BACKGROUND
the nematode caenorhabditis elegans is a widely used model species in contemporary biological research, which covers a number of disciplines including developmental biology, genetics, and evolutionary biology.
many investigations have been of paramount importance for understanding the biology of mammals, but almost all studies in c. elegans, including forward  and reverse ) genetic screens, have been conducted in only a few strains of this organism, of which the canonical strain bristol n <dig> has been the most thoroughly studied.
this severely constrains the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits.
it is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild c. elegans populations.
moreover, the widely used strains, such as n <dig> and cb <dig>  have often been kept under controlled laboratory conditions for decades, and the handling, storage, and maintenance of worms is standardized.
such artificial regimens very likely create multiple bottlenecks that can affect the genotype-phenotype relationship.
for instance, a genetic analysis of wild c. elegans strains showed that the n <dig> alleles of npr- <dig> and glb- <dig> most likely originated as an adaptation to laboratory conditions  <cit> .
genotype-phenotype relations have been studied in model organisms of many species, such as arabidopsis <cit> , drosophila <cit> , and c. elegans <cit> .
for the full appreciation and functional characterization of genes and the genotype-phenotype relations, it is essential to consider the natural context of the species, including analysis of natural isolates, and the interaction of the species with natural challenges.
previous studies on c. elegans have investigated the organism’s response to a wide range of different environmental factors, including exposure to different bacteria  <cit> , ph  <cit> , osmotic pressure  <cit> , chemicals  <cit> , and temperature  <cit> , among others.
as yet, however, these responses have not been tested in natural populations.
c. elegans is an androdioecious species with a low outcrossing rate, leading to homozygous strains in natural isolates  <cit> .
these strains can therefore be treated as haplotypes.
in this study, we investigated variation in genotype-phenotype relations for a total of  <dig> strains, of which  <dig> were freshly isolated from two different sites in france:  <dig> strains from a woodland area in santeuil  from rotting hogweed stems, and  <dig> from an orchard in orsay  from rotting apples.
as an out-group, we used three strains freshly isolated from sites in the netherlands and two strains previously isolated from france.
lastly, the genotypically most diverse laboratory-kept strain cb <dig> and the canonical strain bristol n <dig> were added   <cit> ; these two strains have been used in many studies to uncover genotype-phenotype relations both by comparing strains or by using some type of quantitative trait loci approach  <cit> .
in this study, we provide insight into the genotype-phenotype relations in natural c. elegans populations through analysis of its genomic and transcriptomic variation.
we found that local genetic diversity reflects site-specific signatures of environmental sensing, protein regulation, and the immune defense system.
our results indicate that exploring natural isolates in c. elegans should lead to identification of key components of genotype-phenotype relations compared with studies that are limited to the canonical strain bristol n <dig>
RESULTS
local c. elegans populations are genotypically separable
previous investigations have studied population genetics and genomic diversity in c. elegans, focusing on global  <cit>  or local  <cit>  populations.
cutter showed that there is a lack of geographic distribution of c. elegans genome sequences  <cit> , and andersen et al.
reported that chromosome-scale selective sweeps have acted to reduce genetic variation, and have shaped the global c. elegans population structure in recent history  <cit> .
barrière and félix concluded that local diversity in this organism is high  <cit> .
in all of these papers, diversity in c. elegans was measured as genetic diversity.
to date, very few papers have been published concerning phenotypic variation in wild isolates , and studies used only a small number of isolates  <cit> .
to our knowledge, no studies have been reported on genotype-phenotype relations in wild populations.
as mentioned above, we used wild strains from santeuil and orsay and a number of out-group strains.
all these were genotyped based on the hybridization of genomic c. elegans dna to microarrays , resulting in the identification of  <dig>  polymorphic genes with an absolute ratio of  <dig>  for the mean hybridization intensity .
most of these  were found in only one to three strains , showing that between-strain variation is more abundant than between-site variation.
major hotspots of polymorphic genes were found on chromosomes ii and v and minor on the other chromosomes .
the hotspots of polymorphic genes co-localize with the c-type lectin, nuclear hormone receptor , and math genes, and chemoreceptor gene clusters  <cit> .
in addition, microsatellite loci were used to determine the population structure  <cit>  of the orsay and santeuil strains .
the two isolation sites were genotypically separable.
this was shown by analyses of the presence of gene polymorphisms using principal component analysis  , a distance matrix visualized by an unrooted neighbor-joining  tree , and a minimum spanning network of the microsatellite data .
the minimum spanning network, pca, and nj analyses showed a clear distinction between the santeuil and the orsay strains, with one large genetic group and several smaller genetic groups being identified for both isolation sites.
in pca, the first two principal components capture around 75% of the variation in dna hybridizations.
as shown in figure 2a, n <dig> is in the far right top corner, indicating its genetic difference from all other strains.
moreover, the nj tree showed that the santeuil strains  and orsay strains are different from both n <dig> and cb <dig>  in both the pca and nj analyses, the orsay group  was seen to be genetically less diverse than the santeuil group.
within the main santeuil group , diversity was slightly larger.
furthermore, in the nj tree two small genotypic groups were separate from the main orsay and santeuil groups .
the strains within these separate groups were all from the santeuil site, except for ju <dig>  which is from the orsay site.
the strains from santeuil in group s <dig> were isolated from a single hogweed stem.
similarly, those from s <dig> were also isolated from their own single hogweed stem .
other strains were found on different hogweed stems.
we found that strains isolated from an individual hogweed stem grouped close together, but were not found to form their own separate genotypic groups.
the dutch strains were isolated from two isolation sites, with wn <dig> isolated from one site and wn <dig> and wn <dig> from the other.
the latter two strains grouped together in the nj tree.
one of the french out-group strains, ju <dig>  grouped with the santeuil strains, whereas the other, ju <dig>  was different from the rest of the strains.
n <dig> and cb <dig> were as diverse from the other out-group members as the whole out-group was from the orsay or santeuil strains.
by comparing the four genetic groups  with the out-group, the genes that were polymorphic were identified .
group s <dig> appeared to be the most divergent from the out-group, with  <dig>  genes that differed significantly  =  <dig> ).
the genetic separation between the orsay and santeuil populations was prevalent on all chromosomes .
from the scale of the axes, it can be seen that most chromosomes contributed to the separation between the two isolation sites and the out-group, except for chromosome ii; on this chromosome the santeuil and orsay lines formed one group that was separate only from the out-group.
chromosome v was the largest contributor to the variation between orsay and santeuil, most likely because of the generally higher level of variation among these strains .
of the approximately  <dig>  genes that were different between s <dig>  s <dig>  and s <dig>  around  <dig>  are located on chromosome v. of all the genes on chromosome v, around  <dig> are polymorphic between s <dig>  s <dig>  and s <dig>  this is a significant enrichment  when compared with the other chromosomes, of which  <dig> to 10% of the genes are polymorphic.
chromosomes i and x are under-represented for polymorphic genes .
the detected genotypic diversity between sites is in line with genotyping results from  <dig> markers using amplified fragment length polymorphism analysis, and with microsatellite results from two loci in other local c. elegans populations  <cit> .
however, selective sequencing using restriction site-associated dna tags did not reveal significant local diversity, possibly because of the limited number of different genotypes per location  <cit> .
the four genetic groups  identified by analysis of the ratio intensities were used as input to search for all the genes linked to each isolation site.
this allowed us to identify polymorphic genes by minor hybridization differences  < <dig> ), beyond those  <dig>  found by major hybridization differences .
in this way, we identified  <dig>  genes  that were linked to each isolation site .
of these  <dig>  genes,  <dig>  were already identified as highly polymorphic in the initial analysis on major hybridization differences, and an additional  <dig>  genes associated with isolation were found with only minor hybridization differences.
of the genes with major hybridization differences, around 62% could not be linked to an isolation site, again showing that between-strain variation is more abundant than between-site variation.
genes linked to isolation site were fond to be enriched for the gene classes fbox, math, bath, btb, c-type lectin , serpentine chemoreceptor, and nhr.
to investigate whether specific types of genes are over-represented in the group of genes that could be linked to isolation site  or in the polymorphic genes not linked to isolation site , enrichment analyses were performed .
the linked and unlinked groups were analyzed using three types of annotations: gene class, anatomy terms and gene ontology  terms, to investigate whether certain types of genes were enriched.
for the gene classes, we found that fbox, math, bath, btb, clec, serpentine chemoreceptor, and nhr genes were enriched in the group of genes linked to isolation site .
enrichment analyses were also performed for the genes that were significantly linked to the genetic groups o, s <dig>  s <dig>  and s <dig>  revealing the same gene classes as above .
the enrichment analysis of anatomy terms or go terms did not identify a clear pattern linked to isolation site or genetic group .
aenrichment  of gene classes in the group of genes with variation linked to isolation site  and the group of polymorphic genes that were not linked to isolation site .
bsignificance in –log <dig>
cgene classes with group sizes ≥  <dig>  overlap ≥  <dig> genes, and significance ≥  <dig> .
local c. elegans populations are separable on the basis of their transcriptomes
next, the influence of natural genetic variation on gene expression was studied by measuring the transcript levels of all genes of all strains, corrected for differential hybridization.
an nj tree was constructed based on the rna hybridization data .
this tree showed that the genetic groups o, s <dig>  s <dig>  s <dig>  and the out-group, were also separable based on gene expression level.
again, cb <dig> and n <dig> differed from most of the other natural strains.
isolation site and genetic group influenced the variation in rna levels of  <dig>  and  <dig>  genes, respectively .
most of these genes  were not influenced by dna polymorphisms  or genotype .
expressed genes linked to isolation site are enriched for the gene classes clec, fbxa, bath, and nhr
enrichment analyses were performed for the genes whose rna levels were influenced by isolation site or genetic group .
the gene classes clec, fbxa, bath, and nhr were significantly enriched, thus yielding similar results to our dna-level enrichment analyses.
in addition, several nematode-specific peptide families were also enriched.
together, these results show that at the genomic level, variation between local populations is enriched for the gene classes fbox, math, bath, btb, clec, serpentine chemoreceptor, and nhr, many of which are involved in gene-environment interactions  <cit> .
interestingly, we found that the gene classes clec, fbxa, bath, and nhr were also enriched, with variation linked to isolation site on the transcriptional level, even though the strains that originated from different sites were cultured under the same conditions.
it has been shown that many of these groups of genes are differentially expressed after pathogen exposure, and thus could be involved in the immune response.
for instance, c-type lectin domain-containing proteins  have been repeatedly proposed to contribute to nematode immunity  <cit> .
the immune function of these genes is supported by their specific upregulation in infected c. elegans <cit>  and also by reduced immune phenotypes after rnai knockdown of clec- <dig>  clec- <dig>  clec- <dig>  or clec- <dig> <cit> .
furthermore, f-box proteins  are part of the protein degradation pathway  <cit> .
in this pathway, substrates for degradation are ubiquinated to be recognizable by the 26s proteasome.
taken together, these results show that local genetic diversity reflects site-specific signatures of immune response and protein degradation pathways in c. elegans.
we also found that, in addition to genotypes, transcript profiles can be used to distinguish between local c. elegans populations, and may indicate the functional importance of the identified genes or gene classes in different environments  <cit> .
polymorphic genes are enriched for genes involved in gene-environment interactions
polymorphic genes between the populations were compared using a set of genes extracted from  <dig> different studies on gene expression in c. elegans exposed to biotic and abiotic factors .
in the wild, c. elegans is exposed to many different bacteria.
in studies on the effect on gene expression of various bacteria, such as lactobacillus rhamnosus <cit> , microbacterium.
nematophilum <cit> , drechmeria coniospora <cit> , serratia marcescens <cit> , xenorhabdus nematophila <cit> , and pseudomonas aeruginosa <cit> , c-type lectins were always found to be differentially expressed, as were in most cases the f-box protein genes.
receptors that are used to sense the environment, such as nhrs and serpentine receptors are also frequently differentially expressed when c. elegans is exposed to different bacteria.
in response to abiotic factors such as temperature  <cit> , osmotic stress  <cit>  or ions  <cit> , the c-type lectins and f-box protein genes are also always differentially expressed.
furthermore, the c-type lectins, f-box protein genes, and receptor genes are differentially expressed in the presence of various other substances that can be encountered by wild c. elegans strains, including tryptophan  <cit> , β-naphthoflavone  <cit> , h2s  <cit> , fluoranthene  <cit> , hormones  <cit> , sediment  <cit> , humic substances  <cit> , and pesticides  <cit> .
the other gene classes  that are importaty for the variation seen between the locations at which the wild c. elegans strains were isolated were also found to be differentially expressed in several of the aforementioned environmental studies.
altogether, the differential expression of genes in environmental studies indicates that the genes that are important for the variation between local populations of c. elegans are indeed of significance for interactions with the environment.
local populations are separable for some fitness traits
the next question was whether the genetic polymorphisms between strains could influence fitness trait variation.
c. elegans strains varied significantly in all traits except population size on escherichia coli op <dig> .
as all tests were performed, under standardized laboratory conditions and the variation between strains could be attributed to the genotype, showing that most phenotypic variation has a genetic basis.
a genetic determinant has been found for some of these traits  <cit> .
we found a significant influence of the genetic groups on the population size of c. elegans on bacillus thuringiensis nrrl b-18bt <dig> and on the length/width ratio .
we additionally reconstructed an nj tree using phenotypic trait variation; however, phenotypic variation did not separate the two isolation sites or any of the four genetic groups.
nevertheless, some phenotypes were specific to an isolation site or to certain genetic groups.
even though the two strains with the largest length/width ratio were from santeuil, most worms from santeuil were significantly shorter, had a significantly smaller length/width ratio, and so were stouter than worms from orsay .
in addition, the generation time of worms from santeuil was significantly shorter  .
abbreviations: bt <dig> nematocidal bacillus thuringiensis nrrl b- <dig>  dsm non-nematocidal bacillus thuringiensis dsm- <dig>  na not applicable.
adescribes the number of replicate populations of worms  which were tested per genotype, thus the number of such replicate populations  per isolation site is therefore  <dig> times higher than the replicate populations per genotype .
bsignificant.
cwhen all observations per genotype were used instead of the mean per genotype, the populations from the different isolations sites were significantly different .
local populations are separable with regard to food preference
we then investigated if the wild strains differed from each other in their food preference behavior for naturally co-existing bacteria and for e. coli.
under the laboratory conditions we used, the worms preferred e. coli op <dig> over all other bacteria, followed by erwinia rhapontici, sphingobacterium sp., rhodococcus erythropolis, and lactococcus lactis .
worms from santeuil preferred e. rhapontici  equally to e.
coli., whereas worms from orsay preferred e. coli over e. rhapontici.
this suggests that santeuil worms could have a slight preference for the bacterium species with which they are more likely to be familiar for an overview of the average preference of all strains, see additional file 9b; for the complete dataset, see additional file  <dig>  worksheet q  and see additional file  <dig>  worksheet r .
the most significant differences found between the orsay and santeuil strains were for the bacterial combinations e. coli op50/e.
rhapontici, e. coli op50/r.
erythropolis.
and e. rhapontici/sphingobacterium sp.
.
the canonical strains bristol n <dig> and cb <dig> are genetic outliers
we finally investigated how far the canonical wild types bristol n <dig> and cb <dig> relate to the recently isolated natural strains.
both genotypic and transcriptomic analyses identified bristol n <dig> and cb <dig> as clearly differing from the standing genetic variation of the wild isolated strains.
this also applied when we compared n <dig> and cb <dig> with some of the other out-group strains.
it is assumed that many alleles in cb <dig> and n <dig> are laboratory-derived because both n <dig> and cb <dig> went through multiple phenotypic and genetic bottlenecks over the past decades of laboratory maintenance  <cit> .
together with our results, this shows that the genotype-phenotypes relations in both n <dig> and cb <dig> are likely to be obscured by a number of laboratory-derived alleles with large effect.
this might impede the detection and functional analysis of many other genes and alleles that by themselves have small effects on phenotypes, but together might have a large effect.
CONCLUSIONS
we measured a large variety of phenotypes, including the transcriptome, for multiple wild genotypes in c. elegans collected from different locally separated sites, under the same and constant laboratory conditions.
the wild genotypes could be classified according to their site, based on genotypic and transcriptome analyses.
these differences were also reflected in several fitness traits; however, because of the limited number of populations sampled, we were not able to associate fitness traits to the different sites.
nevertheless our data provide the basis for uncovering site-specific genotypic and phenotypic signatures.
future work should aim to provide insight into genetic drift or adaptation as the major attribute shaping c. elegans local evolution.
most likely, both processes play a role, depending on the gene or genetic element in question.
however, for some gene classes, such as the chemoreceptors, it is tempting to think they are polymorphic as a result of adaptations to specific habitats.
in summary, we have unlocked a wealth of genotype-phenotype relations, indicating that the canonical wild type is a genetic outlier and that its genotype-phenotype characteristics represent a diminutive part of the myriad of interactions present in the wild.

 the nematode caenorhabditis elegans is a widely used model species in contemporary biological research, which covers a number of disciplines including developmental biology, genetics, and evolutionary biology. many investigations have been of paramount importance for understanding the biology of mammals, but almost all studies in c. elegans, including forward and reverse ) genetic screens, have been conducted in only a few strains of this organism, of which the canonical strain bristol n has been the most thoroughly studied. this severely constrains the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. it is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild c. elegans populations. moreover, the widely used strains, such as n and cb have often been kept under controlled laboratory conditions for decades, and the handling, storage, and maintenance of worms is standardized. such artificial regimens very likely create multiple bottlenecks that can affect the genotype-phenotype relationship. for instance, a genetic analysis of wild c. elegans strains showed that the n alleles of npr- and glb- most likely originated as an adaptation to laboratory conditions. genotype-phenotype relations have been studied in model organisms of many species, such as arabidopsis, drosophila, and c. elegans. for the full appreciation and functional characterization of genes and the genotype-phenotype relations, it is essential to consider the natural context of the species, including analysis of natural isolates, and the interaction of the species with natural challenges. previous studies on c. elegans have investigated the organism’s response to a wide range of different environmental factors, including exposure to different bacteria, ph, osmotic pressure, chemicals, and temperature, among others. as yet, however, these responses have not been tested in natural populations. c. elegans is an androdioecious species with a low outcrossing rate, leading to homozygous strains in natural isolates. these strains can therefore be treated as haplotypes. in this study, we investigated variation in genotype-phenotype relations for a total of strains, of which were freshly isolated from two different sites in france: strains from a woodland area in santeuil from rotting hogweed stems, and from an orchard in orsay from rotting apples. as an out-group, we used three strains freshly isolated from sites in the netherlands and two strains previously isolated from france. lastly, the genotypically most diverse laboratory-kept strain cb and the canonical strain bristol n were added; these two strains have been used in many studies to uncover genotype-phenotype relations both by comparing strains or by using some type of quantitative trait loci approach. in this study, we provide insight into the genotype-phenotype relations in natural c. elegans populations through analysis of its genomic and transcriptomic variation. we found that local genetic diversity reflects site-specific signatures of environmental sensing, protein regulation, and the immune defense system. our results indicate that exploring natural isolates in c. elegans should lead to identification of key components of genotype-phenotype relations compared with studies that are limited to the canonical strain bristol n  local c. elegans populations are genotypically separable previous investigations have studied population genetics and genomic diversity in c. elegans, focusing on global or local populations. cutter showed that there is a lack of geographic distribution of c. elegans genome sequences, and andersen et al. reported that chromosome-scale selective sweeps have acted to reduce genetic variation, and have shaped the global c. elegans population structure in recent history. barrière and félix concluded that local diversity in this organism is high. in all of these papers, diversity in c. elegans was measured as genetic diversity. to date, very few papers have been published concerning phenotypic variation in wild isolates, and studies used only a small number of isolates. to our knowledge, no studies have been reported on genotype-phenotype relations in wild populations. as mentioned above, we used wild strains from santeuil and orsay and a number of out-group strains. all these were genotyped based on the hybridization of genomic c. elegans dna to microarrays, resulting in the identification of polymorphic genes with an absolute ratio of for the mean hybridization intensity. most of these were found in only one to three strains, showing that between-strain variation is more abundant than between-site variation. major hotspots of polymorphic genes were found on chromosomes ii and v and minor on the other chromosomes. the hotspots of polymorphic genes co-localize with the c-type lectin, nuclear hormone receptor, and math genes, and chemoreceptor gene clusters. in addition, microsatellite loci were used to determine the population structure of the orsay and santeuil strains. the two isolation sites were genotypically separable. this was shown by analyses of the presence of gene polymorphisms using principal component analysis, a distance matrix visualized by an unrooted neighbor-joining tree, and a minimum spanning network of the microsatellite data. the minimum spanning network, pca, and nj analyses showed a clear distinction between the santeuil and the orsay strains, with one large genetic group and several smaller genetic groups being identified for both isolation sites. in pca, the first two principal components capture around 75% of the variation in dna hybridizations. as shown in figure 2a, n is in the far right top corner, indicating its genetic difference from all other strains. moreover, the nj tree showed that the santeuil strains and orsay strains are different from both n and cb in both the pca and nj analyses, the orsay group was seen to be genetically less diverse than the santeuil group. within the main santeuil group, diversity was slightly larger. furthermore, in the nj tree two small genotypic groups were separate from the main orsay and santeuil groups. the strains within these separate groups were all from the santeuil site, except for ju which is from the orsay site. the strains from santeuil in group s were isolated from a single hogweed stem. similarly, those from s were also isolated from their own single hogweed stem. other strains were found on different hogweed stems. we found that strains isolated from an individual hogweed stem grouped close together, but were not found to form their own separate genotypic groups. the dutch strains were isolated from two isolation sites, with wn isolated from one site and wn and wn from the other. the latter two strains grouped together in the nj tree. one of the french out-group strains, ju grouped with the santeuil strains, whereas the other, ju was different from the rest of the strains. n and cb were as diverse from the other out-group members as the whole out-group was from the orsay or santeuil strains. by comparing the four genetic groups with the out-group, the genes that were polymorphic were identified. group s appeared to be the most divergent from the out-group, with genes that differed significantly = ). the genetic separation between the orsay and santeuil populations was prevalent on all chromosomes. from the scale of the axes, it can be seen that most chromosomes contributed to the separation between the two isolation sites and the out-group, except for chromosome ii; on this chromosome the santeuil and orsay lines formed one group that was separate only from the out-group. chromosome v was the largest contributor to the variation between orsay and santeuil, most likely because of the generally higher level of variation among these strains. of the approximately genes that were different between s s and s around are located on chromosome v. of all the genes on chromosome v, around are polymorphic between s s and s this is a significant enrichment when compared with the other chromosomes, of which to 10% of the genes are polymorphic. chromosomes i and x are under-represented for polymorphic genes. the detected genotypic diversity between sites is in line with genotyping results from markers using amplified fragment length polymorphism analysis, and with microsatellite results from two loci in other local c. elegans populations. however, selective sequencing using restriction site-associated dna tags did not reveal significant local diversity, possibly because of the limited number of different genotypes per location. the four genetic groups identified by analysis of the ratio intensities were used as input to search for all the genes linked to each isolation site. this allowed us to identify polymorphic genes by minor hybridization differences < ), beyond those found by major hybridization differences. in this way, we identified genes that were linked to each isolation site. of these genes, were already identified as highly polymorphic in the initial analysis on major hybridization differences, and an additional genes associated with isolation were found with only minor hybridization differences. of the genes with major hybridization differences, around 62% could not be linked to an isolation site, again showing that between-strain variation is more abundant than between-site variation. genes linked to isolation site were fond to be enriched for the gene classes fbox, math, bath, btb, c-type lectin, serpentine chemoreceptor, and nhr. to investigate whether specific types of genes are over-represented in the group of genes that could be linked to isolation site or in the polymorphic genes not linked to isolation site, enrichment analyses were performed. the linked and unlinked groups were analyzed using three types of annotations: gene class, anatomy terms and gene ontology terms, to investigate whether certain types of genes were enriched. for the gene classes, we found that fbox, math, bath, btb, clec, serpentine chemoreceptor, and nhr genes were enriched in the group of genes linked to isolation site. enrichment analyses were also performed for the genes that were significantly linked to the genetic groups o, s s and s revealing the same gene classes as above. the enrichment analysis of anatomy terms or go terms did not identify a clear pattern linked to isolation site or genetic group. aenrichment of gene classes in the group of genes with variation linked to isolation site and the group of polymorphic genes that were not linked to isolation site. bsignificance in –log cgene classes with group sizes ≥ overlap ≥ genes, and significance ≥. local c. elegans populations are separable on the basis of their transcriptomes next, the influence of natural genetic variation on gene expression was studied by measuring the transcript levels of all genes of all strains, corrected for differential hybridization. an nj tree was constructed based on the rna hybridization data. this tree showed that the genetic groups o, s s s and the out-group, were also separable based on gene expression level. again, cb and n differed from most of the other natural strains. isolation site and genetic group influenced the variation in rna levels of and genes, respectively. most of these genes were not influenced by dna polymorphisms or genotype. expressed genes linked to isolation site are enriched for the gene classes clec, fbxa, bath, and nhr enrichment analyses were performed for the genes whose rna levels were influenced by isolation site or genetic group. the gene classes clec, fbxa, bath, and nhr were significantly enriched, thus yielding similar results to our dna-level enrichment analyses. in addition, several nematode-specific peptide families were also enriched. together, these results show that at the genomic level, variation between local populations is enriched for the gene classes fbox, math, bath, btb, clec, serpentine chemoreceptor, and nhr, many of which are involved in gene-environment interactions. interestingly, we found that the gene classes clec, fbxa, bath, and nhr were also enriched, with variation linked to isolation site on the transcriptional level, even though the strains that originated from different sites were cultured under the same conditions. it has been shown that many of these groups of genes are differentially expressed after pathogen exposure, and thus could be involved in the immune response. for instance, c-type lectin domain-containing proteins have been repeatedly proposed to contribute to nematode immunity. the immune function of these genes is supported by their specific upregulation in infected c. elegans and also by reduced immune phenotypes after rnai knockdown of clec- clec- clec- or clec-. furthermore, f-box proteins are part of the protein degradation pathway. in this pathway, substrates for degradation are ubiquinated to be recognizable by the 26s proteasome. taken together, these results show that local genetic diversity reflects site-specific signatures of immune response and protein degradation pathways in c. elegans. we also found that, in addition to genotypes, transcript profiles can be used to distinguish between local c. elegans populations, and may indicate the functional importance of the identified genes or gene classes in different environments. polymorphic genes are enriched for genes involved in gene-environment interactions polymorphic genes between the populations were compared using a set of genes extracted from different studies on gene expression in c. elegans exposed to biotic and abiotic factors. in the wild, c. elegans is exposed to many different bacteria. in studies on the effect on gene expression of various bacteria, such as lactobacillus rhamnosus, microbacterium. nematophilum, drechmeria coniospora, serratia marcescens, xenorhabdus nematophila, and pseudomonas aeruginosa, c-type lectins were always found to be differentially expressed, as were in most cases the f-box protein genes. receptors that are used to sense the environment, such as nhrs and serpentine receptors are also frequently differentially expressed when c. elegans is exposed to different bacteria. in response to abiotic factors such as temperature, osmotic stress or ions, the c-type lectins and f-box protein genes are also always differentially expressed. furthermore, the c-type lectins, f-box protein genes, and receptor genes are differentially expressed in the presence of various other substances that can be encountered by wild c. elegans strains, including tryptophan, β-naphthoflavone, h2s, fluoranthene, hormones, sediment, humic substances, and pesticides. the other gene classes that are importaty for the variation seen between the locations at which the wild c. elegans strains were isolated were also found to be differentially expressed in several of the aforementioned environmental studies. altogether, the differential expression of genes in environmental studies indicates that the genes that are important for the variation between local populations of c. elegans are indeed of significance for interactions with the environment. local populations are separable for some fitness traits the next question was whether the genetic polymorphisms between strains could influence fitness trait variation. c. elegans strains varied significantly in all traits except population size on escherichia coli op. as all tests were performed, under standardized laboratory conditions and the variation between strains could be attributed to the genotype, showing that most phenotypic variation has a genetic basis. a genetic determinant has been found for some of these traits. we found a significant influence of the genetic groups on the population size of c. elegans on bacillus thuringiensis nrrl b-18bt and on the length/width ratio. we additionally reconstructed an nj tree using phenotypic trait variation; however, phenotypic variation did not separate the two isolation sites or any of the four genetic groups. nevertheless, some phenotypes were specific to an isolation site or to certain genetic groups. even though the two strains with the largest length/width ratio were from santeuil, most worms from santeuil were significantly shorter, had a significantly smaller length/width ratio, and so were stouter than worms from orsay. in addition, the generation time of worms from santeuil was significantly shorter. abbreviations: bt nematocidal bacillus thuringiensis nrrl b- dsm non-nematocidal bacillus thuringiensis dsm- na not applicable. adescribes the number of replicate populations of worms which were tested per genotype, thus the number of such replicate populations per isolation site is therefore times higher than the replicate populations per genotype. bsignificant. cwhen all observations per genotype were used instead of the mean per genotype, the populations from the different isolations sites were significantly different. local populations are separable with regard to food preference we then investigated if the wild strains differed from each other in their food preference behavior for naturally co-existing bacteria and for e. coli. under the laboratory conditions we used, the worms preferred e. coli op over all other bacteria, followed by erwinia rhapontici, sphingobacterium sp., rhodococcus erythropolis, and lactococcus lactis. worms from santeuil preferred e. rhapontici equally to e. coli., whereas worms from orsay preferred e. coli over e. rhapontici. this suggests that santeuil worms could have a slight preference for the bacterium species with which they are more likely to be familiar for an overview of the average preference of all strains, see additional file 9b; for the complete dataset, see additional file worksheet q and see additional file worksheet r. the most significant differences found between the orsay and santeuil strains were for the bacterial combinations e. coli op50/e. rhapontici, e. coli op50/r. erythropolis. and e. rhapontici/sphingobacterium sp.. the canonical strains bristol n and cb are genetic outliers we finally investigated how far the canonical wild types bristol n and cb relate to the recently isolated natural strains. both genotypic and transcriptomic analyses identified bristol n and cb as clearly differing from the standing genetic variation of the wild isolated strains. this also applied when we compared n and cb with some of the other out-group strains. it is assumed that many alleles in cb and n are laboratory-derived because both n and cb went through multiple phenotypic and genetic bottlenecks over the past decades of laboratory maintenance. together with our results, this shows that the genotype-phenotypes relations in both n and cb are likely to be obscured by a number of laboratory-derived alleles with large effect. this might impede the detection and functional analysis of many other genes and alleles that by themselves have small effects on phenotypes, but together might have a large effect.  we measured a large variety of phenotypes, including the transcriptome, for multiple wild genotypes in c. elegans collected from different locally separated sites, under the same and constant laboratory conditions. the wild genotypes could be classified according to their site, based on genotypic and transcriptome analyses. these differences were also reflected in several fitness traits; however, because of the limited number of populations sampled, we were not able to associate fitness traits to the different sites. nevertheless our data provide the basis for uncovering site-specific genotypic and phenotypic signatures. future work should aim to provide insight into genetic drift or adaptation as the major attribute shaping c. elegans local evolution. most likely, both processes play a role, depending on the gene or genetic element in question. however, for some gene classes, such as the chemoreceptors, it is tempting to think they are polymorphic as a result of adaptations to specific habitats. in summary, we have unlocked a wealth of genotype-phenotype relations, indicating that the canonical wild type is a genetic outlier and that its genotype-phenotype characteristics represent a diminutive part of the myriad of interactions present in the wild. 
it is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild c. elegans populations. this hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. these results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. we used multiple wild c. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. comparing polymorphic genes between the populations with a set of genes extracted from different studies on gene expression in c. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation. we found that wild c. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in c. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild. gene-environment interactionsgenotype-phenotype relationswild c. elegans strainstranscriptomic diversity research on the nematode caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. the separation between the isolation sites was prevalent on all chromosomes, but chromosome v was the largest contributor to this variation. the genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains.
 analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. research on the nematode caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. this hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. it is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild c. elegans populations.  we used multiple wild c. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. the genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains. the separation between the isolation sites was prevalent on all chromosomes, but chromosome v was the largest contributor to this variation. these results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. comparing polymorphic genes between the populations with a set of genes extracted from different studies on gene expression in c. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation.  we found that wild c. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in c. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild. gene-environment interactionsgenotype-phenotype relationswild c. elegans strainstranscriptomic diversity 