemblies in the fasta format. it is also possible to perform blast searches against all ests. development of single nucleotide polymorphism markers in guppies for identification of polymorphisms, we used ests to design pcr primers for amplification from genomic dna of different strains. we primarily amplified 3' utr sequences, because they are less conserved than coding sequences and not interrupted by introns. the 3' utrs are typically shorter than in mammals, as has been reported for other lower vertebrates. this somewhat reduces the usefulness of 3' utrs for identification of snps by resequencing of genomic dna from different strains. several est libraries were constructed and some consisted of size-selected sub fractions. in the different libraries, between and % of the cdna sequences had 3' utrs longer than bp and those were given priority. the 3' end of the coding sequence was included where required to produce fragments about to bp in length. in many instances, pcr amplification and sequencing revealed that the 3' ends of coding regions contained short introns. a comparison of ests from different strains reveals that, as expected, coding sequences are less polymorphic than the 3' utrs. some primers that were designed to flank an intron were also used, yet their efficient design required prediction of the most likely exon-intron boundaries by alignment of the ests to genomic sequences from other fish species, and the length of the resulting pcr products was unpredictable. aside from snps, 3' utrs and introns also contained short insertions and deletions at lower frequency. of these indels, about % were found as parts of either short tandem repeat polymorphisms or of homopolymer stretches. a second mysql database was established for the management of the strain-specific markers and was linked to the est database. from the marker database information can be retrieved on the reference clone, the primer pairs used, type, and position of polymorphisms between genomic sequences of the different guppy strains. the marker database can also accommodate information on available assays for these polymorphisms. so far, these are mainly maldi tof assays developed for high throughput detection of snp markers. all multiple alignments of genomic and cdna sequences that had been generated for polymorphism detection were loaded into the multiple snp query tool, a database used for snp assay development, especially for design of strain-specific assays. from unique ests that were successfully re-sequenced in strains of guppy, we could identify est linked polymorphic loci. about 75% of the polymorphisms were identified by comparison of only four populations, originally collected in the quare, tranquille and upper aripo rivers, and in central cumaná. the geographically most distant among these are the quare and the cumaná guppies and we found these to be most genetically divergent. as shown in table the evaluation of polymorphic ests using the msqt, allowed for the selection of assays for markers that are each linked to a different gene product and that distinguish between quare and cumaná populations. a similar or slightly smaller number of assays were predicted to distinguish other pairs of ecotypes. for analysis of genetic crosses between different feral guppy strains, these est-linked snp markers can be supplemented by about microsatellite markers previously described in guppies. numbers above the diagonal refer to the total number of polymorphisms in the dataset, numbers in bold refer to polymorphisms representing one distinct est each. blast searches with the guppy ests selected for marker development were performed against the tetraodon nigroviridis genome. the number of hits for each chromosome was approximately proportionate to the length of each t. nigroviridis chromosome and indicated that at least two markers corresponding to each tetraodon chromosome had been identified. another 38% of these sequences aligned to the 40% of the tetraodon sequences that were not yet assigned to one of its chromosomes. similarly, guppy ests were aligned to genomic sequences of zebrafish, fugu and medaka, to facilitate prediction of potential coding sequences in regions of interest for mapping, genomic walking, and for prediction of exon-intron boundaries. since a certain degree of synteny between different fish species has been previously described, a higher marker density in regions of interest for genetic mapping may be achieved by ortholog cloning based on synteny predictions on genes in the guppy. however, even between close relatives, this approach will not work perfectly, given the fact that chromosome numbers are variable and chromosomal rearrangements occur frequently. molecular phylogeny of the guppy previous molecular phylogenetic studies on guppies were primarily based on mitochondrial sequences. the molecular resources we developed enable studies on molecular evolution of nuclear genes, e.g. the identification of genes that have rapidly diversified and are potentially under positive selection, as exemplified by a study comparing genes encoding the long wave length sensitive opsins to other opsins. additional applications include studies on molecular phylogeny of the guppy. coding sequences of orthologous expressed nuclear genes from nine different fish species, including the guppy, were identified by reciprocal blast. for phylogenetic analysis, these sequences were concatenated and a bionj tree was reconstructed, using splitstree software, version. the selected genes include highly conserved ribosomal proteins as well as metabolic enzymes and transcription factors. the number of teleost species in this analysis was limited by available sequence data from so-called non-model species, since the intersection of orthologous genes got smaller the more non-model species were included. the resulting topology was essentially the same when only four coding sequences were concatenated. by convergence tests using increasing numbers of nuclear genes we showed that the pairwise distance statistics and the bootstrap values further improved upon addition of more sequences. this showed that a stable tree topology requires seven or more genes. furthermore, the topology of the phylogenetic tree was the same when maximum likelihood or maximum parsimony estimation was used instead of bionj. this is in agreement with previously published comparisons of reconstruction methods, showing that bionj is not inferior to maximum likelihood. this tree confirms previous phylogenetic studies on poeciliid fishes, based on morphological criteria and on genomic sequences. in the future, intraspecific polymorphisms found in nuclear genes of feral guppy populations of different origins will be compared to previous phylogenies of different guppy strains. this will enable investigation of population structure and mapping of quantitative adaptive traits.  we established a non-normalized est library of the guppy from embryos and six different adult tissues, containing entries with three-fold redundancy on average. we show that re-sequencing of 3' utrs from genomic dna of different guppy strains is a powerful strategy to identify polymorphisms existing between feral guppies of geographically different origins. sequence information of nuclear coding genes is sufficient to reconstruct a robust teleost phylogeny including the guppy poecilia reticulata. 
the guppy, poecilia reticulata, is a well-known model organism for studying inheritance and variation of male ornamental traits as well as adaptation to different river habitats. about ests were annotated according to blastn and blastx results and the sequence information from the 3' utrs was exploited to generate pcr primers for re-sequencing of genomic dna from different wild type strains. by comparison of est-linked genomic sequences from at least four different ecotypes, about polymorphisms were identified, representing about distinct genes.
 the guppy, poecilia reticulata, is a well-known model organism for studying inheritance and variation of male ornamental traits as well as adaptation to different river habitats. however, genomic resources for studying this important model were not previously widely available.  with the aim of generating molecular markers for genetic mapping of the guppy, cdna libraries were constructed from embryos and different adult organs to generate expressed sequence tags. about ests were annotated according to blastn and blastx results and the sequence information from the 3' utrs was exploited to generate pcr primers for re-sequencing of genomic dna from different wild type strains. by comparison of est-linked genomic sequences from at least four different ecotypes, about polymorphisms were identified, representing about distinct genes. two interconnected mysql databases were built to organize the ests and markers, respectively. a robust phylogeny of the guppy was reconstructed, based on different nuclear genes.  our est and marker databases provide useful tools for genetic mapping and phylogenetic studies of the guppy. 