BACKGROUND
the white campion, silene latifolia poiret, a member of the plant family caryophyllaceae, is a dioecious herb.
the species is diploid, has a large nuclear genome size  and a haploid chromosome number of  <dig>  sex is determined genetically by heteromorphic sex chromosomes that were first described by blackburn  <cit>  and winge  <cit> .
as in humans, females are homogametic, xx, and males are the heterogametic sex, xy.
the sex chromosomes are the largest chromosomes and contribute substantially to the large genome size of this species.
although dioecy has evolved many times in different plant lineages  <cit> , well differentiated, heteromorphic sex chromosomes are relatively rare in plants.
over the last decades, silene latifolia has become a model organism in plant ecology and evolution.
major research avenues include for example the evolution of heteromorphic sex chromosomes in plants  <cit> , sexual dimorphism  <cit> , plant-pathogen  <cit>  and plant pollinator interactions  <cit> , invasive plant biology  <cit> , hybridization and introgression  <cit> , and habitat adaptation  <cit> .
to address these ecological and evolutionary questions, a diverse set of molecular markers has been used to date in silene latifolia.
only recently have the first genomic simple sequence repeats , also known as microsatellites, been identified and used  <cit> .
current limitations of the available markers include the problems that the widely used aflps, and formerly rapds, are anonymous and dominant markers.
while aflp markers have been used successfully for linkage mapping in the related silene vulgaris, the resulting maps derived from the maternal and paternal parent, respectively, could not be joined into a single, unifying map, because of the limited information on coupling phase provided by dominant markers  <cit> .
similarly, a recent genome scan analysis for markers under selection identified several aflp markers that carried the signature of selection  <cit> , but characterization of the outlier markers failed to identify transcribed genes.
to overcome such limitations, we have embarked on an expressed sequence tag  project to identify transcribed genes in s. latifolia and to characterize simple sequence repeats in these expressed genes.
ssr markers identified in est sequences are known as est-ssrs.
ssrs are tandemly repeated tracts of dna composed of 1– <dig> base pair  long units.
they are ubiquitous in prokaryotes and eukaryotes  <cit> , both in coding and noncoding regions, and are usually characterized by a high degree of length polymorphism.
ssr markers are useful for a variety of applications, because of their multiallelic nature, codominant inheritance, relative abundance, and good genome coverage.
the conservation of flanking sequences in the vicinity of the repeat motifs often permits the genotyping of related species with a single primer set  <cit> .
microsatellites have proven to be an extremely valuable tool for genome mapping in many organisms  <cit> , but their applications span over different areas ranging from ancient and forensic dna studies to population genetics and conservation/management of biological resources  <cit> .
moreover, microsatellites, due to their large amount of variability, have the potential to be informative about gene and genome duplication, but only recently have been used for these topics  <cit> .
expressed sequence tags  are sequenced portions of messenger rna.
in recent years, est projects have been initiated for numerous plant and animal species, and have generated a vast amount of sequence information that can be used for gene discovery, functional genetic studies, and marker development  <cit> .
ssrs are relatively common in expressed genes, mainly in the 5' and 3' untranslated regions .
such est-ssrs, or genic ssrs, have several advantages compared to other molecular markers.
first, studies in plants, animals, and fungi have shown that est-ssrs are often more widely transferable between species, and even genera, than genomic ssr  <cit> .
second, because they are located in transcribed genes, the identification of outlier est-ssrs loci in genome scan analyses may directly identify candidates for genes under selection  <cit> .
by comparing the est sequence to protein sequence databases, it may further be possible to shed light on the functional identity of the gene.
third, the increased likelihood for cross-species amplification and the codominant nature of est-ssrs make them ideal markers for comparative mapping  <cit> .
fourth, est-ssrs often display reduced levels of polymorphism compared to genomic ssrs  <cit> , which may facilitate genotyping and allow a more accurate estimates of allele frequencies in population genetic studies compare to hypervariable loci.
finally, the ease with which est-ssrs can be mapped may facilitate the identification of new genes that are linked to traits of particular interest, such as to the sex chromosomes in s. latifolia.
as codominant markers, est-ssrs are expected to segregate according to mendel's laws in crosses between individuals, and mendelian inheritance of alleles is a requirement for population genetic analyses.
an earlier review of microsatellite inheritance studies found that mendelian inheritance was almost never rejected for diploid vertebrate species  <cit> .
however, there is increasing evidence of what appear to be "non-mendelian" patterns of inheritance of microsatellites  <cit> .
because relatively few studies report tests for mendelian inheritance, it is still unclear how common non-mendelian inheritance is.
a large fraction of "non-mendelian" ratios of alleles in offspring of experimental crosses is apparently caused by null alleles  <cit> .
potential causes of non-mendelian behaviour include sex linkage, physical association with genes under strong selection, transposable elements, or processes such as non-disjunction or meiotic drive that act during meiosis  <cit> .
to use est-ssr loci for population genetics, it is thus essential that mendelian segregation be verified in controlled crosses.
we constructed an est library from floral tissue of male and female s. latifolia with the aim to identify expressed genes and to develop polymorphic molecular markers.
floral tissue was chosen because we are interested in the genetic basis of sex determination in dioecious silene, the evolution of plant sex chromosomes, and floral isolation between s. latifolia and the closely related s. dioica.
in this paper we first describe our est library and show that this library is a rich source of genes that may be involved in flower development and the control of floral trait differences between male and female plants, but also between s. latifolia and related species.
second, we describe a set of newly characterized est-ssr loci and provide information about their polymorphism, mendelian inheritance, and transferability to other silene species.
finally, we report on the utility of these markers for comparative mapping.
RESULTS
est library characterization
random 5' sequencing of our directional cdna library resulted in  <dig> high quality sequences with an average length of  <dig> nucleotides.
assembly using tgicl resulted in  <dig> unigenes, consisting of  <dig> singlets and  <dig> contigs.
average unigene length  was shorter than average contig length .
most contigs  contained  <dig> ests.
only  <dig> contigs  contained  <dig> or more sequences and the largest number of sequences per contig was  <dig> .
* /unigene #  <cit>
during pre-processing, all sequences were searched for the tags identifying three different pools.
of the  <dig> high quality sequences,  <dig> were from pool a ,  <dig> from pool b , and  <dig> from pool c .
in  <dig> sequences  the adaptor could not be retrieved.
despite the small number of ests per contig, most contigs  consisting of four or more ests contained sequences from more than one tissue pool.
only  <dig> such contigs were made up by sequences from a single tissue pool.
of these,  <dig> contigs combined sequences from pool b, and one contig each combined sequences from pools a and c.
est annotation and functional classification
we used blastx to annotate our silene latifolia unigene sequences.
<dig>  of the unigenes matched genes in other species with an expectation value of 1e- <dig> or better in a search against the ncbi nr protein database .
best hits in blastx searches were mainly to vitis vinifera , arabidopsis thaliana , populus trichocarpa , and oryza sativa .
out of our  <dig> unigenes, only twenty-six had a best hit with a silene species .
several unigenes identified in our est library have been identified as putative homologs of arabidopsis genes that are implicated in floral development .
gene ontology   <cit>  annotation was performed with blast2go.
in total,  <dig> unigenes were annotated with  <dig> go terms.
at least one biological process was proposed for  <dig> unigenes, a cellular component for  <dig> unigenes, and  <dig> unigenes were annotated with at least one molecular function.
there were  <dig> sequences with annotations for all three go categories  and  <dig> unigenes had annotations for at least  <dig> categories.
the relative frequencies of go hits are shown in fig.
<dig>
characterization of microsatellite motifs
we identified  <dig> sequences containing one or more microsatellite motifs in the library screen.
the observed frequencies of di-, tri-, tetra-, penta- and hexa-repeats were  <dig> % ,  <dig> % ,  <dig> % ,  <dig> %  and  <dig> % , respectively.
the  <dig> di-nucleotide repeat sequences consisted of /n, /n and /n.
among the di-nucleotide repeats there was a distinct predominance of /n repeats , with low frequencies of other di-nucleotide repeats, n and n .
<dig> tri-nucleotide repeat motifs were recognized, which represents the most frequent repeat unit .
their motifs included /n, /n, /n, /n, /n, /n, /n, /n, /n, and /n.
of these, the motif /n was the most frequent , followed by /n , /n , /n , and /n .
/n, /n, /n, / and / showed a very low frequency.
tetra-nt repeat motifs were identified in  <dig> different clones ; these included /n, /n, /n, /n, /n, /n, /n, /n, /n, /n, /n, /n and /n.
the most frequent 4-nt repeat motifs were /n, /n, /n, /n, but their frequency was low .
we identified  <dig> and  <dig> different penta or hexa-nt ssr motifs, most of them were found only once.
identification of polymorphic markers
among the  <dig> ssr-containing unigenes we selected  <dig> that had long enough sequences flanking the ssr to design primer pairs.
<dig> primer pairs that were not likely to form internal secondary structures were designed and tested by amplifying template dna from s. latifolia.
after some optimization,  <dig> of these primer pairs were successfully amplified.
the other  <dig> primer pairs failed.
among the working primer pairs,  <dig> produced pcr products of the expected size,  <dig> produced pcr fragments that were considerably longer than expected and the rest produced multiple bands.
finally, about 66% of the primers that were initially designed appeared to amplify the expected product as judged from agarose-gel electrophoresis.
however, when these pcr products were subsequently analyzed on a capillary sequencer, some of them were not scorable due to excessive "stutter bands".
finally,  <dig> primer pairs remained .
in order to obtain information on the putative identities and functions of the genes containing est-ssrs, the corresponding unigene sequences were subjected to blastx searches against the arabidopsis thaliana refseq database.
about 90% of est-ssrs matched arabidopsis genes with an expectation value of 1e- <dig> or better .
the position of the ssr motifs was unambiguously identified for  <dig> out of the  <dig> loci.
for five loci, the two methods used to infer the ssr position disagreed and for one locus, no similarity was found to known arabidopsis genes.
of the  <dig> loci,  <dig> are located in protein coding sequences ,  <dig> are located in the 5' utr, and  <dig> in the 3' utr.
the level of polymorphism as estimated from the polymorphic information content  was higher in loci located in untranslated regions  than in loci located in coding regions .
est-ssr polymorphism
we surveyed the allelic variability of the markers by genotyping individuals from a natural population of s. latifolia.
most microsatellite loci showed allelic polymorphism.
the number of alleles per locus varied from  <dig> to  <dig> in the panel of  <dig> individuals, and the pic values ranged from  <dig>  to  <dig>  with a mean value of  <dig> .
the mean number of alleles per locus was  <dig>  alleles, the mean observed heterozygosity was  <dig> , and the mean expected heretozygosity was  <dig>  .
a small proportion of the microsatellite markers  amplified more than two alleles in some individuals, indicating that these primer pairs may be amplifying duplicated loci.
duplicated loci can share alleles of the same length so that alleles cannot unambiguously be assigned to one locus or the other.
therefore we were hesitant to use these loci for population genetic analysis.
however, it is possible to use these duplicated loci in other applications such as gene mapping or the study of gene duplication.
additional file  <dig> lists the repeat unit found in the original est sequence, together with the primer sequences that were used to pcr amplify the microsatellite loci; additionally, allele size ranges, genomic position of ssr and the number of alleles observed among the samples studied are given for each locus.
exact tests for hardy-weinberg equilibrium revealed that the majority of these microsatellites were in hwe, but  <dig> loci  showed significant departure from hwe  after bonferroni correction .
of these,  <dig> loci revealed a heterozygote deficit and one a heterozygote excess.
segregation analysis
of the  <dig> est-ssr loci developed in this study,  <dig> were polymorphic in an experimental interspecific cross between s. latifolia and s. dioica.
these loci were tested for mendelian segregation in  <dig> progeny.
null alleles were deduced at some loci where unexpected progeny genotypes could be explained only by null alleles in the parents.
as indicated by the x <dig> contingency test, most ssrs segregated in mendelian ratios, but  <dig> loci showed significant segregation distortion after bonferroni correction for multiple testing .
*significant deviation from expected mendelian ratio after bonferroni correction
a indicates an inferred null allele
<dig> f <dig> offspring from a cross between silene latifolia and s. dioica were genotyped and segregation ratios tested.
transferability of est-ssr loci
among the  <dig> microsatellite primers tested for amplification in other silene species, 93% amplified a product of expected size in s. dioica, 90% in s. diclinis, 63% in s. nutans, 57% in s. acaulis and 47% of the primer pairs were transferable to s. vulgaris, s. colpophylla and s. ciliata .
- no amplification
+ duplicated locus
utility of est-ssrs for mapping
linkage mapping based on  <dig> markers polymorphic in an interspecific cross between s. latifolia and s. dioica led to the identification of  <dig> linkage groups when a lod value of  <dig>  was employed.
these  <dig> linkage groups encompassed  <dig> est-ssrs.
<dig> markers remained unlinked.
these unlinked markers are located on other linkage groups that harbour only one or few weakly linked est-ssrs, as indicated by the fact that in combination with dominant aflp markers, all est-ssr markers map to one of  <dig> linkage groups , which corresponds to the haploid chromosome number in dioecious silene.
discussion
the white campion, silene latifolia, has long been used as a model organism for a wide range of ecological and evolutionary questions.
despite this great interest in the species, no systematic attempts have been made to characterize large numbers of genes across the species' genome and to identify gene-specific markers that can be used for a wide range of research questions and can also be transferred to closely related species.
our est library and the molecular markers derived from this library may therefore provide a valuable molecular tool for further studies on this ecological model organism.
est library annotation
our normalized floral cdna library displayed a low observed redundancy and was efficient to identify a large number of previously uncharacterized genes in s. latifolia that represent all major categories in the gene ontology  classification.
this confirms that even a limited est dataset represents a valuable resource for molecular non-model organisms  <cit> .
the great majority of unigenes identified in the present study  had a significant similarity with genes in other plant species.
most similarities were found to vitis vinifera, arabidopsis thaliana, and populus trichocarpa.
these species are all core eudicots and belong to the rosids, whereas silene belongs to the core eudicot clade caryophyllales  <cit> .
the fact that similarities were most often found to these particular plant species is a consequence of the fact that all these species have fully sequenced genomes and large est databases, and does not reflect their phylogenetic proximity to silene.
similarities with genes from these species provided some insights into the identities of silene genes.
however, blast-based annotations, especially of short est sequences, can be misleading when hits are due to domain homologies, rather than homology to orthologs  <cit> .
the few hits  to silene sequences available in genbank reflect the lack of sequence information for this genus and emphasizes the value of the est dataset developed in the present study.
the great majority of contigs with of more than four est reads combined sequences derived from more than one tissue.
of the  <dig> contigs for which all ests were derived from a single tissue, four contained sequences expressed in male flower buds .
two of these contigs had strong similarities with genes that were previously found to be expressed exclusively in males.
one contig had high similarity  to mros <dig>  <cit>  and another one was similar  to men- <dig>  <cit> .
to what extend the other genes that were found to be expressed in either males  or females  in the present study are indeed sex-specifically expressed remains to be tested experimentally.
floral development genes
we identified several unigenes that are putative homologs of arabidopsis genes that are involved in floral development .
these include genes that perceive or respond to environmental signals such as early flowering  <dig> , and stress enhanced protein  <dig> , and transcription factors that control floral development such as sepallata  <dig> , myb domain protein  <dig> , and zinc finger family proteins.
the transition to flowering in plants is regulated by environmental factors such as temperature and light.
day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock.
the gene early flowering  <dig>  is involved in photoperiod perception and circadian regulation  <cit> .
the expression of stress enhanced protein  <dig> is induced specifically by light stress and is specific, because other physiological stresses such as cold, heat, or salt do not promote accumulation of sep <dig> transcripts  <cit> .
sepallata  genes form a subfamily of mads-box transcription factors that are critical for a number of developmental processes.
in particular, the sepallata  genes play an important role in controlling the development of floral organs in flowering plants.
in arabidopsis thaliana, sep <dig>  sep  <dig>  sep <dig> and sep <dig> are required for specifying the identity of all four whorls of floral organs, and for the floral meristem determination  <cit> .
myb proteins are transcription factors that are characterize by a myb  domain.
myb <dig> is specifically expressed in flowers in a. thaliana and directly activates the expression of genes involved in the phenylpropanoid metabolism  <cit> .
analysis of expression patterns of putative homologs of these arabidopsis genes in s. latifolia will reveal to what extend their functions are conserved in silene and may help to elucidate their roles in silene flower development.
ssr frequency and distribution
in this study we found trinucleotide repeats  to be the most common ssr type in ests of silene latifolia.
this is in agreement with a majority of studies that report tnrs as the most abundant class of ssrs in plant ests  <cit> , in contrast to recent studies in actinidia  <cit>  and picea species  <cit>  wherein dinucleotide repeats  were found to be the most abundant class of est-ssrs.
interestingly, dnrs have been reported to be the most abundant ssrs in ests of many animal species such as medaka, fundulus, zebrafish, and xiphophorus  <cit> .
among the trimeric motifs, n, n and n were the most common  in s. latifolia.
in rice, 60% of est-derived microsatellite sequences were n, n, n and n  <cit> , and in maize n and n were most abundant  <cit> .
n was also the most common motif in sugarcane  <cit> .
in contrast, the motifs n and n represented 60% of all microsatellite motifs of the dicotyledon arabidopsis  <cit> .
the motif n was found to be rare in barley, rice, maize and sugarcane, as well as in arabidopsis, and was not found here in s. latifolia.
a possible reason for its rarity is that taa-based variants code for stop codons have a direct effect on protein synthesis  <cit> .
of the dimeric repeats, the motif n was the most common in our dataset with 76% of dinucleotide repeats, whereas no n motif was found.
in plants, tc and ctt repeats  were found to be typical of transcribed regions and to occur with high frequency in the 5' utrs.
it has also been suggested that the high level of the n motif is due to its translation into ala and leu, depending on the reading frame  <cit> ; ala and leu are present in proteins at high frequencies of 8% and 10%, respectively.
n repeats have been reported to be very abundant in the genomic sequences of plants  <cit> , but they were relatively rare  in our silene est sequences.
the deficiency of at-ssrs in our est sequences is in accordance with reports from rice  <cit> , arabidopsis  <cit>  and maize  <cit> .
overall, gc-rich ssr motifs were less frequent in silene ests than gc-poor motifs.
this was most evident in the relative abundance of /n and deficiency of /n repeat motifs among the dnars/tnrs, respectively, identified in this study.
interestingly, a similar difference in ssr motif in ests has been reported earlier, and seems to be a common feature of the dicotyledon species  <cit> .
est-ssr marker polymorphism
the majority of s. latifolia est-ssrs generated high-quality amplification products, suggesting that ests are ideally suited for specific primer design.
in this study, pcr amplification was successful for 82% of the primer pairs designed from ests.
among the primer pairs that amplified, we noticed that in some cases the amplification product was substantially larger than expected from the est sequence analysis.
this increase in product size was most likely due to the presence of introns and large insertions in the corresponding genomic sequence.
other primer pairs  failed to amplify a pcr product.
generally, inconsistent amplification or amplification failure of est-ssr loci may arise as a result of factors such as the presence of introns that are too large for efficient amplification, the use of poor quality sequences for primer design, and mutational substitutions, insertions or deletions within the priming site  <cit> .
levels of polymorphism detected with est-ssrs have been compared in several studies to those revealed by genomic ssrs.
in most cases, the latter were found to be more polymorphic  <cit> .
our est-ssrs revealed relatively low levels of polymorphism in the s. latifolia population surveyed as indicated by the average number of alleles per locus  and the average he  and ho .
a study based on genomic ssrs  <cit>  detected substantially higher levels of polymorphism in ssrs, with 25– <dig> alleles per locus and he between  <dig>  to  <dig>  and ho between  <dig>  to  <dig> .
all polymorphic loci developed in the present study were tested for deviations from hardy-weinberg equilibrium .
we observed significant deviations from hwe at six loci  after bonferroni correction in a natural s. latifolia population.
the fact that only a minority of surveyed loci revealed deviations from hwe indicates that the investigated population overall is in hardy-weinberg equilibrium, and that deviations at individual loci are most likely due to locus-specific effects, and not due to biological factors such as inbreeding or genetic drift, which would affect all loci.
null alleles are known to be a major cause of heterozygote deficiencies observed in ssr analyses of animal and plant populations  <cit> .
null alleles most commonly arise from point mutations in the sequence flanking the repeat region  <cit> , which reduces or prevents primer annealing.
null alleles can also be generated via differential amplification of size-variant alleles  <cit> .
due to the competitive nature of the pcr process, shorter alleles often amplify more efficiently than larger ones, such that only the smaller of two alleles may be detected from a heterozygous individual.
in general, null alleles complicate the interpretation of microsatellite data because of the reduced level of observed heterozygosity  <cit> .
problems with null alleles can be ameliorated by improvements in primer design  <cit> .
in addition to these technical problems, several population genetic phenomena may give the false impression that microsatellite null alleles are present in a given study.
biological factors such as wahlund effect or inbreeding, for example, can cause significant heterozygote deficits relative to hwe that might be misconstrued as evidence for null alleles  <cit> .
the use of a large number of ssr loci may help to distinguish between locus-specific problems and biological processes leading to heterozygote deficiencies.
a further potential cause of deviations from hardy-weinberg expectations involves sex-linkage.
the divergence between the x and y chromosomes in species with heterogametic males  often leads to the phenomenon that only one allele is amplified in the heterogametic sex, although sex chromosomes typically evolved from ancestral autosomes.
thus, if sex-linkage remains unrecognized at a locus, an associated locus-specific heterozygote deficit may be wrongly interpreted as indicative of null alleles.
indeed, one locus that maps to linkage group  <dig>  which corresponds to the sex chromosome, reveals significant deviation from hwe after bonferroni correction.
mendelian segregation of est-ssr markers
we used plants from an experimental cross to assess mendelian inheritance of our est-ssr loci.
28% of the loci that were polymorphic in the cross showed a significant deviation from expected mendelian ratios after bonferrroni correction .
segregation distortion may be caused by technical problems, including null alleles, but may also have a biological basis, such as gametic selection, embryogenesis, seed set or sex linkage.
in addition, segregation distortion may occur as a consequence of the divergence between the two species.
although markers displaying segregation distortion may complicate linkage analysis  <cit> , distorted loci can often be mapped, and the mapping of distorted markers may help to identify genes that have important biological functions  <cit> .
transferability of est-ssr markers
by virtue of the sequence conservation of transcribed regions of the genome, a significant portion of the primer pairs designed from est-ssrs is expected to function in distantly related species.
transferability of est-derived markers over different taxonomic levels has been demonstrated earlier  <cit> .
in our study, the majority of our  <dig> est-derived ssr loci from s. latifolia revealed cross-species amplification with alleles of comparable sizes in one or several of the tested silene species.
as expected, the transferability of the markers was higher for s. dioica and s. diclinis than for the other species, because of their close phylogenetic relatedness to s. latifolia.
high amplification success suggests that the flanking regions of these loci are sufficiently conserved, and that these loci can be used for comparative analyses of genetic diversity in the genus silene.
in addition, these genic ssrs are good candidates for the development of conserved orthologous markers for linkage mapping and qtl analyses in different silene species  <cit> .
est-ssr markers for comparative mapping
the abundance of microsatellites in transcribed regions of the genome and the level of polymorphism of these markers make est libraries a valuable source of markers for genetic mapping.
the high proportion of informative markers  found in the present study for an interspecific cross between two closely related dioecious silene species, and the identification of  <dig> out of  <dig> expected linkage groups, including the sex chromosome, reveal that these est-ssr loci are valuable markers for linkage mapping in s. latifolia and related dioecious species.
perhaps the most important feature of the est-ssr markers for comparative linkage mapping is that they are transferable also to more distantly related species.
the value of the transferability of such markers to related species for the purpose of comparative mapping has been demonstrated in several studies in wheat, rye and rice  <cit> .
our study shows that  <dig> to 47% of est-ssr primer pairs designed for s. latifolia will also yield amplicons in s. dioica, s. diclinis, s. vulgaris, s. colpophylla and s. ciliata and thus provide valuable markers for comparative linkage mapping in these species.
with these markers, new insights can, for example, be gained into the independent evolution of sex chromosomes from ancestral autosomes in the genus silene, a topic that has recently received great interest  <cit> .
moreover, the est-ssr loci that were found to map to the s. latifolia sex chromosomes represent a set of newly identified sex-linked genes that are now being used to further explore the divergence between the x and y chromosomes in this species.
CONCLUSIONS
only few microsatellite markers have to date been described in the literature for silene species  <cit>  and all of these are genomic ssrs for which no information on transferability to other species is available.
thus, the set of  <dig> est-ssr markers reported in this study represents an important resource for future studies on s. latifolia and other species of this highly diverse genus.
most notably, these est-ssrs will allow to perform comparative analyses of population structure, help with the identification of loci under selection in population genomic studies, and facilitate comparative linkage mapping in the genus silene.

 the white campion, silene latifolia poiret, a member of the plant family caryophyllaceae, is a dioecious herb. the species is diploid, has a large nuclear genome size and a haploid chromosome number of sex is determined genetically by heteromorphic sex chromosomes that were first described by blackburn and winge. as in humans, females are homogametic, xx, and males are the heterogametic sex, xy. the sex chromosomes are the largest chromosomes and contribute substantially to the large genome size of this species. although dioecy has evolved many times in different plant lineages, well differentiated, heteromorphic sex chromosomes are relatively rare in plants. over the last decades, silene latifolia has become a model organism in plant ecology and evolution. major research avenues include for example the evolution of heteromorphic sex chromosomes in plants, sexual dimorphism, plant-pathogen and plant pollinator interactions, invasive plant biology, hybridization and introgression, and habitat adaptation. to address these ecological and evolutionary questions, a diverse set of molecular markers has been used to date in silene latifolia. only recently have the first genomic simple sequence repeats, also known as microsatellites, been identified and used. current limitations of the available markers include the problems that the widely used aflps, and formerly rapds, are anonymous and dominant markers. while aflp markers have been used successfully for linkage mapping in the related silene vulgaris, the resulting maps derived from the maternal and paternal parent, respectively, could not be joined into a single, unifying map, because of the limited information on coupling phase provided by dominant markers. similarly, a recent genome scan analysis for markers under selection identified several aflp markers that carried the signature of selection, but characterization of the outlier markers failed to identify transcribed genes. to overcome such limitations, we have embarked on an expressed sequence tag project to identify transcribed genes in s. latifolia and to characterize simple sequence repeats in these expressed genes. ssr markers identified in est sequences are known as est-ssrs. ssrs are tandemly repeated tracts of dna composed of 1– base pair long units. they are ubiquitous in prokaryotes and eukaryotes, both in coding and noncoding regions, and are usually characterized by a high degree of length polymorphism. ssr markers are useful for a variety of applications, because of their multiallelic nature, codominant inheritance, relative abundance, and good genome coverage. the conservation of flanking sequences in the vicinity of the repeat motifs often permits the genotyping of related species with a single primer set. microsatellites have proven to be an extremely valuable tool for genome mapping in many organisms, but their applications span over different areas ranging from ancient and forensic dna studies to population genetics and conservation/management of biological resources. moreover, microsatellites, due to their large amount of variability, have the potential to be informative about gene and genome duplication, but only recently have been used for these topics. expressed sequence tags are sequenced portions of messenger rna. in recent years, est projects have been initiated for numerous plant and animal species, and have generated a vast amount of sequence information that can be used for gene discovery, functional genetic studies, and marker development. ssrs are relatively common in expressed genes, mainly in the 5' and 3' untranslated regions. such est-ssrs, or genic ssrs, have several advantages compared to other molecular markers. first, studies in plants, animals, and fungi have shown that est-ssrs are often more widely transferable between species, and even genera, than genomic ssr. second, because they are located in transcribed genes, the identification of outlier est-ssrs loci in genome scan analyses may directly identify candidates for genes under selection. by comparing the est sequence to protein sequence databases, it may further be possible to shed light on the functional identity of the gene. third, the increased likelihood for cross-species amplification and the codominant nature of est-ssrs make them ideal markers for comparative mapping. fourth, est-ssrs often display reduced levels of polymorphism compared to genomic ssrs, which may facilitate genotyping and allow a more accurate estimates of allele frequencies in population genetic studies compare to hypervariable loci. finally, the ease with which est-ssrs can be mapped may facilitate the identification of new genes that are linked to traits of particular interest, such as to the sex chromosomes in s. latifolia. as codominant markers, est-ssrs are expected to segregate according to mendel's laws in crosses between individuals, and mendelian inheritance of alleles is a requirement for population genetic analyses. an earlier review of microsatellite inheritance studies found that mendelian inheritance was almost never rejected for diploid vertebrate species. however, there is increasing evidence of what appear to be "non-mendelian" patterns of inheritance of microsatellites. because relatively few studies report tests for mendelian inheritance, it is still unclear how common non-mendelian inheritance is. a large fraction of "non-mendelian" ratios of alleles in offspring of experimental crosses is apparently caused by null alleles. potential causes of non-mendelian behaviour include sex linkage, physical association with genes under strong selection, transposable elements, or processes such as non-disjunction or meiotic drive that act during meiosis. to use est-ssr loci for population genetics, it is thus essential that mendelian segregation be verified in controlled crosses. we constructed an est library from floral tissue of male and female s. latifolia with the aim to identify expressed genes and to develop polymorphic molecular markers. floral tissue was chosen because we are interested in the genetic basis of sex determination in dioecious silene, the evolution of plant sex chromosomes, and floral isolation between s. latifolia and the closely related s. dioica. in this paper we first describe our est library and show that this library is a rich source of genes that may be involved in flower development and the control of floral trait differences between male and female plants, but also between s. latifolia and related species. second, we describe a set of newly characterized est-ssr loci and provide information about their polymorphism, mendelian inheritance, and transferability to other silene species. finally, we report on the utility of these markers for comparative mapping.  est library characterization random 5' sequencing of our directional cdna library resulted in high quality sequences with an average length of nucleotides. assembly using tgicl resulted in unigenes, consisting of singlets and contigs. average unigene length was shorter than average contig length. most contigs contained ests. only contigs contained or more sequences and the largest number of sequences per contig was. * /unigene # during pre-processing, all sequences were searched for the tags identifying three different pools. of the high quality sequences, were from pool a, from pool b, and from pool c. in sequences the adaptor could not be retrieved. despite the small number of ests per contig, most contigs consisting of four or more ests contained sequences from more than one tissue pool. only such contigs were made up by sequences from a single tissue pool. of these, contigs combined sequences from pool b, and one contig each combined sequences from pools a and c. est annotation and functional classification we used blastx to annotate our silene latifolia unigene sequences. of the unigenes matched genes in other species with an expectation value of 1e- or better in a search against the ncbi nr protein database. best hits in blastx searches were mainly to vitis vinifera, arabidopsis thaliana, populus trichocarpa, and oryza sativa. out of our unigenes, only twenty-six had a best hit with a silene species. several unigenes identified in our est library have been identified as putative homologs of arabidopsis genes that are implicated in floral development. gene ontology annotation was performed with blast2go. in total, unigenes were annotated with go terms. at least one biological process was proposed for unigenes, a cellular component for unigenes, and unigenes were annotated with at least one molecular function. there were sequences with annotations for all three go categories and unigenes had annotations for at least categories. the relative frequencies of go hits are shown in fig. characterization of microsatellite motifs we identified sequences containing one or more microsatellite motifs in the library screen. the observed frequencies of di-, tri-, tetra-, penta- and hexa-repeats were %, %, %, % and %, respectively. the di-nucleotide repeat sequences consisted of /n, /n and /n. among the di-nucleotide repeats there was a distinct predominance of /n repeats, with low frequencies of other di-nucleotide repeats, n and n. tri-nucleotide repeat motifs were recognized, which represents the most frequent repeat unit. their motifs included /n, /n, /n, /n, /n, /n, /n, /n, /n, and /n. of these, the motif /n was the most frequent, followed by /n, /n, /n, and /n. /n, /n, /n, / and / showed a very low frequency. tetra-nt repeat motifs were identified in different clones; these included /n, /n, /n, /n, /n, /n, /n, /n, /n, /n, /n, /n and /n. the most frequent 4-nt repeat motifs were /n, /n, /n, /n, but their frequency was low. we identified and different penta or hexa-nt ssr motifs, most of them were found only once. identification of polymorphic markers among the ssr-containing unigenes we selected that had long enough sequences flanking the ssr to design primer pairs. primer pairs that were not likely to form internal secondary structures were designed and tested by amplifying template dna from s. latifolia. after some optimization, of these primer pairs were successfully amplified. the other primer pairs failed. among the working primer pairs, produced pcr products of the expected size, produced pcr fragments that were considerably longer than expected and the rest produced multiple bands. finally, about 66% of the primers that were initially designed appeared to amplify the expected product as judged from agarose-gel electrophoresis. however, when these pcr products were subsequently analyzed on a capillary sequencer, some of them were not scorable due to excessive "stutter bands". finally, primer pairs remained. in order to obtain information on the putative identities and functions of the genes containing est-ssrs, the corresponding unigene sequences were subjected to blastx searches against the arabidopsis thaliana refseq database. about 90% of est-ssrs matched arabidopsis genes with an expectation value of 1e- or better. the position of the ssr motifs was unambiguously identified for out of the loci. for five loci, the two methods used to infer the ssr position disagreed and for one locus, no similarity was found to known arabidopsis genes. of the loci, are located in protein coding sequences, are located in the 5' utr, and in the 3' utr. the level of polymorphism as estimated from the polymorphic information content was higher in loci located in untranslated regions than in loci located in coding regions. est-ssr polymorphism we surveyed the allelic variability of the markers by genotyping individuals from a natural population of s. latifolia. most microsatellite loci showed allelic polymorphism. the number of alleles per locus varied from to in the panel of individuals, and the pic values ranged from to with a mean value of. the mean number of alleles per locus was alleles, the mean observed heterozygosity was, and the mean expected heretozygosity was. a small proportion of the microsatellite markers amplified more than two alleles in some individuals, indicating that these primer pairs may be amplifying duplicated loci. duplicated loci can share alleles of the same length so that alleles cannot unambiguously be assigned to one locus or the other. therefore we were hesitant to use these loci for population genetic analysis. however, it is possible to use these duplicated loci in other applications such as gene mapping or the study of gene duplication. additional file lists the repeat unit found in the original est sequence, together with the primer sequences that were used to pcr amplify the microsatellite loci; additionally, allele size ranges, genomic position of ssr and the number of alleles observed among the samples studied are given for each locus. exact tests for hardy-weinberg equilibrium revealed that the majority of these microsatellites were in hwe, but loci showed significant departure from hwe after bonferroni correction. of these, loci revealed a heterozygote deficit and one a heterozygote excess. segregation analysis of the est-ssr loci developed in this study, were polymorphic in an experimental interspecific cross between s. latifolia and s. dioica. these loci were tested for mendelian segregation in progeny. null alleles were deduced at some loci where unexpected progeny genotypes could be explained only by null alleles in the parents. as indicated by the x contingency test, most ssrs segregated in mendelian ratios, but loci showed significant segregation distortion after bonferroni correction for multiple testing. *significant deviation from expected mendelian ratio after bonferroni correction a indicates an inferred null allele f offspring from a cross between silene latifolia and s. dioica were genotyped and segregation ratios tested. transferability of est-ssr loci among the microsatellite primers tested for amplification in other silene species, 93% amplified a product of expected size in s. dioica, 90% in s. diclinis, 63% in s. nutans, 57% in s. acaulis and 47% of the primer pairs were transferable to s. vulgaris, s. colpophylla and s. ciliata. - no amplification + duplicated locus utility of est-ssrs for mapping linkage mapping based on markers polymorphic in an interspecific cross between s. latifolia and s. dioica led to the identification of linkage groups when a lod value of was employed. these linkage groups encompassed est-ssrs. markers remained unlinked. these unlinked markers are located on other linkage groups that harbour only one or few weakly linked est-ssrs, as indicated by the fact that in combination with dominant aflp markers, all est-ssr markers map to one of linkage groups, which corresponds to the haploid chromosome number in dioecious silene. discussion the white campion, silene latifolia, has long been used as a model organism for a wide range of ecological and evolutionary questions. despite this great interest in the species, no systematic attempts have been made to characterize large numbers of genes across the species' genome and to identify gene-specific markers that can be used for a wide range of research questions and can also be transferred to closely related species. our est library and the molecular markers derived from this library may therefore provide a valuable molecular tool for further studies on this ecological model organism. est library annotation our normalized floral cdna library displayed a low observed redundancy and was efficient to identify a large number of previously uncharacterized genes in s. latifolia that represent all major categories in the gene ontology classification. this confirms that even a limited est dataset represents a valuable resource for molecular non-model organisms. the great majority of unigenes identified in the present study had a significant similarity with genes in other plant species. most similarities were found to vitis vinifera, arabidopsis thaliana, and populus trichocarpa. these species are all core eudicots and belong to the rosids, whereas silene belongs to the core eudicot clade caryophyllales. the fact that similarities were most often found to these particular plant species is a consequence of the fact that all these species have fully sequenced genomes and large est databases, and does not reflect their phylogenetic proximity to silene. similarities with genes from these species provided some insights into the identities of silene genes. however, blast-based annotations, especially of short est sequences, can be misleading when hits are due to domain homologies, rather than homology to orthologs. the few hits to silene sequences available in genbank reflect the lack of sequence information for this genus and emphasizes the value of the est dataset developed in the present study. the great majority of contigs with of more than four est reads combined sequences derived from more than one tissue. of the contigs for which all ests were derived from a single tissue, four contained sequences expressed in male flower buds. two of these contigs had strong similarities with genes that were previously found to be expressed exclusively in males. one contig had high similarity to mros and another one was similar to men-. to what extend the other genes that were found to be expressed in either males or females in the present study are indeed sex-specifically expressed remains to be tested experimentally. floral development genes we identified several unigenes that are putative homologs of arabidopsis genes that are involved in floral development. these include genes that perceive or respond to environmental signals such as early flowering, and stress enhanced protein, and transcription factors that control floral development such as sepallata, myb domain protein, and zinc finger family proteins. the transition to flowering in plants is regulated by environmental factors such as temperature and light. day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. the gene early flowering is involved in photoperiod perception and circadian regulation. the expression of stress enhanced protein is induced specifically by light stress and is specific, because other physiological stresses such as cold, heat, or salt do not promote accumulation of sep transcripts. sepallata genes form a subfamily of mads-box transcription factors that are critical for a number of developmental processes. in particular, the sepallata genes play an important role in controlling the development of floral organs in flowering plants. in arabidopsis thaliana, sep sep sep and sep are required for specifying the identity of all four whorls of floral organs, and for the floral meristem determination. myb proteins are transcription factors that are characterize by a myb domain. myb is specifically expressed in flowers in a. thaliana and directly activates the expression of genes involved in the phenylpropanoid metabolism. analysis of expression patterns of putative homologs of these arabidopsis genes in s. latifolia will reveal to what extend their functions are conserved in silene and may help to elucidate their roles in silene flower development. ssr frequency and distribution in this study we found trinucleotide repeats to be the most common ssr type in ests of silene latifolia. this is in agreement with a majority of studies that report tnrs as the most abundant class of ssrs in plant ests, in contrast to recent studies in actinidia and picea species wherein dinucleotide repeats were found to be the most abundant class of est-ssrs. interestingly, dnrs have been reported to be the most abundant ssrs in ests of many animal species such as medaka, fundulus, zebrafish, and xiphophorus. among the trimeric motifs, n, n and n were the most common in s. latifolia. in rice, 60% of est-derived microsatellite sequences were n, n, n and n, and in maize n and n were most abundant. n was also the most common motif in sugarcane. in contrast, the motifs n and n represented 60% of all microsatellite motifs of the dicotyledon arabidopsis. the motif n was found to be rare in barley, rice, maize and sugarcane, as well as in arabidopsis, and was not found here in s. latifolia. a possible reason for its rarity is that taa-based variants code for stop codons have a direct effect on protein synthesis. of the dimeric repeats, the motif n was the most common in our dataset with 76% of dinucleotide repeats, whereas no n motif was found. in plants, tc and ctt repeats were found to be typical of transcribed regions and to occur with high frequency in the 5' utrs. it has also been suggested that the high level of the n motif is due to its translation into ala and leu, depending on the reading frame; ala and leu are present in proteins at high frequencies of 8% and 10%, respectively. n repeats have been reported to be very abundant in the genomic sequences of plants, but they were relatively rare in our silene est sequences. the deficiency of at-ssrs in our est sequences is in accordance with reports from rice, arabidopsis and maize. overall, gc-rich ssr motifs were less frequent in silene ests than gc-poor motifs. this was most evident in the relative abundance of /n and deficiency of /n repeat motifs among the dnars/tnrs, respectively, identified in this study. interestingly, a similar difference in ssr motif in ests has been reported earlier, and seems to be a common feature of the dicotyledon species. est-ssr marker polymorphism the majority of s. latifolia est-ssrs generated high-quality amplification products, suggesting that ests are ideally suited for specific primer design. in this study, pcr amplification was successful for 82% of the primer pairs designed from ests. among the primer pairs that amplified, we noticed that in some cases the amplification product was substantially larger than expected from the est sequence analysis. this increase in product size was most likely due to the presence of introns and large insertions in the corresponding genomic sequence. other primer pairs failed to amplify a pcr product. generally, inconsistent amplification or amplification failure of est-ssr loci may arise as a result of factors such as the presence of introns that are too large for efficient amplification, the use of poor quality sequences for primer design, and mutational substitutions, insertions or deletions within the priming site. levels of polymorphism detected with est-ssrs have been compared in several studies to those revealed by genomic ssrs. in most cases, the latter were found to be more polymorphic. our est-ssrs revealed relatively low levels of polymorphism in the s. latifolia population surveyed as indicated by the average number of alleles per locus and the average he and ho. a study based on genomic ssrs detected substantially higher levels of polymorphism in ssrs, with 25– alleles per locus and he between to and ho between to. all polymorphic loci developed in the present study were tested for deviations from hardy-weinberg equilibrium. we observed significant deviations from hwe at six loci after bonferroni correction in a natural s. latifolia population. the fact that only a minority of surveyed loci revealed deviations from hwe indicates that the investigated population overall is in hardy-weinberg equilibrium, and that deviations at individual loci are most likely due to locus-specific effects, and not due to biological factors such as inbreeding or genetic drift, which would affect all loci. null alleles are known to be a major cause of heterozygote deficiencies observed in ssr analyses of animal and plant populations. null alleles most commonly arise from point mutations in the sequence flanking the repeat region, which reduces or prevents primer annealing. null alleles can also be generated via differential amplification of size-variant alleles. due to the competitive nature of the pcr process, shorter alleles often amplify more efficiently than larger ones, such that only the smaller of two alleles may be detected from a heterozygous individual. in general, null alleles complicate the interpretation of microsatellite data because of the reduced level of observed heterozygosity. problems with null alleles can be ameliorated by improvements in primer design. in addition to these technical problems, several population genetic phenomena may give the false impression that microsatellite null alleles are present in a given study. biological factors such as wahlund effect or inbreeding, for example, can cause significant heterozygote deficits relative to hwe that might be misconstrued as evidence for null alleles. the use of a large number of ssr loci may help to distinguish between locus-specific problems and biological processes leading to heterozygote deficiencies. a further potential cause of deviations from hardy-weinberg expectations involves sex-linkage. the divergence between the x and y chromosomes in species with heterogametic males often leads to the phenomenon that only one allele is amplified in the heterogametic sex, although sex chromosomes typically evolved from ancestral autosomes. thus, if sex-linkage remains unrecognized at a locus, an associated locus-specific heterozygote deficit may be wrongly interpreted as indicative of null alleles. indeed, one locus that maps to linkage group which corresponds to the sex chromosome, reveals significant deviation from hwe after bonferroni correction. mendelian segregation of est-ssr markers we used plants from an experimental cross to assess mendelian inheritance of our est-ssr loci. 28% of the loci that were polymorphic in the cross showed a significant deviation from expected mendelian ratios after bonferrroni correction. segregation distortion may be caused by technical problems, including null alleles, but may also have a biological basis, such as gametic selection, embryogenesis, seed set or sex linkage. in addition, segregation distortion may occur as a consequence of the divergence between the two species. although markers displaying segregation distortion may complicate linkage analysis, distorted loci can often be mapped, and the mapping of distorted markers may help to identify genes that have important biological functions. transferability of est-ssr markers by virtue of the sequence conservation of transcribed regions of the genome, a significant portion of the primer pairs designed from est-ssrs is expected to function in distantly related species. transferability of est-derived markers over different taxonomic levels has been demonstrated earlier. in our study, the majority of our est-derived ssr loci from s. latifolia revealed cross-species amplification with alleles of comparable sizes in one or several of the tested silene species. as expected, the transferability of the markers was higher for s. dioica and s. diclinis than for the other species, because of their close phylogenetic relatedness to s. latifolia. high amplification success suggests that the flanking regions of these loci are sufficiently conserved, and that these loci can be used for comparative analyses of genetic diversity in the genus silene. in addition, these genic ssrs are good candidates for the development of conserved orthologous markers for linkage mapping and qtl analyses in different silene species. est-ssr markers for comparative mapping the abundance of microsatellites in transcribed regions of the genome and the level of polymorphism of these markers make est libraries a valuable source of markers for genetic mapping. the high proportion of informative markers found in the present study for an interspecific cross between two closely related dioecious silene species, and the identification of out of expected linkage groups, including the sex chromosome, reveal that these est-ssr loci are valuable markers for linkage mapping in s. latifolia and related dioecious species. perhaps the most important feature of the est-ssr markers for comparative linkage mapping is that they are transferable also to more distantly related species. the value of the transferability of such markers to related species for the purpose of comparative mapping has been demonstrated in several studies in wheat, rye and rice. our study shows that to 47% of est-ssr primer pairs designed for s. latifolia will also yield amplicons in s. dioica, s. diclinis, s. vulgaris, s. colpophylla and s. ciliata and thus provide valuable markers for comparative linkage mapping in these species. with these markers, new insights can, for example, be gained into the independent evolution of sex chromosomes from ancestral autosomes in the genus silene, a topic that has recently received great interest. moreover, the est-ssr loci that were found to map to the s. latifolia sex chromosomes represent a set of newly identified sex-linked genes that are now being used to further explore the divergence between the x and y chromosomes in this species.  only few microsatellite markers have to date been described in the literature for silene species and all of these are genomic ssrs for which no information on transferability to other species is available. thus, the set of est-ssr markers reported in this study represents an important resource for future studies on s. latifolia and other species of this highly diverse genus. most notably, these est-ssrs will allow to perform comparative analyses of population structure, help with the identification of loci under selection in population genomic studies, and facilitate comparative linkage mapping in the genus silene. 
in the present study we constructed a normalized est library from floral tissue of silene latifolia with the aim to identify expressed genes and to develop polymorphic molecular markers. the polymorphism and transferability of est-ssr markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus silene. tests for crossamplification of the est-ssr loci in other silene species established their applicability to related species. the newly characterized genes and gene-derived markers from our silene est library represent a valuable genetic resource for future studies on silene latifolia and related species. we found sequences containing one or more ssr motifs. one class of markers derived from est sequences are simple sequence repeat markers, also known as est-ssrs. expressed sequence tag databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. the inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. another important feature of est-ssr markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. a total of microsatellite loci were identified from ests having sufficient flanking sequences for primer design.
 expressed sequence tag databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. one class of markers derived from est sequences are simple sequence repeat markers, also known as est-ssrs. these are useful in plant genetic and evolutionary studies because they are located in transcribed genes and a putative function can often be inferred from homology searches. another important feature of est-ssr markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. in the present study we constructed a normalized est library from floral tissue of silene latifolia with the aim to identify expressed genes and to develop polymorphic molecular markers.  we obtained a total of high quality sequences from a normalized silene cdna library. these represent unigenes, with 73% of unigenes matching genes in other species. we found sequences containing one or more ssr motifs. more than 60% of these ssrs were trinucleotides. a total of microsatellite loci were identified from ests having sufficient flanking sequences for primer design. the inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. tests for crossamplification of the est-ssr loci in other silene species established their applicability to related species.  the newly characterized genes and gene-derived markers from our silene est library represent a valuable genetic resource for future studies on silene latifolia and related species. the polymorphism and transferability of est-ssr markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus silene. 