BACKGROUND
salmonidae  are of economic and environmental importance, leading to a high level of interest in many different areas of biology.
of the sixty-six species in this family  <cit> , atlantic salmon  has been used as a model for studies in several areas including osmoregulation, environmental toxicology, immunology, growth, physiology, and genomics  <cit> .
both s. salar and the closely related rainbow trout  are commonly used as important sentinel species to monitor the health of aquatic environments  <cit> .
conservation and enhancement of wild stocks of these fish continues to be the subject of very large internationally concerned groups  <cit> .
basic biological knowledge of s. salar serves as a foundation for improving fish health, conserving wild stocks, and increasing the commercial sustainability of aquaculture.
recent efforts in genomics have provided new tools to address fundamental questions regarding fish health, ecology, physiology, and genetics, as well as allowing investigation of post-tetraploidization genome remodelling  <cit> .
detailed efforts to annotate the entire complement of s. salar genes will greatly facilitate a better understanding of all aspects of salmonid biology.
the study of salmonid genomes is made more difficult and biologically interesting because of a whole genome duplication  that occurred through an autotetraploidization event in the common ancestor of salmonids between 25- <dig> million years ago  <cit> .
extant salmonids are currently in a pseudotetraploid state and are in the process of reverting to a stable diploid state  <cit> .
though many of the gene duplicates from the wgd have been lost through deletion events or by being converted into pseudogenes, many sets of paralogs remain.
as a result, there are practical problems in distinguishing among alleles, recent segmental duplications, gene family members, and duplications arising from the wgd.
experimentally, s. salar genes have proven challenging to characterize because of the complexities resulting from assembling large numbers of partial mrna sequences represented by expressed sequence tags  obtained from these duplicated and other closely related sequences.
in addition, interspersed repeat sequences  <cit>  can lead to the formation of incorrect assemblies of genomic sequences and transcripts .
to resolve these potential errors, a gene containing coding sequence  flanked by 5' and 3' untranslated regions  coming from a single, completely characterized cdna clone provides an important reference sequence representing a single allele of a single gene.
the expansion of reference clone resources are particularly important not only in identifying other potential alleles and gene duplicates that are so pervasive in the pseudotetraploid salmonids, but also in studying fundamental genetic rates and modes of evolutionary change.
relatively few organisms and lineages have been used to examine the evolution of duplicated genes following a wgd.
morin et al.
<cit>  investigated the selective pressures acting on paralogs in xenopus laevis, which resulted from allotetraploidization, and found wide-spread purifying selection but with some relaxation of pressure relative to orthologs in the diploid xenopus tropicalis.
maere et al.
<cit>  studied substitution rates and found certain functional categories of genes that were selectively lost after genome duplication events in arabidopsis thaliana.
in a larger scale study, conant and wagner  <cit>  researched the genomes of a number of different organisms that have undergone wgds, testing for asymmetric divergence of paralogs which they found in  <dig> - 30% of duplicates.
looking at asymmetrically evolving paralogs in yeast, turunen et al.
<cit>  recently presented evidence for relaxation of selective pressures.
furthermore, in another examination of the wgd in s. cerevisiae, positive selection was detected in a substantial portion of paralogs  <cit> .
these studies examined the ratio of amino acid changing substitutions to silent substitutions  to measure evolutionary rates.
the present study incorporates some of these approaches to identify evolutionary patterns in the genome of s. salar.
since there is not a large number of examples of post-tetraploidization evolution available for study, the wgd in salmonids becomes an important area for research.
of the few organisms studied, some have been examined by a number of research groups.
since this is one of the first studies examining the post-tetraploidization evolutionary patterns in the salmonid genome, it is our hope that other groups in addition to our own will expand on the work presented here, incorporating growing datasets and using a wide variety of phylogenetic and evolutionary methods.
characterizing evolutionary changes in polyploid genomes requires comparison to a pre-wgd out-group species so that differences in substitution rates with respect to an ancestral genomic state can be determined.
ishiguro et al., lopez et al., and li et al.
<cit>  report that the order esociformes is the closest non-polyploid sister group to the salmoniformes.
karyotypic data  <cit>  and c-values of ~ <dig>  -  <dig>  pg in salmonids and ~ <dig>  -  <dig>  pg in esocids  <cit>  are consistent with the occurrence of the wgd after the divergence of esocids and salmonids.
in particular, studying northern pike  as a representative of the order would provide an opportunity to continue building upon existing efforts.
as there were only  <dig> core nucleotide sequences,  <dig> protein sequences, and  <dig>  est sequences  <cit>  available for northern pike prior to this study, it was necessary to expand sequence information of this species before a more thorough analysis of salmonid gene duplications could be done.
the objectives of this study were to: 1) obtain a large number of full-length reference cdna clone sequences; 2) expand the transcriptomic resources  of e. lucius; and 3) identify evolutionary patterns of duplicated genes in the autotetraploid s. salar species.
RESULTS
full-length cdna library construction and analysis
the majority of existing est data from s. salar came from highly normalized cdna libraries that were full-length  biased  <cit> .
to specifically identify more full-length transcripts, a protocol for enrichment of 5'-caped mrna was employed which prevents truncated mrna from being reverse-transcribed, followed by transfer of intact double-stranded cdnas directly into the library vector using invitrogen gateway® recombination cloning  <cit> .
starting from s. salar brain, head kidney, and spleen tissues, three non-normalized, size selected, full-length libraries were constructed.
mrnas were size-selected for  <dig> to  <dig>  bp ,  <dig>  to  <dig>  bp  and > <dig>  bp .
<dig> ,  <dig> , and  <dig>  clones from rgg, rgh, and rgf, respectively, were bi-directionally sequenced.
for the short insert library ,  <dig>  sequences were obtained and assembled into  <dig>  transcripts .
this library had the fewest novel transcripts and had the highest redundancy in terms of identified sequences.
the majority of transcripts in this library were identified as hemoglobins, ribosomal protein genes or other genes previously seen in our existing est dataset  <cit> .
for the mid-sized insert library ,  <dig>  sequences were obtained and assembled into  <dig>  transcripts .
while the sequence diversity of this library was higher, nearly all complete transcripts had been previously identified  <cit> .
for the large-sized insert library ,  <dig>  sequences were obtained and assembled into  <dig>  transcripts .
this library contained the highest number of novel transcripts.
identification of s. salar full-length cdna contigs from existing est assemblies
starting with a  <dig>  s. salar est assembly  <cit>  ,  <dig>  contigs  were compared to the swissprot protein database  <cit>   and  <dig>  unique transcripts were identified.
<dig>  of these were potential flcdna contigs as determined by similarity comparisons to known proteins .
these assembled sequences represent potential full-length transcripts with significant similarity to swissprot protein sequences.
<dig>   of these contigs contained complete orfs and  <dig>  of these could be represented by a single completely characterized, non-redundant clone.
these clone sequences are consistent with contig consensus sequences representing two or more different clones and were provisionally designated as reference flcdnas.
e. lucius ests
a full-length biased, normalized cdna library from e. lucius head kidney, spleen, heart and gill tissues was constructed and  <dig>  clones were bi-directionally sequenced.
<dig>  sequences averaging  <dig> bp were obtained and, with the previously available  <dig>  est sequences  <cit> , assembled into  <dig>  contigs .
blastx analysis  <cit>  revealed a total of  <dig>  unique transcripts with strong swissprot protein similarity .
using the same method outlined in figure  <dig> part  <dig> for s. salar,  <dig>  were identified as potential full-length transcripts.
after orf analysis ,  <dig>  flcdna contigs contained sequences corresponding to full-length proteins  and  <dig>  non-redundant reference clones were identified.
reference full-length cdna identification using individual clone assembly
paired 5' and 3' sequence reads from short and mid-sized insert flcdna libraries from s. salar  were assembled individually to yield  <dig>  rgg and  <dig>  rgh cdna clone sequences.
these sequences were selected for further full-length, orf and non-redundancy analysis .
the short-insert library  yielded  <dig> new, full-length protein reference clone sequences.
the midrange insert library  yielded  <dig> new flcdna reference clone sequences.
the low yields of novel reference clones likely reflect similar clone insert sizes obtained from previous cdna library characterizations.
the assembled  <dig>  transcripts from the large-insert full-length cdna library  from s. salar were initially examined for non-redundancy with existing full-length reference genes and potential for representing a full-length gene .
based on partial reference flcdna characterization,  <dig>  clones were chosen for complete characterization using primer walking methods.
once these clones were completely sequenced,  <dig>  were shown to have significant swissprot similarity.
of those clones,  <dig>  represented novel non-redundant s. salar transcripts with complete orfs that corresponded to known proteins.
clones whose inserts contained full 5' annotation  and a proper orf  were designated as reference flcdna clones.
in total,  <dig>  non-redundant reference flcdnas  were characterized from the three flcdna libraries of rgf, rgg, and rgh.
reference full-length cdna assessment
the 5' utrs, orfs and 3' utrs for the  <dig>  reference clones were characterized and the results are summarized in figure  <dig>  the mean reference flcdna length for  <dig>  s. salar sequences  is  <dig>  +/-  <dig> bp , and ranges from  <dig> to  <dig>  bp.
of these sequences the mean 5' utr and 3' utr is  <dig> +/-  <dig> bp and  <dig> +/-  <dig> bp, respectively.
the mean reference orf is  <dig> +/-  <dig> bp.
similar analysis of e. lucius reference flcdnas  shows a mean length of  <dig>  +/-  <dig> bp, ranging from  <dig> to  <dig>  bp.
mean utrs in the 5' and 3' regions are  <dig> +/-  <dig> bp and  <dig> +/-  <dig> bp, respectively.
the mean reference orf is  <dig> +/-  <dig> bp.
the utr results are comparable with efforts from groups which have compiled mrna utr databases.
in one such study of utrs for a variety of species belonging to the 'fish' category, these species were shown to have an average 5' utr of  <dig> bp, while 3' utrs averaged  <dig> bp  <cit> .
these results indicate that the utrs from this study are consistent with full-length sequences.
while similar studies in this area are available for non-fish flcdnas, comparisons among more closely related organisms are lacking  <cit> .
a contig confirmation and uniqueness study was performed on each reference flcdna sequence set and the results are outlined in table  <dig>  a comparison was done on each corresponding contig set in an attempt to demonstrate which reference flcdnas could be confirmed by an existing contig.
reference flcdnas from s. salar full-length libraries were not included in the contig assembly and therefore possibly contain novel cdna inserts.
there were  <dig>  reference flcdnas that could be confirmed by a contig sequence for s. salar.
all e. lucius clones were included in its contig assembly.
as a result, the entire  <dig>  reference flcdna set could therefore be confirmed by a contig sequence.
reference flcdnas were confirmed, using blastn, against their corresponding contig set.
the remainder of the reference flcdnas are represented by clones that are similar to and consistent with swissprot entries and are unique to the full-length libraries characterized in this study.
s. salar and e. lucius alignments
flcdna transcripts from s. salar were used to identify protein coding regions for an analysis of silent and amino acid changing substitution rates in duplicated genes.
the coding sequences were translated and used as queries in a tblastn comparison to the nucleotide database consisting of all s. salar and e. lucius est assemblies.
contig sequences corresponding to the tblastn hits were organized into clusters, then translated, resulting in a cluster of nucleotide sequences and a corresponding cluster of protein translations for each full-length gene.
a common region of alignment with respect to the translated orf was found for the dna sequences and the corresponding proteins based on length and quality of alignment criteria.
a final screening process was performed to prevent allelic or distant homolog comparisons.
<dig> clusters contained the necessary one sequence from e. lucius and two sequences from s. salar.
these sequences and alignments are given in additional file  <dig>
non-synonymous , synonymous  and ω  values were calculated for the  <dig> individual gene trees to investigate patterns of evolution.
a value for ω that is <  <dig> over the alignment is indicative of purifying selection .
a value for ω that is >  <dig> is indicative of diversifying or positive selection  <cit> .
pairwise comparisons to determine ds and ω
for each gene cluster, the corrected number of synonymous substitutions per synonymous site  was determined by comparing the e. lucius gene to each of the duplicate s. salar genes  and the s. salar duplicate genes to each other .
for each branch, the frequency of ds values is plotted in figure 4a.
s. salar gene duplicate ds values  have a median value of  <dig>  and the e. lucius to s. salar ds values  have a median value of  <dig> .
this difference confirms that the salmonid genome duplication occurred more recently than the separation of e. lucius and s. salar lineages.
the ratios of non-synonymous to synonymous substitution rates, or ω values, were in a similar manner calculated for each gene set and the frequency of ω values presented in figure 4b.
the median ω for e. lucius to each of the duplicate s. salar genes  is  <dig>  and the median ω for the duplicate s. salar genes  is  <dig> .
the low ω values for all three sets of pairwise comparisons indicates an average of 7- <dig> synonymous substitutions for every non-synonymous substitution.
this ratio confirms that purifying selection is the predominant evolutionary force in these genes.
in most cases, both copies of these genes appear to have had their original functions retained based on the relatively low ratio of substitutions and high similarity between sequences.
it is worth noting however, that poorly aligning and therefore potentially more divergent regions are trimmed from the overall alignments.
therefore, these estimates may be on the conservative side.
the paralogous comparisons  produced ω values that were generally larger than the orthologous comparisons .
upon using a kruskal-wallis test to compare distributions, both sets of orthologous comparisons were found to be significantly different from the paralogous comparisons  while orthologous sets were not significantly different with respect to each other .
therefore, while there is a large variance in the level of selection among the different genes, this result supports a small but significant relaxation in the level of selection pressure following gene duplication.
this result is consistent with a comparison of  <dig> gene duplicates in the polyploid xenopus laevis  <cit> .
ds and ω for tree segments
to more closely examine the effects of evolutionary pressures before and after duplication of the salmonid genome , the substitution rates and ratios were separated into three tree segments .
this subdivision was accomplished by using all three pairwise comparisons to calculate ds values and ω values from the occurrence of the genome duplication  to each extant gene .
this calculation provides an approximation for the number of substitutions before and after the duplication event.
the post-duplication branches  yielded median ds values of  <dig>  and  <dig>  and the pre-duplication branch yielded a median ds value of  <dig> .
e. lucius sequences clearly diverge from the s. salar paralogs to a much greater extent than the s. salar paralogs diverge from each other.
this again is consistent with the wgd occurring in salmonids but not in ecosids.
the few values that are less than zero are presumably a result of the variation in the divergence of the three sequences with respect to one another and chance convergent substitutions.
to pursue the observation in figure 4b showing a relaxation of selection following gene duplication  and operating under the assumption that one duplicate could be retained and preserve its original function, freeing the other to diverge  <cit> , the two post-duplicate branches were separated into two groups; one member of each pair represented the slow branch  and the other member represented the fast branch .
while this method of sorting duplicate genes is somewhat arbitrary, the results can be reviewed with respect to an ancestral branch leading to e. lucius.
selection values  for the fast and slow duplicates along with the ancestral branch were calculated for each gene set and the frequency of ω values plotted in figure 4d.
in the slow s. salar branch  the median ω was  <dig>  and in the ancestral branch  the median ω was  <dig> .
while the slow branch and pre-duplication branch do differ significantly from each other in terms of the means of the ranks of the data , the slow branch has a very similar median ω to the pre-duplication condition.
the fast branch, on the other hand, has a much higher average ω  than both of the other branches.
these results are consistent with the view that after the wgd there was little change in the evolutionary rate for one member of the pair; though in many cases, the rate of incorporation of non-synonymous changes increased dramatically for the other member.
despite a high level of variation, these data suggest that there is some asymmetry in evolutionary pressures on paralogs.
gene ontology analysis
in order to determine if there were ontological categories that were enriched for gene pairs that were subjected to more asymmetrical rates of evolution than others, gene ontology terms  <cit>  were found for two groups of gene sets: those that had high fold differences  in the ω values found for the two s. salar branches and those that had low or no fold differences .
the results are shown in table  <dig>  for the most part, the two groups are populated by categories with similar proportions.
a few notable exceptions include a larger proportion of genes involved in nucleic acid metabolic processes  in the high fold-change group relative to the low.
likewise, the low fold-change group has a higher proportion of "other" metabolic processes  such as lipid and carbohydrate metabolism.
however, the relatively low number of genes in most categories limits the possibilities in this study of correlating ontological terms with specific patterns of evolution.
gene ontology terms are given for gene trios in which there is a high fold-change in ω between paralogs  or a low fold-change in ω .
the proportions of genes contained within gene ontology categories are compared between the two groups.
nucleic acid metabolism  is more highly represented in asymmetrically evolving gene duplicates.
discussion
the objectives of this study were to: 1) characterize a large unambiguous set of reference gene sequences to compare with alleles and duplicates in s. salar, genes in other salmonid species, and genes in more distantly related fish species; 2) expand genomic resources for a representative member of the closest non-tetraploidized fish group  to provide a reference for the study of wgd in salmonids; and 3) identify patterns of change in the evolution of duplicated genes in the autotetraploid s. salar.
genome duplications have a profound impact on the physiology, reproductive biology, ecology and evolution of a species.
salmonids   <cit>  are one of the most economically important and most studied groups of fish.
a purported wgd in their common ancestor between 25- <dig> million years ago  <cit> , after the separation from esocids, plays a prominent role in understanding the biology of this group.
the different salmonid species are currently in the process of reverting to a stable diploid state through deletions and rearrangements  <cit> .
in the absence of a completed genome, there is a significant problem in distinguishing among numerous duplicates, alleles and other very similar sequences.
the integrity of genes resulting from assembling large numbers of partial mrna sequences  remains open to question.
to resolve this problem, a collection of reference genes containing cds and flanking utrs coming from single, completely characterized cdna clones provides an essential resource in gene identification, future genome annotation, and the study of evolutionary patterns.
an analysis of existing est data from s. salar led to estimates of  <dig>  flcdna contig consensus sequences.
however, these contigs may represent an amalgamation of many unique transcript products with high similarity, rather than a single unique allele.
<dig>  s. salar reference flcdna clones were determined in this study to resolve this issue.
these flcdna sequences represent a significant community resource, adding to the current knowledge base on salmonid biology.
these sequences can also serve as scaffolding with which to aid in genomic sequencing and creation of physical maps in other salmonids.
the increasing popularity of microarrays in gene expression experiments has allowed for more precise control of probe design and information on full-length sequences enables probes to be optimally designed with higher specificity.
an increase in probe-binding specificity reduces unwanted cross-species interactions.
salmonid research benefits from a fuller characterization of s. salar genes.
to expand evolutionary studies of salmonids,  <dig>  e. lucius ests were obtained and when combined with the existing  <dig>  est sequences ,  <dig>  contigs and  <dig>  flcdna reference sequences were identified.
this resource not only provides an important initial genetic foundation for the study of pike throughout north america, europe and asia, but also provides essential information on a diploid reference species for the study of wgd in salmonids.
in this study, the flcdna sequences from s. salar along with homologous data from e. lucius were used to analyze evolutionary trends in some of the genes in the pseudotetraploid genome of s. salar.
while the salmon genome may still be in the process of returning to a stable diploid state, it is evident that many gene duplicates have been retained.
the peak in figure 4c indicates a collection of genes that arose from a duplication event after the separation of esocids from a salmonid ancestor.
these genes are likely to be still active because the data for this study are based on mrna, est-derived sequences.
it is interesting to note that both morin et al.
<cit>  and the present study started with approximately  <dig>  full-length transcripts.
by selecting a subset of sequence clusters that fulfil alignment and homology criteria, both studies ended up with only 400- <dig> gene sets.
this ~4% yield is due in part to the strict criteria for usable sequences as well as the more limited e. lucius dataset from which to draw sequences.
further investigation should be undertaken to determine if both x. laevis and s. salar have retained similar proportions of gene duplicates, which would be of great interest in understanding responses to tetraploidization events.
the numerous other species in the salmonid family provide an opportunity to facilitate finer analysis of the genome duplication, once additional data have been gathered for them.
moreover, the additional gene sets that contained more than two s. salar sequences in addition to the e. lucius ortholog  could be studied to gain an understanding of some of the smaller scale duplication events or potentially more ancestral wgds.
over the last century, many individuals and groups have developed ideas about gene duplication in evolution and its importance in expanding on existing biological functions  <cit> .
a central model that has been strongly supported by ohno  <cit>  states that a duplicate gene can accumulate mutations and become non-functional  or diverge to a novel function  while the other duplicate keeps its original function.
other models have been proposed including sub-functionalization  <cit> , where both duplicates accumulate mutations resulting in complementary expression, leaving each copy with its own sub-function.
it is of interest to look for signatures of different types of selection in order to better understand models that may be directing the fates of one or both paralogs.
based on the observation that the genes under investigation are in fact being transcribed to some degree in s. salar, it would be expected that purifying selection would be acting on both duplicates.
the vast majority of ω values that are presented  are much less than one.
it is apparent that negative selection is the predominant force in this evolutionary process as was also found in the similar analysis done by morin et al.
<cit> .
however, relative to the state of the genes before the duplication, there is significant relaxation of selective pressure on at least some of the paralogs, suggesting reduced constraints.
this relaxation is consistent with the idea that having redundancy in the genome will result in increased freedom for divergence  <cit> .
these trends could facilitate neo-functionalization or modification of existing functionality taking place in some of the paralogs.
data in this study provide evidence that selection constraints are not acting on both gene duplicates to the same extent.
in a number of the  <dig> gene sets examined, one paralog may be relaxed, while it appears that the other is maintained to roughly the same degree as the pre-duplication single-copy gene.
this asymmetrical pattern of evolution has recently been observed in specific hox clusters in s. salar  <cit>  as well as in earlier genome-wide studies in other organisms such as drosophila melanogaster and caenorhabditis elegans  <cit> .
the question that results from this observation centers around the fate of these duplicate genes that are operating under relaxed selection.
the paralogs that were studied are still being transcribed and are presumably functional  and have not been subject to non-functionalization.
duplicates that were deleted since the wgd would not be observed and neither would the presumably large number of duplicates that have become pseudogenes.
conclusive evidence for a general trend of positive selection was not found for the set of genes, since nearly all ω values were much less than one, though there were a few higher post-duplication values that suggested some duplicates may have been influenced by directional selection.
the few genes that did have an ω value greater than one showed no enrichment for an ontological category .
turunen et al.
<cit>  looked at asymmetrically evolving gene duplicates in yeast and found evidence for relaxation of selective pressure, sub-functionalization, and even neo-functionalization, though the average ω was significantly less than one.
therefore, it is not surprising that a strong signature of diversifying selection was not detected.
positive selection that may have occurred over a small region or short period of time could be masked by a larger overall pattern of negative selection.
for example, once a neo-functionalization event has occurred, purifying selection would act to maintain that new function in the long term.
indeed, hughes et al.
<cit>  reported 30- <dig> million years of divergence to be the upper limit of detection of positive selection in eukaryotes using dn/ds analysis.
looking at a variety of salmonid species in a comparative fashion could enable a higher resolution study of changes in evolutionary pressures and may provide more clues as to the events that took place in the duplicated genes soon after the tetraploidization.
in addition, other groups  <cit>  have studied polyploidization in xenopus species using some alternative methods that may be applicable to s. salar in future efforts.
one example was using transversion rates at four-fold synonymous codon positions  to measure evolutionary divergence, though saturation of mutations at synonymous sites was not a problem for the present study.
functional gene groups defined by gene ontology terms were found for s. salar gene duplicates that displayed either substantial or very small to no differences in selection constraints .
the proportions of genes falling into the defined categories were generally quite similar .
however, one interesting result was the higher percentage of genes involved in nucleic acid metabolic processes   in the group of gene sets in which a large difference in selection constraints was identified.
in this case, the conclusion that nucleic acid metabolism genes were more often present in the asymmetrical group than the symmetrical group would be consistent with earlier studies, which found that nucleic acid processing and nucleoside metabolism functional groups were selectively lost after whole genome duplications in x. laevis and a. thaliana.
this suggests that nucleic acid processing and nucleoside metabolism functional groups of genes may have a greater chance of conferring dosage sensitivity  <cit> .
CONCLUSIONS
during the rediploidization after a wgd in the common ancestor of the salmonids, many gene duplicates were retained.
there is strong evidence that purifying selection is the predominant force acting on these gene duplicates.
however, there is also evidence that this selection has been relaxed significantly in genes after the duplication.
furthermore, the relaxation of selection occurred in an asymmetrical manner, preferentially allowing the divergence of one duplicate over the other.
though more research is needed to gain a higher resolution picture of the fates of the retained duplicates, these results add to the body of knowledge surrounding models of evolution following genome duplications and shed more light on the complex salmonid genome.

 salmonidae are of economic and environmental importance, leading to a high level of interest in many different areas of biology. of the sixty-six species in this family, atlantic salmon has been used as a model for studies in several areas including osmoregulation, environmental toxicology, immunology, growth, physiology, and genomics. both s. salar and the closely related rainbow trout are commonly used as important sentinel species to monitor the health of aquatic environments. conservation and enhancement of wild stocks of these fish continues to be the subject of very large internationally concerned groups. basic biological knowledge of s. salar serves as a foundation for improving fish health, conserving wild stocks, and increasing the commercial sustainability of aquaculture. recent efforts in genomics have provided new tools to address fundamental questions regarding fish health, ecology, physiology, and genetics, as well as allowing investigation of post-tetraploidization genome remodelling. detailed efforts to annotate the entire complement of s. salar genes will greatly facilitate a better understanding of all aspects of salmonid biology. the study of salmonid genomes is made more difficult and biologically interesting because of a whole genome duplication that occurred through an autotetraploidization event in the common ancestor of salmonids between 25- million years ago. extant salmonids are currently in a pseudotetraploid state and are in the process of reverting to a stable diploid state. though many of the gene duplicates from the wgd have been lost through deletion events or by being converted into pseudogenes, many sets of paralogs remain. as a result, there are practical problems in distinguishing among alleles, recent segmental duplications, gene family members, and duplications arising from the wgd. experimentally, s. salar genes have proven challenging to characterize because of the complexities resulting from assembling large numbers of partial mrna sequences represented by expressed sequence tags obtained from these duplicated and other closely related sequences. in addition, interspersed repeat sequences can lead to the formation of incorrect assemblies of genomic sequences and transcripts. to resolve these potential errors, a gene containing coding sequence flanked by 5' and 3' untranslated regions coming from a single, completely characterized cdna clone provides an important reference sequence representing a single allele of a single gene. the expansion of reference clone resources are particularly important not only in identifying other potential alleles and gene duplicates that are so pervasive in the pseudotetraploid salmonids, but also in studying fundamental genetic rates and modes of evolutionary change. relatively few organisms and lineages have been used to examine the evolution of duplicated genes following a wgd. morin et al. investigated the selective pressures acting on paralogs in xenopus laevis, which resulted from allotetraploidization, and found wide-spread purifying selection but with some relaxation of pressure relative to orthologs in the diploid xenopus tropicalis. maere et al. studied substitution rates and found certain functional categories of genes that were selectively lost after genome duplication events in arabidopsis thaliana. in a larger scale study, conant and wagner researched the genomes of a number of different organisms that have undergone wgds, testing for asymmetric divergence of paralogs which they found in - 30% of duplicates. looking at asymmetrically evolving paralogs in yeast, turunen et al. recently presented evidence for relaxation of selective pressures. furthermore, in another examination of the wgd in s. cerevisiae, positive selection was detected in a substantial portion of paralogs. these studies examined the ratio of amino acid changing substitutions to silent substitutions to measure evolutionary rates. the present study incorporates some of these approaches to identify evolutionary patterns in the genome of s. salar. since there is not a large number of examples of post-tetraploidization evolution available for study, the wgd in salmonids becomes an important area for research. of the few organisms studied, some have been examined by a number of research groups. since this is one of the first studies examining the post-tetraploidization evolutionary patterns in the salmonid genome, it is our hope that other groups in addition to our own will expand on the work presented here, incorporating growing datasets and using a wide variety of phylogenetic and evolutionary methods. characterizing evolutionary changes in polyploid genomes requires comparison to a pre-wgd out-group species so that differences in substitution rates with respect to an ancestral genomic state can be determined. ishiguro et al., lopez et al., and li et al. report that the order esociformes is the closest non-polyploid sister group to the salmoniformes. karyotypic data and c-values of ~ - pg in salmonids and ~ - pg in esocids are consistent with the occurrence of the wgd after the divergence of esocids and salmonids. in particular, studying northern pike as a representative of the order would provide an opportunity to continue building upon existing efforts. as there were only core nucleotide sequences, protein sequences, and est sequences available for northern pike prior to this study, it was necessary to expand sequence information of this species before a more thorough analysis of salmonid gene duplications could be done. the objectives of this study were to: 1) obtain a large number of full-length reference cdna clone sequences; 2) expand the transcriptomic resources of e. lucius; and 3) identify evolutionary patterns of duplicated genes in the autotetraploid s. salar species.  full-length cdna library construction and analysis the majority of existing est data from s. salar came from highly normalized cdna libraries that were full-length biased. to specifically identify more full-length transcripts, a protocol for enrichment of 5'-caped mrna was employed which prevents truncated mrna from being reverse-transcribed, followed by transfer of intact double-stranded cdnas directly into the library vector using invitrogen gateway® recombination cloning. starting from s. salar brain, head kidney, and spleen tissues, three non-normalized, size selected, full-length libraries were constructed. mrnas were size-selected for to bp, to bp and > bp.,, and clones from rgg, rgh, and rgf, respectively, were bi-directionally sequenced. for the short insert library, sequences were obtained and assembled into transcripts. this library had the fewest novel transcripts and had the highest redundancy in terms of identified sequences. the majority of transcripts in this library were identified as hemoglobins, ribosomal protein genes or other genes previously seen in our existing est dataset. for the mid-sized insert library, sequences were obtained and assembled into transcripts. while the sequence diversity of this library was higher, nearly all complete transcripts had been previously identified. for the large-sized insert library, sequences were obtained and assembled into transcripts. this library contained the highest number of novel transcripts. identification of s. salar full-length cdna contigs from existing est assemblies starting with a s. salar est assembly, contigs were compared to the swissprot protein database and unique transcripts were identified. of these were potential flcdna contigs as determined by similarity comparisons to known proteins. these assembled sequences represent potential full-length transcripts with significant similarity to swissprot protein sequences. of these contigs contained complete orfs and of these could be represented by a single completely characterized, non-redundant clone. these clone sequences are consistent with contig consensus sequences representing two or more different clones and were provisionally designated as reference flcdnas. e. lucius ests a full-length biased, normalized cdna library from e. lucius head kidney, spleen, heart and gill tissues was constructed and clones were bi-directionally sequenced. sequences averaging bp were obtained and, with the previously available est sequences, assembled into contigs. blastx analysis revealed a total of unique transcripts with strong swissprot protein similarity. using the same method outlined in figure part for s. salar, were identified as potential full-length transcripts. after orf analysis, flcdna contigs contained sequences corresponding to full-length proteins and non-redundant reference clones were identified. reference full-length cdna identification using individual clone assembly paired 5' and 3' sequence reads from short and mid-sized insert flcdna libraries from s. salar were assembled individually to yield rgg and rgh cdna clone sequences. these sequences were selected for further full-length, orf and non-redundancy analysis. the short-insert library yielded new, full-length protein reference clone sequences. the midrange insert library yielded new flcdna reference clone sequences. the low yields of novel reference clones likely reflect similar clone insert sizes obtained from previous cdna library characterizations. the assembled transcripts from the large-insert full-length cdna library from s. salar were initially examined for non-redundancy with existing full-length reference genes and potential for representing a full-length gene. based on partial reference flcdna characterization, clones were chosen for complete characterization using primer walking methods. once these clones were completely sequenced, were shown to have significant swissprot similarity. of those clones, represented novel non-redundant s. salar transcripts with complete orfs that corresponded to known proteins. clones whose inserts contained full 5' annotation and a proper orf were designated as reference flcdna clones. in total, non-redundant reference flcdnas were characterized from the three flcdna libraries of rgf, rgg, and rgh. reference full-length cdna assessment the 5' utrs, orfs and 3' utrs for the reference clones were characterized and the results are summarized in figure the mean reference flcdna length for s. salar sequences is +/- bp, and ranges from to bp. of these sequences the mean 5' utr and 3' utr is +/- bp and +/- bp, respectively. the mean reference orf is +/- bp. similar analysis of e. lucius reference flcdnas shows a mean length of +/- bp, ranging from to bp. mean utrs in the 5' and 3' regions are +/- bp and +/- bp, respectively. the mean reference orf is +/- bp. the utr results are comparable with efforts from groups which have compiled mrna utr databases. in one such study of utrs for a variety of species belonging to the'fish' category, these species were shown to have an average 5' utr of bp, while 3' utrs averaged bp. these results indicate that the utrs from this study are consistent with full-length sequences. while similar studies in this area are available for non-fish flcdnas, comparisons among more closely related organisms are lacking. a contig confirmation and uniqueness study was performed on each reference flcdna sequence set and the results are outlined in table a comparison was done on each corresponding contig set in an attempt to demonstrate which reference flcdnas could be confirmed by an existing contig. reference flcdnas from s. salar full-length libraries were not included in the contig assembly and therefore possibly contain novel cdna inserts. there were reference flcdnas that could be confirmed by a contig sequence for s. salar. all e. lucius clones were included in its contig assembly. as a result, the entire reference flcdna set could therefore be confirmed by a contig sequence. reference flcdnas were confirmed, using blastn, against their corresponding contig set. the remainder of the reference flcdnas are represented by clones that are similar to and consistent with swissprot entries and are unique to the full-length libraries characterized in this study. s. salar and e. lucius alignments flcdna transcripts from s. salar were used to identify protein coding regions for an analysis of silent and amino acid changing substitution rates in duplicated genes. the coding sequences were translated and used as queries in a tblastn comparison to the nucleotide database consisting of all s. salar and e. lucius est assemblies. contig sequences corresponding to the tblastn hits were organized into clusters, then translated, resulting in a cluster of nucleotide sequences and a corresponding cluster of protein translations for each full-length gene. a common region of alignment with respect to the translated orf was found for the dna sequences and the corresponding proteins based on length and quality of alignment criteria. a final screening process was performed to prevent allelic or distant homolog comparisons. clusters contained the necessary one sequence from e. lucius and two sequences from s. salar. these sequences and alignments are given in additional file non-synonymous, synonymous and ω values were calculated for the individual gene trees to investigate patterns of evolution. a value for ω that is < over the alignment is indicative of purifying selection. a value for ω that is > is indicative of diversifying or positive selection. pairwise comparisons to determine ds and ω for each gene cluster, the corrected number of synonymous substitutions per synonymous site was determined by comparing the e. lucius gene to each of the duplicate s. salar genes and the s. salar duplicate genes to each other. for each branch, the frequency of ds values is plotted in figure 4a. s. salar gene duplicate ds values have a median value of and the e. lucius to s. salar ds values have a median value of. this difference confirms that the salmonid genome duplication occurred more recently than the separation of e. lucius and s. salar lineages. the ratios of non-synonymous to synonymous substitution rates, or ω values, were in a similar manner calculated for each gene set and the frequency of ω values presented in figure 4b. the median ω for e. lucius to each of the duplicate s. salar genes is and the median ω for the duplicate s. salar genes is. the low ω values for all three sets of pairwise comparisons indicates an average of 7- synonymous substitutions for every non-synonymous substitution. this ratio confirms that purifying selection is the predominant evolutionary force in these genes. in most cases, both copies of these genes appear to have had their original functions retained based on the relatively low ratio of substitutions and high similarity between sequences. it is worth noting however, that poorly aligning and therefore potentially more divergent regions are trimmed from the overall alignments. therefore, these estimates may be on the conservative side. the paralogous comparisons produced ω values that were generally larger than the orthologous comparisons. upon using a kruskal-wallis test to compare distributions, both sets of orthologous comparisons were found to be significantly different from the paralogous comparisons while orthologous sets were not significantly different with respect to each other. therefore, while there is a large variance in the level of selection among the different genes, this result supports a small but significant relaxation in the level of selection pressure following gene duplication. this result is consistent with a comparison of gene duplicates in the polyploid xenopus laevis. ds and ω for tree segments to more closely examine the effects of evolutionary pressures before and after duplication of the salmonid genome, the substitution rates and ratios were separated into three tree segments. this subdivision was accomplished by using all three pairwise comparisons to calculate ds values and ω values from the occurrence of the genome duplication to each extant gene. this calculation provides an approximation for the number of substitutions before and after the duplication event. the post-duplication branches yielded median ds values of and and the pre-duplication branch yielded a median ds value of. e. lucius sequences clearly diverge from the s. salar paralogs to a much greater extent than the s. salar paralogs diverge from each other. this again is consistent with the wgd occurring in salmonids but not in ecosids. the few values that are less than zero are presumably a result of the variation in the divergence of the three sequences with respect to one another and chance convergent substitutions. to pursue the observation in figure 4b showing a relaxation of selection following gene duplication and operating under the assumption that one duplicate could be retained and preserve its original function, freeing the other to diverge, the two post-duplicate branches were separated into two groups; one member of each pair represented the slow branch and the other member represented the fast branch. while this method of sorting duplicate genes is somewhat arbitrary, the results can be reviewed with respect to an ancestral branch leading to e. lucius. selection values for the fast and slow duplicates along with the ancestral branch were calculated for each gene set and the frequency of ω values plotted in figure 4d. in the slow s. salar branch the median ω was and in the ancestral branch the median ω was. while the slow branch and pre-duplication branch do differ significantly from each other in terms of the means of the ranks of the data, the slow branch has a very similar median ω to the pre-duplication condition. the fast branch, on the other hand, has a much higher average ω than both of the other branches. these results are consistent with the view that after the wgd there was little change in the evolutionary rate for one member of the pair; though in many cases, the rate of incorporation of non-synonymous changes increased dramatically for the other member. despite a high level of variation, these data suggest that there is some asymmetry in evolutionary pressures on paralogs. gene ontology analysis in order to determine if there were ontological categories that were enriched for gene pairs that were subjected to more asymmetrical rates of evolution than others, gene ontology terms were found for two groups of gene sets: those that had high fold differences in the ω values found for the two s. salar branches and those that had low or no fold differences. the results are shown in table for the most part, the two groups are populated by categories with similar proportions. a few notable exceptions include a larger proportion of genes involved in nucleic acid metabolic processes in the high fold-change group relative to the low. likewise, the low fold-change group has a higher proportion of "other" metabolic processes such as lipid and carbohydrate metabolism. however, the relatively low number of genes in most categories limits the possibilities in this study of correlating ontological terms with specific patterns of evolution. gene ontology terms are given for gene trios in which there is a high fold-change in ω between paralogs or a low fold-change in ω. the proportions of genes contained within gene ontology categories are compared between the two groups. nucleic acid metabolism is more highly represented in asymmetrically evolving gene duplicates. discussion the objectives of this study were to: 1) characterize a large unambiguous set of reference gene sequences to compare with alleles and duplicates in s. salar, genes in other salmonid species, and genes in more distantly related fish species; 2) expand genomic resources for a representative member of the closest non-tetraploidized fish group to provide a reference for the study of wgd in salmonids; and 3) identify patterns of change in the evolution of duplicated genes in the autotetraploid s. salar. genome duplications have a profound impact on the physiology, reproductive biology, ecology and evolution of a species. salmonids are one of the most economically important and most studied groups of fish. a purported wgd in their common ancestor between 25- million years ago, after the separation from esocids, plays a prominent role in understanding the biology of this group. the different salmonid species are currently in the process of reverting to a stable diploid state through deletions and rearrangements. in the absence of a completed genome, there is a significant problem in distinguishing among numerous duplicates, alleles and other very similar sequences. the integrity of genes resulting from assembling large numbers of partial mrna sequences remains open to question. to resolve this problem, a collection of reference genes containing cds and flanking utrs coming from single, completely characterized cdna clones provides an essential resource in gene identification, future genome annotation, and the study of evolutionary patterns. an analysis of existing est data from s. salar led to estimates of flcdna contig consensus sequences. however, these contigs may represent an amalgamation of many unique transcript products with high similarity, rather than a single unique allele. s. salar reference flcdna clones were determined in this study to resolve this issue. these flcdna sequences represent a significant community resource, adding to the current knowledge base on salmonid biology. these sequences can also serve as scaffolding with which to aid in genomic sequencing and creation of physical maps in other salmonids. the increasing popularity of microarrays in gene expression experiments has allowed for more precise control of probe design and information on full-length sequences enables probes to be optimally designed with higher specificity. an increase in probe-binding specificity reduces unwanted cross-species interactions. salmonid research benefits from a fuller characterization of s. salar genes. to expand evolutionary studies of salmonids, e. lucius ests were obtained and when combined with the existing est sequences, contigs and flcdna reference sequences were identified. this resource not only provides an important initial genetic foundation for the study of pike throughout north america, europe and asia, but also provides essential information on a diploid reference species for the study of wgd in salmonids. in this study, the flcdna sequences from s. salar along with homologous data from e. lucius were used to analyze evolutionary trends in some of the genes in the pseudotetraploid genome of s. salar. while the salmon genome may still be in the process of returning to a stable diploid state, it is evident that many gene duplicates have been retained. the peak in figure 4c indicates a collection of genes that arose from a duplication event after the separation of esocids from a salmonid ancestor. these genes are likely to be still active because the data for this study are based on mrna, est-derived sequences. it is interesting to note that both morin et al. and the present study started with approximately full-length transcripts. by selecting a subset of sequence clusters that fulfil alignment and homology criteria, both studies ended up with only 400- gene sets. this ~4% yield is due in part to the strict criteria for usable sequences as well as the more limited e. lucius dataset from which to draw sequences. further investigation should be undertaken to determine if both x. laevis and s. salar have retained similar proportions of gene duplicates, which would be of great interest in understanding responses to tetraploidization events. the numerous other species in the salmonid family provide an opportunity to facilitate finer analysis of the genome duplication, once additional data have been gathered for them. moreover, the additional gene sets that contained more than two s. salar sequences in addition to the e. lucius ortholog could be studied to gain an understanding of some of the smaller scale duplication events or potentially more ancestral wgds. over the last century, many individuals and groups have developed ideas about gene duplication in evolution and its importance in expanding on existing biological functions. a central model that has been strongly supported by ohno states that a duplicate gene can accumulate mutations and become non-functional or diverge to a novel function while the other duplicate keeps its original function. other models have been proposed including sub-functionalization, where both duplicates accumulate mutations resulting in complementary expression, leaving each copy with its own sub-function. it is of interest to look for signatures of different types of selection in order to better understand models that may be directing the fates of one or both paralogs. based on the observation that the genes under investigation are in fact being transcribed to some degree in s. salar, it would be expected that purifying selection would be acting on both duplicates. the vast majority of ω values that are presented are much less than one. it is apparent that negative selection is the predominant force in this evolutionary process as was also found in the similar analysis done by morin et al.. however, relative to the state of the genes before the duplication, there is significant relaxation of selective pressure on at least some of the paralogs, suggesting reduced constraints. this relaxation is consistent with the idea that having redundancy in the genome will result in increased freedom for divergence. these trends could facilitate neo-functionalization or modification of existing functionality taking place in some of the paralogs. data in this study provide evidence that selection constraints are not acting on both gene duplicates to the same extent. in a number of the gene sets examined, one paralog may be relaxed, while it appears that the other is maintained to roughly the same degree as the pre-duplication single-copy gene. this asymmetrical pattern of evolution has recently been observed in specific hox clusters in s. salar as well as in earlier genome-wide studies in other organisms such as drosophila melanogaster and caenorhabditis elegans. the question that results from this observation centers around the fate of these duplicate genes that are operating under relaxed selection. the paralogs that were studied are still being transcribed and are presumably functional and have not been subject to non-functionalization. duplicates that were deleted since the wgd would not be observed and neither would the presumably large number of duplicates that have become pseudogenes. conclusive evidence for a general trend of positive selection was not found for the set of genes, since nearly all ω values were much less than one, though there were a few higher post-duplication values that suggested some duplicates may have been influenced by directional selection. the few genes that did have an ω value greater than one showed no enrichment for an ontological category. turunen et al. looked at asymmetrically evolving gene duplicates in yeast and found evidence for relaxation of selective pressure, sub-functionalization, and even neo-functionalization, though the average ω was significantly less than one. therefore, it is not surprising that a strong signature of diversifying selection was not detected. positive selection that may have occurred over a small region or short period of time could be masked by a larger overall pattern of negative selection. for example, once a neo-functionalization event has occurred, purifying selection would act to maintain that new function in the long term. indeed, hughes et al. reported 30- million years of divergence to be the upper limit of detection of positive selection in eukaryotes using dn/ds analysis. looking at a variety of salmonid species in a comparative fashion could enable a higher resolution study of changes in evolutionary pressures and may provide more clues as to the events that took place in the duplicated genes soon after the tetraploidization. in addition, other groups have studied polyploidization in xenopus species using some alternative methods that may be applicable to s. salar in future efforts. one example was using transversion rates at four-fold synonymous codon positions to measure evolutionary divergence, though saturation of mutations at synonymous sites was not a problem for the present study. functional gene groups defined by gene ontology terms were found for s. salar gene duplicates that displayed either substantial or very small to no differences in selection constraints. the proportions of genes falling into the defined categories were generally quite similar. however, one interesting result was the higher percentage of genes involved in nucleic acid metabolic processes in the group of gene sets in which a large difference in selection constraints was identified. in this case, the conclusion that nucleic acid metabolism genes were more often present in the asymmetrical group than the symmetrical group would be consistent with earlier studies, which found that nucleic acid processing and nucleoside metabolism functional groups were selectively lost after whole genome duplications in x. laevis and a. thaliana. this suggests that nucleic acid processing and nucleoside metabolism functional groups of genes may have a greater chance of conferring dosage sensitivity.  during the rediploidization after a wgd in the common ancestor of the salmonids, many gene duplicates were retained. there is strong evidence that purifying selection is the predominant force acting on these gene duplicates. however, there is also evidence that this selection has been relaxed significantly in genes after the duplication. furthermore, the relaxation of selection occurred in an asymmetrical manner, preferentially allowing the divergence of one duplicate over the other. though more research is needed to gain a higher resolution picture of the fates of the retained duplicates, these results add to the body of knowledge surrounding models of evolution following genome duplications and shed more light on the complex salmonid genome. 
extensive new genomic resources have recently become available for atlantic salmon, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. full-length reference genes were characterized in s. salar and can be used to identify alleles and gene family members. a further reference full-length clones were annotated from northern pike ests. pairwise dn/ds comparisons within each of sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. from existing expressed sequence tag resources and three new full-length cdna libraries, reference quality full-length gene insert clones were identified for atlantic salmon. salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids.
 salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. this duplication greatly impacts species diversification, functional specialization, and adaptation. extensive new genomic resources have recently become available for atlantic salmon, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution.  from existing expressed sequence tag resources and three new full-length cdna libraries, reference quality full-length gene insert clones were identified for atlantic salmon. a further reference full-length clones were annotated from northern pike ests. pairwise dn/ds comparisons within each of sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates.  full-length reference genes were characterized in s. salar and can be used to identify alleles and gene family members. comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. in addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate. 