BACKGROUND
the clone-based sequencing and assembly of the first high quality mouse genome, from the inbred c57bl/6j strain, revealed over  <dig>  protein coding genes  <cit> .
approximately three quarters of these are direct 1: <dig> orthologues of genes identified in the human genome, but a number of gene families show striking rodent-specific expansions.
these are enriched in genes associated with reproduction, reflecting an influence of sexual competition on the evolution of the mouse genome  <cit> .
mouse sexual behaviour and physiology is strongly regulated by pheromones: biochemical signals emitted by conspecifics that directly influence the behaviour or physiology of a receiving animal  <cit> .
accordingly, families of genes encoding protein pheromones are among those expanded in rodents  <cit> .
this is mirrored by extensive species-specific expansions  in gene families encoding rodent vomeronasal receptors   <cit> , which are putative receptors for pheromones  <cit> .
the rodent vr repertoire consists of three distantly related families of g-protein coupled receptor, each expressed in sensory neurons of the vomeronasal organ in the nose: v1rs, v2rs and formyl-peptide receptors   <cit> .
each vomeronasal sensory neuron  expresses a very restricted sub-set of vrs, typically one or a few of the same sub-family, thereby patterning each neuron to detect a limited number of ligands  <cit> .
with a few exceptions  <cit> , direct relationships between specific vrs and their pheromone ligands remain unresolved.
however some general trends have emerged: neurons expressing v1rs appear tuned to detect small volatile chemicals  <cit> , those expressing v2rs have been shown to detect proteinaceous ligands  <cit> , while those expressing fprs are stimulated by disease and inflammation-related peptides  <cit> .
in addition, a significant subset vsns detect chemical signals emitted by sympatric and predatory species, suggesting some vrs may be specifically tuned to allo-specific, instead of conspecific, cues  <cit> .
the number of vrs identified in the c57bl/6j genome steadily increased with each draft genome assembly release, but has stabilised as the genome nears completion  <cit> .
recent analyses find  <dig> functional v1r genes  <cit> ,  <dig> functional v2r genes  <cit>  and  <dig> fpr genes  distributed across the mouse genome in tightly clustered arrays.
when their sequences are compared, the vrs within each cluster tend to fall within phylogenetically related clades, indicating that they expanded by localised duplication events  <cit> .
comparisons of vr clades across a range of mammals show that each species has a “semi-private” repertoire, consistent with a functional role as receptors for species-specific signals such as pheromones  <cit> .
although the variation in vr repertoire between divergent species is well documented  <cit> , much less is known about the microevolution of vr repertoires between very closely related species.
an analysis of  <dig> v1rs between m. m. musculus and m. spretus revealed dynamic modulation in evolutionary pressures, including examples of positive selection and lineage-specific pseudogenisation  <cit> .
similarly, a recent study of  <dig> v1rs in m. m. musculus and m. m. domesticus found evidence of genes shaped by negative selection and random drift, with a small proportion having evidence of positive selection  <cit> .
thus different rodent vr genes may be evolving under very different selective pressures, perhaps depending on the functional nature of their ligands; though whether these correlate within vr families, clades or even clusters is unknown.
our understanding of intra-specific vr variation is even more limited.
on one hand vrs may be expected to be under stabilising selection within a species, favouring low variation in receptor characteristics to ensure essential chemo signals  are accurately detected and transduced.
on the other hand, sexual selection driven by individual recognition may favour variation between individuals in both chemo signals and receptors  <cit> .
genome wide studies of copy number variation have reported significant variation in the size of vr clusters within inbred laboratory strains of mice  <cit> , though accurate quantification of vr content and sequence level analyses of specific receptors are lacking.
however, the value of using highly inbred, domesticated lab mice to study pheromone signalling has also been questioned  <cit> .
pheromone signalling may be largely redundant in laboratory mice releasing vr genes from selective constraint; alternatively these strains may offer a snapshot of the vr variation within the founder population, frozen by generations of inbreeding  <cit> .
it is also possible that the very process of laboratory domestication has artificially shaped pheromone receptor content or variation, as was recently demonstrated in c. elegans <cit> .
the development of massively parallel sequencing  now offers the opportunity to rapidly and affordably resequence genomes from multiple individuals of the same species  <cit> .
recently the mouse genomes project produced high coverage genome sequence of  <dig> inbred strains of mouse, including several laboratory-derived strains and wild-derived m. musculus musculus, m. musculus domesticus and m. musculus castaneus strains  <cit> .
these are widely considered to be three subspecies of mus musculus.
m. m. musculus ranges across eastern europe and northern asia, domesticus is found throughout western europe, the middle east and north africa, whereas castaneus extends throughout south and east asia .
the laboratory-derived mouse strains are overwhelmingly domesticus in origin, with minor genomic contributions from castaneusx and musculus   <cit> .
the mouse genomes project also sequenced the genome of a wild-derived mus spretus strain  <cit> .
m. spretus is a distinct species of short-tailed mouse with a sympatric range to domesticus, and serves as a genetic outgroup to the mus musculus subspecies.
here we interrogate the full complement of coding vr gene sequences in these  <dig> strains, compared to the c57bl/6j reference.
we show that mps can accurately resolve over half of mouse vrs, but that copy number variation  and non-specific short read mapping confounds complete repertoire analysis.
we identify divergent patterns in the number and distribution of single-nucleotide polymorphisms  within genes, clades and clusters of vrs, as well as between strains, and present evidence that the functional vomeronasal repertoire may vary significantly between mus species and subspecies.
RESULTS
generating an accurate dataset of vr snp variation
we collected snp data in the open reading frames of  <dig> genes across  <dig> strains , each encoding a potentially functional vr identified in the c57bl/6j reference genome.
we then adopted a conservative parsing strategy to ensure accurate and statistically robust data, removing genes on the basis of ambiguous read calls, read mapping quality and incomplete gene coverage .
in total  <dig> vr genes  were excluded leaving a dataset of  <dig> full-length vrs:  <dig> v1rs,  <dig> v2rs and  <dig> fprs.
within the coding region of these genes we identified  <dig>  snps that differ from the c57bl/6j reference sequence .
to assess the accuracy of our dataset, we compared vr sequences identified by mps with those generated by traditional long-read, capillary sequencing.
recently kurzweil et al.
reported the sequencing of a cluster of v1r genes and pseudogenes from a spret/eij bac library  <cit> .
we identified nine bac sequenced v1r genes  that was also in our parsed dataset.
assuming no error in the bac sequencing, we calculate a false positive snp call rate of  <dig> %, with a false negative rate of  <dig> %.
this equates to  <dig> % accuracy in base-pair calling.
we were also able to identify four genes  that we had excluded for failing to meet our quality threshold.
in these genes the false-positive and false-negative snp rates are significantly higher, 5% and  <dig> % respectively, which supports the use of our parsing strategy to generate an accurate, albeit conservative, snp catalogue for this study.
vr snp distribution among mouse strains
the  <dig> sequenced strains were carefully selected to ensure maximum utility among the research community, and include  <dig> common lab strains, as well as strains more recently derived from wild-caught mice of other mus species and subspecies  <cit> .
we first determined whether the distribution of snps in vrs showed unusual distribution patterns.
as expected, wild-derived strains harbour a greater number of vr snps than classical laboratory strains, each in proportions similar to the rest of the genome   <cit> .
the musculus  and castaneus  wild-derived strains  have approximately  <dig> times more snps in vrs.
the spretus strain , an entirely distinct mus species, has approximately  <dig> times more .
however, the total number of snps overestimates the diversity of unique vr alleles within the strains of mice as many variants are likely to be shared, particularly between laboratory-derived strains.
we therefore determined the repertoire of private vr snps within each strain .
again, the distribution of private snps closely mirrors that reported across the whole genome  <cit> .
there are very few private snps in vrs from lab-derived strains, but a large proportion of the vr snps in wild-derived strains are private; the number increasing with greater evolutionary distance from largely domesticus-derived reference strain .
strikingly, wsb/eij, the mus domesticus wild-derived strain  <cit> , has a similar number of total vr snps to the lab strains but a greater proportion of these  are private .
this difference illustrates the restricted genomic diversity in the vrs of classical laboratory-derived strains compared to wild-derived mice, and mirrors a similar restriction in diversity of vr ligand expression  <cit> .
together these may impact on the suitability of laboratory strains for studying the neural mechanisms underpinning social recognition, but do wild-derived strains better approximate the vr repertoire of wild individuals?
to test this we utilised a recently published dataset of  <dig> v1r gene sequences, each from  <dig> m. m. musculus, and  <dig> m. m. domesticus wild isolates  <cit> , and asked whether each wild mouse has a vr haplotype that matches the musculus  and domesticus  wild-derived strains .
we found that on average half of the isolates had at least one allele from its respective wild-derived strain .
we identified only one vr gene, vmn1r <dig> , in which no wild isolate matched the sequence from any of the  <dig> inbred strains in this study.
functional variation in vrs
snps within the coding region of genes can be divided into two classes based on functional consequence: those that are predicted to alter the resultant amino acid sequence  and those that do not influence the sequence of the protein product .
we identified  <dig> non-synonymous snps, 57% of the total, and found no difference in the ratio of non-synonymous to synonymous snps between wild-derived and lab strains .
however the density of non-synonymous snps in vrs is relatively high: on average one was found every  <dig> codons of vr gene, which is  <dig>  times more frequent than when calculated across the whole coding genome .
this relative enrichment is specific to non-synonymous snps, as the frequency of synonymous snps in vrs is similar to that of the genome average .
a large increase in non-synonymous snps could indicate that vr genes have been completely released from selective constraint in domesticated strains, in which case non-synonymous snps would randomly accumulate throughout the length of vr genes; or that the restraint is weakened in which case some evidence of selection may remain apparent in regions of the gene.
we therefore ascertained whether the distribution of variation is uniform within vr domains.
v1rs and fprs are members of the class a rhodopsin-like receptor superfamily  <cit> .
they are encoded by small, typically single exon, genes and dominated by the core seventrans-membrane domains with only a small extracellular domain  <cit> .
v2rs belong to the class c glutamate-like receptor superfamily; they are multi-exonic and are characterised by a long n-terminal extracellular domain.
this region is predicted to be involved in ligand recognition and, in contrast to the highly conserved seven trans-membrane domains, is highly variable between different members of the v2r family   <cit> .
we divided  <dig> v2r genes from  <dig> strains into n-terminal and trans-membrane domains, and calculated the ratio of non-synonymous to synonymous snps for each.
in both laboratory-derived and wild-derived strains, we found a statistically significant bias in functional variation towards the n-terminal domains .
this is consistent with an enhanced selective constraint on the conserved seven trans-membrane domains and suggests that v2rs, at least, are not entirely redundant in domesticated mice.
to further assess the functional constraint on vrs, we next determined how many non-synonymous snps created a termination codon that results in a premature truncation of the reading frame, potentially rendering the mature receptor non-functional.
we identified  <dig> new termination codons that collectively truncate  <dig> vr genes .
some truncating snps are shared, especially among laboratory strains; but when aggregated we found that  <dig> % of the vr genes analysed are truncated in at least one strain.
shifts in reading frame, as a result of insertions or deletions of a few nucleotides , are also likely to result in a non-functional protein.
we that found that a further  <dig> % of the genes analysed are frame-shifted in at least one strain .
we were also able to identify evidence of larger , lineage-specific deletions in  <dig> vrs and lineage-specific duplications in  <dig> vrs .
taken together, it is clear that the functional repertoire of pheromone receptors varies significantly between inbred strains of mice and the c57bl/6j vr reference set.
snp distribution between and within vr families
cellular expression of the three known sub-families of vomeronasal receptors, v1r, v2r and fprs is segregated within the vno, but it is not yet known whether the behaviours they regulate are functionally distinct and therefore under different selective pressures.
we therefore analysed the sequences of each sub-family independently to assess for differences in genomic variation, and considered only those snps with the potential to have a functional consequence.
after normalisation for gene length, we find the mean abundance of non-synonymous snps across all strains to be very similar between v1rs and v2rs in both lab-derived strains  and wild-derived strains .
the small family of fprs have increased mean abundances in lab strains, but the increase is not significant and largely the consequence of one unusually variable outlier, fpr-rs <dig>  .
as we found no evidence of differences between entire vr sub-families we next tested whether there was statistical correlation in variation within sub-families, based on the hypothesis that closely related receptors are likely to be activated by closely related ligands that may, in turn, regulate similar types of behaviours.
considering wild-derived and lab-derived strains independently, principal component analyses  found highly significant correlations between non-synonymous snp distribution and phylogeny within lab strain v2rs .
for example, over 90% of the v2r variation in lab-derived strains can be represented by four principal components .
grouping of vrs by cluster is apparent by the first two components in pca, as shown by v2rb and v2rc, which are highly conserved among lab-derived strains, and v2ra <dig>  which is highly variable .
similar correlations can be observed within v1rs and wild-derived strains  and even within closely related receptor clades, such as the two non-contiguous vr clusters that together generate v2ra <dig>  vmn2r57- <dig> on chromosome 7and vmn2r90- <dig> on chromosome  <dig> .
physically clustered vrs may therefore act as “units” of variation, with constituent receptor genes sharing characteristics of snp accumulation or resistance.
this implies that at least some clusters of vrs were shaped by contrasting selective pressures before or during strain derivation, but can these differences be reconciled with what is currently known about vr function?
snp distribution between functional classes of v2r
recently isogai et al.
undertook a comprehensive survey of immediate early gene upregulation in vsns after exposure to olfactory stimuli derived from conspecifics and a diverse range of other animals.
they then identified the vrs expressed in these vsns and showed that individual v2rs appear specifically tuned to detect the nature of the signal emitter, whereas v1rs are activated by cues from multiple species  <cit> .
although both the specific chemosignals and the behavioural consequences of detection remain unresolved, these groups of v2rs represent the first functional classification of vrs in mice.
the receptors can be classed as those that detect conspecific odour cues , predators  and sympatric non-predatory rodents   <cit> .
we reasoned that these functional classes of v2r would likely be under divergent selective pressures within and between species/subspecies of mouse.
indeed, within the strains that represent different species/subspecies, the v2rs that detect sympatric cues differ significantly more from the c57bl/6j reference than those that detect either conspecific  or predator cues  .
however, within domesticus strains there is no significant difference in variation by function .
discussion
massively parallel sequencing of vrs
to our knowledge, this is the first application of mps to specifically resequence and analyse vomeronasal receptor genes.
therefore we first assessed whether current technology was capable of accurately resolving the sequences of these highly homologous, clustered sequences.
across entire genomes, 77-87% of reference sequence can be resolved, depending on the strain  <cit> .
in contrast we were able to resolve between 50-60% of known coding vr sequence with high accuracy.
while some of the unresolved sequence is certainly a consequence of strain-specific vr deletions or duplications, the majority is likely due to the challenge of uniquely mapping short sequence reads to highly repetitive genes  <cit> .
two strands of evidence support this conclusion: firstly the vr clades with the highest sequence homology are enriched in the unresolved sequence, and these are also typically unresolved across all strains.
secondly in c57bl/6nj, a sub-line of the c57bl/6j reference strain that has just one snp difference over 300 kb of vr sequence, only 60% resolution was achieved.
it is improbable that such a large proportion of the vr repertoire would differ between such closely related sub-strains while virtually no snps were accumulated.
instead it is likely that 60% resolution is a baseline value for mapping reads to vrs in this dataset.
in comparison, we were able to resolve only 50% of the spretus vrs, which may indicate that as much as one tenth of the domesticus functional vr repertoire is missing in spretus .
mps is therefore currently only partially effective for accurately resequencing vrs in mouse.
however, as mapping success is largely influenced by read length, the rapid development of mps technology suggests that more of the vr repertoire will be resolved in future resequencing efforts  <cit> .
considerations in using inbred strains for behavioural research
domestication by hundreds of generations of inbreeding has resulted in strains of laboratory mouse that display constrained innate behavioural responses when compared to wild  relatives  <cit> .
we find that variation within vrs to be similarly restricted within lab-derived strains, reinforcing the genomic limitation of these strains in studies of olfactory-mediated mouse behaviour, especially those that involve individual recognition or the detection of genetic relatedness by olfaction  <cit> .
nevertheless, inbred lab-derived strains do differ from one another in some olfactory and/or pheromone mediated behaviours  <cit> .
strain-specific variance in genetically-encoded pheromone production will certainly account for some of these differences  <cit> , but we also find evidence of unusual strain-specific differences in vrs that may influence behaviour.
for example, in some cases unusual clustering of rare privates snp scan be observed.
129p2/olahsd has no private snps in v1rs but 8% of the v2r snps in that strain are private and almost all are clustered within a single vr clade, v2ra <dig>  furthermore 129p2/olahsd, which serves as the background strain for many knockout mice lines  <cit> , also has over 5% of the c57bl/6j functional vr repertoire either truncated, frame-shifted or deleted.
it is important to stress that the truncating snps and frame-shifting indels described here do not definitively denote pseudogenisation, as alternative splicing or termination codons near the 3′ end of genes may still result in fully functional receptors being produced.
nor can we confidently report this the upper limit of vr pseudogenisation, as other snps that result in non-conservative amino-acid substitutions may also result in functionally disabled genes and are not recorded here.
nevertheless, these data will assist strain selection and control when conducting or comparing experiments on olfactory-mediated behaviour in lab mice.
the vr variation reported here may also explain conflicting reports in the literature about the effectiveness of some putative pheromones  <cit> .
selection on vrs in inbred mice
we found that the distribution of snps in lab-derived strains is not random, but correlated with vr clade and genomic clusters, suggesting at least some vrs remain under selective constraint during the process of domestication by humans.
it is tempting to speculate that those clades with the least variation mediate critical pheromone-mediated behaviours, such as reproduction or maternal care, which are essential even in a laboratory environment.
however the paucity of vrs with defined ligands or functions makes it impossible to draw firm conclusions at this time.
of the vrs with known ligands  <cit> , vmn2r <dig>  was not resolved in this study.
two others, vmn2r <dig>  and vmn1r <dig> , are highly conserved among lab-derived strains.
the fprs display a range of variation, but it is presently unclear what behaviours these receptors mediate.
it is apparent, however, that the receptors in the atypical v2rc clade, which may act as functional co-receptors with other v2rs  <cit> , are among the most conserved vrs.
as a corollary to selection for essential vrs, receptors that are widely inactivated may be either disadvantageous  to domesticated environments.
recently a study demonstrated examples of acquired pheromone resistance during domestication of c. elegans, by deletion of dauer pheromone receptor genes  <cit> .
similar pressures may have acted on vrs during mouse domestication, especially as a number of pheromone-mediated behaviours, such as aggression or inbreeding avoidance, are likely to be disadvantageous in a laboratory environment.
unfortunately, because the reference genome is also from a laboratory-derived inbred strain, this study is not well suited to detect such deletions.
however mapping the vr sequences described here to a de novo genome assembly of a wild-derived domesticus mouse genome would permit the identification of novel vrs missing in domesticated mice.
vrs between species and subspecies of mice
we used four wild-derived strains as representatives of “wild” species and subspecies of mus: m. musculus musculus, m. musculus domesticusm.
musculus castaneus and m. spretus.
these strains are also inbred to homozygosity, albeit through fewer generations, and therefore under-represent the true genomic variation in wild-caught mice.
nevertheless here we have shown that they faithfully capture many of the pheromone receptor gene sequences found in wild individuals  <cit> , supporting their use as genetically controlled, “pseudo-wild” animals to investigate vr function.
however, some wild-derived inbred strains have genomic contamination with classical strains  <cit>  and our data reveals specific examples of this when compared with sub-sets of vrs from wild-caught mice  <cit> .
most strikingly, the entire v1ra and v1rb receptor cluster in the musculus wild-derived strain  are identical to the c57bl/6j reference  and do not match any of the alleles from wild musculus individuals .
even with such cases of introgression between subspecies, we were able to identify significant differences in vr variation depending on the nature of their ligand.
most species/subspecies of mus will encounter olfactory signals from similar predatory species that activate subsets of vrs resulting in defensive behaviours  <cit> .
there is relatively little coding variation in those receptors between domesticus and non-domesticus derived strains, consistent with strong selective pressure.
in contrast, some vrs are tuned to detect cues from other sympatric mouse subspecies  <cit> .
the functions of these receptors are unknown, but they may mediate a behavioural barrier that contributes to reproductive isolation between subspecies  <cit> .
we found that, collectively, these receptors are more variable in non-domesticus derived strains than those detecting predators or conspecifics.
vmn1r <dig>  has been proposed as a candidate for mediating subspecific recognition between these subspecies  <cit> .
the gene was not included in the analysis described above, but we did identify the same  <dig> snp differences between musculus and domesticus alleles reported previously.
unexpectedly, four laboratory-derived strains  also share the presumably introgressed musculus allele while the others have the domesticus allele .
thus it may now be possible to exploit these introgressions to delineate a function for this unusually variable receptor in subspecific recognition, using more commonly available laboratory-derived mice.
CONCLUSIONS
here we have resolved, analysed and compared the full coding sequences of over  <dig> pheromone receptors of unknown function, from  <dig> strains of inbred mice using massively parallel sequencing data of whole genomes.
we describe complex patterns of non-random sequence variation that indicate these receptors are under divergent selective pressures that correlate with proposed ligand, phylogeny, chromosomal clustering, and protein domain.
moreover, we demonstrate that vr protein sequences are unusually variable between mice, and that the repertoire of functional genes differs significantly, mirroring the inter-individual diversity in pheromone ligands.
together this supports a two-component model in which differences in both receptor and pheromone ligand sequence  may together genetically encode a diverse range of innate responses.
we anticipate, as vrs are increasingly deorphanised in mice, the data described here will be invaluable for investigation into the genetic basis of behavioural differences.

 the clone-based sequencing and assembly of the first high quality mouse genome, from the inbred c57bl/6j strain, revealed over protein coding genes. approximately three quarters of these are direct 1: orthologues of genes identified in the human genome, but a number of gene families show striking rodent-specific expansions. these are enriched in genes associated with reproduction, reflecting an influence of sexual competition on the evolution of the mouse genome. mouse sexual behaviour and physiology is strongly regulated by pheromones: biochemical signals emitted by conspecifics that directly influence the behaviour or physiology of a receiving animal. accordingly, families of genes encoding protein pheromones are among those expanded in rodents. this is mirrored by extensive species-specific expansions in gene families encoding rodent vomeronasal receptors, which are putative receptors for pheromones. the rodent vr repertoire consists of three distantly related families of g-protein coupled receptor, each expressed in sensory neurons of the vomeronasal organ in the nose: v1rs, v2rs and formyl-peptide receptors. each vomeronasal sensory neuron expresses a very restricted sub-set of vrs, typically one or a few of the same sub-family, thereby patterning each neuron to detect a limited number of ligands. with a few exceptions, direct relationships between specific vrs and their pheromone ligands remain unresolved. however some general trends have emerged: neurons expressing v1rs appear tuned to detect small volatile chemicals, those expressing v2rs have been shown to detect proteinaceous ligands, while those expressing fprs are stimulated by disease and inflammation-related peptides. in addition, a significant subset vsns detect chemical signals emitted by sympatric and predatory species, suggesting some vrs may be specifically tuned to allo-specific, instead of conspecific, cues. the number of vrs identified in the c57bl/6j genome steadily increased with each draft genome assembly release, but has stabilised as the genome nears completion. recent analyses find functional v1r genes, functional v2r genes and fpr genes distributed across the mouse genome in tightly clustered arrays. when their sequences are compared, the vrs within each cluster tend to fall within phylogenetically related clades, indicating that they expanded by localised duplication events. comparisons of vr clades across a range of mammals show that each species has a “semi-private” repertoire, consistent with a functional role as receptors for species-specific signals such as pheromones. although the variation in vr repertoire between divergent species is well documented, much less is known about the microevolution of vr repertoires between very closely related species. an analysis of v1rs between m. m. musculus and m. spretus revealed dynamic modulation in evolutionary pressures, including examples of positive selection and lineage-specific pseudogenisation. similarly, a recent study of v1rs in m. m. musculus and m. m. domesticus found evidence of genes shaped by negative selection and random drift, with a small proportion having evidence of positive selection. thus different rodent vr genes may be evolving under very different selective pressures, perhaps depending on the functional nature of their ligands; though whether these correlate within vr families, clades or even clusters is unknown. our understanding of intra-specific vr variation is even more limited. on one hand vrs may be expected to be under stabilising selection within a species, favouring low variation in receptor characteristics to ensure essential chemo signals are accurately detected and transduced. on the other hand, sexual selection driven by individual recognition may favour variation between individuals in both chemo signals and receptors. genome wide studies of copy number variation have reported significant variation in the size of vr clusters within inbred laboratory strains of mice, though accurate quantification of vr content and sequence level analyses of specific receptors are lacking. however, the value of using highly inbred, domesticated lab mice to study pheromone signalling has also been questioned. pheromone signalling may be largely redundant in laboratory mice releasing vr genes from selective constraint; alternatively these strains may offer a snapshot of the vr variation within the founder population, frozen by generations of inbreeding. it is also possible that the very process of laboratory domestication has artificially shaped pheromone receptor content or variation, as was recently demonstrated in c. elegans. the development of massively parallel sequencing now offers the opportunity to rapidly and affordably resequence genomes from multiple individuals of the same species. recently the mouse genomes project produced high coverage genome sequence of inbred strains of mouse, including several laboratory-derived strains and wild-derived m. musculus musculus, m. musculus domesticus and m. musculus castaneus strains. these are widely considered to be three subspecies of mus musculus. m. m. musculus ranges across eastern europe and northern asia, domesticus is found throughout western europe, the middle east and north africa, whereas castaneus extends throughout south and east asia. the laboratory-derived mouse strains are overwhelmingly domesticus in origin, with minor genomic contributions from castaneusx and musculus. the mouse genomes project also sequenced the genome of a wild-derived mus spretus strain. m. spretus is a distinct species of short-tailed mouse with a sympatric range to domesticus, and serves as a genetic outgroup to the mus musculus subspecies. here we interrogate the full complement of coding vr gene sequences in these strains, compared to the c57bl/6j reference. we show that mps can accurately resolve over half of mouse vrs, but that copy number variation and non-specific short read mapping confounds complete repertoire analysis. we identify divergent patterns in the number and distribution of single-nucleotide polymorphisms within genes, clades and clusters of vrs, as well as between strains, and present evidence that the functional vomeronasal repertoire may vary significantly between mus species and subspecies.  generating an accurate dataset of vr snp variation we collected snp data in the open reading frames of genes across strains, each encoding a potentially functional vr identified in the c57bl/6j reference genome. we then adopted a conservative parsing strategy to ensure accurate and statistically robust data, removing genes on the basis of ambiguous read calls, read mapping quality and incomplete gene coverage. in total vr genes were excluded leaving a dataset of full-length vrs: v1rs, v2rs and fprs. within the coding region of these genes we identified snps that differ from the c57bl/6j reference sequence. to assess the accuracy of our dataset, we compared vr sequences identified by mps with those generated by traditional long-read, capillary sequencing. recently kurzweil et al. reported the sequencing of a cluster of v1r genes and pseudogenes from a spret/eij bac library. we identified nine bac sequenced v1r genes that was also in our parsed dataset. assuming no error in the bac sequencing, we calculate a false positive snp call rate of %, with a false negative rate of %. this equates to % accuracy in base-pair calling. we were also able to identify four genes that we had excluded for failing to meet our quality threshold. in these genes the false-positive and false-negative snp rates are significantly higher, 5% and % respectively, which supports the use of our parsing strategy to generate an accurate, albeit conservative, snp catalogue for this study. vr snp distribution among mouse strains the sequenced strains were carefully selected to ensure maximum utility among the research community, and include common lab strains, as well as strains more recently derived from wild-caught mice of other mus species and subspecies. we first determined whether the distribution of snps in vrs showed unusual distribution patterns. as expected, wild-derived strains harbour a greater number of vr snps than classical laboratory strains, each in proportions similar to the rest of the genome. the musculus and castaneus wild-derived strains have approximately times more snps in vrs. the spretus strain, an entirely distinct mus species, has approximately times more. however, the total number of snps overestimates the diversity of unique vr alleles within the strains of mice as many variants are likely to be shared, particularly between laboratory-derived strains. we therefore determined the repertoire of private vr snps within each strain. again, the distribution of private snps closely mirrors that reported across the whole genome. there are very few private snps in vrs from lab-derived strains, but a large proportion of the vr snps in wild-derived strains are private; the number increasing with greater evolutionary distance from largely domesticus-derived reference strain. strikingly, wsb/eij, the mus domesticus wild-derived strain, has a similar number of total vr snps to the lab strains but a greater proportion of these are private. this difference illustrates the restricted genomic diversity in the vrs of classical laboratory-derived strains compared to wild-derived mice, and mirrors a similar restriction in diversity of vr ligand expression. together these may impact on the suitability of laboratory strains for studying the neural mechanisms underpinning social recognition, but do wild-derived strains better approximate the vr repertoire of wild individuals? to test this we utilised a recently published dataset of v1r gene sequences, each from m. m. musculus, and m. m. domesticus wild isolates, and asked whether each wild mouse has a vr haplotype that matches the musculus and domesticus wild-derived strains. we found that on average half of the isolates had at least one allele from its respective wild-derived strain. we identified only one vr gene, vmn1r, in which no wild isolate matched the sequence from any of the inbred strains in this study. functional variation in vrs snps within the coding region of genes can be divided into two classes based on functional consequence: those that are predicted to alter the resultant amino acid sequence and those that do not influence the sequence of the protein product. we identified non-synonymous snps, 57% of the total, and found no difference in the ratio of non-synonymous to synonymous snps between wild-derived and lab strains. however the density of non-synonymous snps in vrs is relatively high: on average one was found every codons of vr gene, which is times more frequent than when calculated across the whole coding genome. this relative enrichment is specific to non-synonymous snps, as the frequency of synonymous snps in vrs is similar to that of the genome average. a large increase in non-synonymous snps could indicate that vr genes have been completely released from selective constraint in domesticated strains, in which case non-synonymous snps would randomly accumulate throughout the length of vr genes; or that the restraint is weakened in which case some evidence of selection may remain apparent in regions of the gene. we therefore ascertained whether the distribution of variation is uniform within vr domains. v1rs and fprs are members of the class a rhodopsin-like receptor superfamily. they are encoded by small, typically single exon, genes and dominated by the core seventrans-membrane domains with only a small extracellular domain. v2rs belong to the class c glutamate-like receptor superfamily; they are multi-exonic and are characterised by a long n-terminal extracellular domain. this region is predicted to be involved in ligand recognition and, in contrast to the highly conserved seven trans-membrane domains, is highly variable between different members of the v2r family. we divided v2r genes from strains into n-terminal and trans-membrane domains, and calculated the ratio of non-synonymous to synonymous snps for each. in both laboratory-derived and wild-derived strains, we found a statistically significant bias in functional variation towards the n-terminal domains. this is consistent with an enhanced selective constraint on the conserved seven trans-membrane domains and suggests that v2rs, at least, are not entirely redundant in domesticated mice. to further assess the functional constraint on vrs, we next determined how many non-synonymous snps created a termination codon that results in a premature truncation of the reading frame, potentially rendering the mature receptor non-functional. we identified new termination codons that collectively truncate vr genes. some truncating snps are shared, especially among laboratory strains; but when aggregated we found that % of the vr genes analysed are truncated in at least one strain. shifts in reading frame, as a result of insertions or deletions of a few nucleotides, are also likely to result in a non-functional protein. we that found that a further % of the genes analysed are frame-shifted in at least one strain. we were also able to identify evidence of larger, lineage-specific deletions in vrs and lineage-specific duplications in vrs. taken together, it is clear that the functional repertoire of pheromone receptors varies significantly between inbred strains of mice and the c57bl/6j vr reference set. snp distribution between and within vr families cellular expression of the three known sub-families of vomeronasal receptors, v1r, v2r and fprs is segregated within the vno, but it is not yet known whether the behaviours they regulate are functionally distinct and therefore under different selective pressures. we therefore analysed the sequences of each sub-family independently to assess for differences in genomic variation, and considered only those snps with the potential to have a functional consequence. after normalisation for gene length, we find the mean abundance of non-synonymous snps across all strains to be very similar between v1rs and v2rs in both lab-derived strains and wild-derived strains. the small family of fprs have increased mean abundances in lab strains, but the increase is not significant and largely the consequence of one unusually variable outlier, fpr-rs. as we found no evidence of differences between entire vr sub-families we next tested whether there was statistical correlation in variation within sub-families, based on the hypothesis that closely related receptors are likely to be activated by closely related ligands that may, in turn, regulate similar types of behaviours. considering wild-derived and lab-derived strains independently, principal component analyses found highly significant correlations between non-synonymous snp distribution and phylogeny within lab strain v2rs. for example, over 90% of the v2r variation in lab-derived strains can be represented by four principal components. grouping of vrs by cluster is apparent by the first two components in pca, as shown by v2rb and v2rc, which are highly conserved among lab-derived strains, and v2ra which is highly variable. similar correlations can be observed within v1rs and wild-derived strains and even within closely related receptor clades, such as the two non-contiguous vr clusters that together generate v2ra vmn2r57- on chromosome 7and vmn2r90- on chromosome. physically clustered vrs may therefore act as “units” of variation, with constituent receptor genes sharing characteristics of snp accumulation or resistance. this implies that at least some clusters of vrs were shaped by contrasting selective pressures before or during strain derivation, but can these differences be reconciled with what is currently known about vr function? snp distribution between functional classes of v2r recently isogai et al. undertook a comprehensive survey of immediate early gene upregulation in vsns after exposure to olfactory stimuli derived from conspecifics and a diverse range of other animals. they then identified the vrs expressed in these vsns and showed that individual v2rs appear specifically tuned to detect the nature of the signal emitter, whereas v1rs are activated by cues from multiple species. although both the specific chemosignals and the behavioural consequences of detection remain unresolved, these groups of v2rs represent the first functional classification of vrs in mice. the receptors can be classed as those that detect conspecific odour cues, predators and sympatric non-predatory rodents. we reasoned that these functional classes of v2r would likely be under divergent selective pressures within and between species/subspecies of mouse. indeed, within the strains that represent different species/subspecies, the v2rs that detect sympatric cues differ significantly more from the c57bl/6j reference than those that detect either conspecific or predator cues. however, within domesticus strains there is no significant difference in variation by function. discussion massively parallel sequencing of vrs to our knowledge, this is the first application of mps to specifically resequence and analyse vomeronasal receptor genes. therefore we first assessed whether current technology was capable of accurately resolving the sequences of these highly homologous, clustered sequences. across entire genomes, 77-87% of reference sequence can be resolved, depending on the strain. in contrast we were able to resolve between 50-60% of known coding vr sequence with high accuracy. while some of the unresolved sequence is certainly a consequence of strain-specific vr deletions or duplications, the majority is likely due to the challenge of uniquely mapping short sequence reads to highly repetitive genes. two strands of evidence support this conclusion: firstly the vr clades with the highest sequence homology are enriched in the unresolved sequence, and these are also typically unresolved across all strains. secondly in c57bl/6nj, a sub-line of the c57bl/6j reference strain that has just one snp difference over 300 kb of vr sequence, only 60% resolution was achieved. it is improbable that such a large proportion of the vr repertoire would differ between such closely related sub-strains while virtually no snps were accumulated. instead it is likely that 60% resolution is a baseline value for mapping reads to vrs in this dataset. in comparison, we were able to resolve only 50% of the spretus vrs, which may indicate that as much as one tenth of the domesticus functional vr repertoire is missing in spretus. mps is therefore currently only partially effective for accurately resequencing vrs in mouse. however, as mapping success is largely influenced by read length, the rapid development of mps technology suggests that more of the vr repertoire will be resolved in future resequencing efforts. considerations in using inbred strains for behavioural research domestication by hundreds of generations of inbreeding has resulted in strains of laboratory mouse that display constrained innate behavioural responses when compared to wild relatives. we find that variation within vrs to be similarly restricted within lab-derived strains, reinforcing the genomic limitation of these strains in studies of olfactory-mediated mouse behaviour, especially those that involve individual recognition or the detection of genetic relatedness by olfaction. nevertheless, inbred lab-derived strains do differ from one another in some olfactory and/or pheromone mediated behaviours. strain-specific variance in genetically-encoded pheromone production will certainly account for some of these differences, but we also find evidence of unusual strain-specific differences in vrs that may influence behaviour. for example, in some cases unusual clustering of rare privates snp scan be observed. 129p2/olahsd has no private snps in v1rs but 8% of the v2r snps in that strain are private and almost all are clustered within a single vr clade, v2ra furthermore 129p2/olahsd, which serves as the background strain for many knockout mice lines, also has over 5% of the c57bl/6j functional vr repertoire either truncated, frame-shifted or deleted. it is important to stress that the truncating snps and frame-shifting indels described here do not definitively denote pseudogenisation, as alternative splicing or termination codons near the 3′ end of genes may still result in fully functional receptors being produced. nor can we confidently report this the upper limit of vr pseudogenisation, as other snps that result in non-conservative amino-acid substitutions may also result in functionally disabled genes and are not recorded here. nevertheless, these data will assist strain selection and control when conducting or comparing experiments on olfactory-mediated behaviour in lab mice. the vr variation reported here may also explain conflicting reports in the literature about the effectiveness of some putative pheromones. selection on vrs in inbred mice we found that the distribution of snps in lab-derived strains is not random, but correlated with vr clade and genomic clusters, suggesting at least some vrs remain under selective constraint during the process of domestication by humans. it is tempting to speculate that those clades with the least variation mediate critical pheromone-mediated behaviours, such as reproduction or maternal care, which are essential even in a laboratory environment. however the paucity of vrs with defined ligands or functions makes it impossible to draw firm conclusions at this time. of the vrs with known ligands, vmn2r was not resolved in this study. two others, vmn2r and vmn1r, are highly conserved among lab-derived strains. the fprs display a range of variation, but it is presently unclear what behaviours these receptors mediate. it is apparent, however, that the receptors in the atypical v2rc clade, which may act as functional co-receptors with other v2rs, are among the most conserved vrs. as a corollary to selection for essential vrs, receptors that are widely inactivated may be either disadvantageous to domesticated environments. recently a study demonstrated examples of acquired pheromone resistance during domestication of c. elegans, by deletion of dauer pheromone receptor genes. similar pressures may have acted on vrs during mouse domestication, especially as a number of pheromone-mediated behaviours, such as aggression or inbreeding avoidance, are likely to be disadvantageous in a laboratory environment. unfortunately, because the reference genome is also from a laboratory-derived inbred strain, this study is not well suited to detect such deletions. however mapping the vr sequences described here to a de novo genome assembly of a wild-derived domesticus mouse genome would permit the identification of novel vrs missing in domesticated mice. vrs between species and subspecies of mice we used four wild-derived strains as representatives of “wild” species and subspecies of mus: m. musculus musculus, m. musculus domesticusm. musculus castaneus and m. spretus. these strains are also inbred to homozygosity, albeit through fewer generations, and therefore under-represent the true genomic variation in wild-caught mice. nevertheless here we have shown that they faithfully capture many of the pheromone receptor gene sequences found in wild individuals, supporting their use as genetically controlled, “pseudo-wild” animals to investigate vr function. however, some wild-derived inbred strains have genomic contamination with classical strains and our data reveals specific examples of this when compared with sub-sets of vrs from wild-caught mice. most strikingly, the entire v1ra and v1rb receptor cluster in the musculus wild-derived strain are identical to the c57bl/6j reference and do not match any of the alleles from wild musculus individuals. even with such cases of introgression between subspecies, we were able to identify significant differences in vr variation depending on the nature of their ligand. most species/subspecies of mus will encounter olfactory signals from similar predatory species that activate subsets of vrs resulting in defensive behaviours. there is relatively little coding variation in those receptors between domesticus and non-domesticus derived strains, consistent with strong selective pressure. in contrast, some vrs are tuned to detect cues from other sympatric mouse subspecies. the functions of these receptors are unknown, but they may mediate a behavioural barrier that contributes to reproductive isolation between subspecies. we found that, collectively, these receptors are more variable in non-domesticus derived strains than those detecting predators or conspecifics. vmn1r has been proposed as a candidate for mediating subspecific recognition between these subspecies. the gene was not included in the analysis described above, but we did identify the same snp differences between musculus and domesticus alleles reported previously. unexpectedly, four laboratory-derived strains also share the presumably introgressed musculus allele while the others have the domesticus allele. thus it may now be possible to exploit these introgressions to delineate a function for this unusually variable receptor in subspecific recognition, using more commonly available laboratory-derived mice.  here we have resolved, analysed and compared the full coding sequences of over pheromone receptors of unknown function, from strains of inbred mice using massively parallel sequencing data of whole genomes. we describe complex patterns of non-random sequence variation that indicate these receptors are under divergent selective pressures that correlate with proposed ligand, phylogeny, chromosomal clustering, and protein domain. moreover, we demonstrate that vr protein sequences are unusually variable between mice, and that the repertoire of functional genes differs significantly, mirroring the inter-individual diversity in pheromone ligands. together this supports a two-component model in which differences in both receptor and pheromone ligand sequence may together genetically encode a diverse range of innate responses. we anticipate, as vrs are increasingly deorphanised in mice, the data described here will be invaluable for investigation into the genetic basis of behavioural differences. 
the mouse reference genome has over functional vrs arranged in highly homologous clusters, but the vast majority are of unknown function. approximately half of the vr genes that we investigated could be successfully resolved, and those that were unambiguously mapped resulted in an extremely accurate dataset. collectively vrs have over twice the coding sequence variation of the genome average; but we identify striking non-random distribution of these variants within and between genes, clusters, clades and functional classes of vrs. vomeronasal receptors, expressed in sensory neurons of the vomeronasal organ, are thought to bind pheromones and mediate innate behaviours. together our results suggest that the unusually variable vr repertoires of mice have a significant role in encoding differences in olfactory-mediated responses and behaviours.
 vomeronasal receptors, expressed in sensory neurons of the vomeronasal organ, are thought to bind pheromones and mediate innate behaviours. the mouse reference genome has over functional vrs arranged in highly homologous clusters, but the vast majority are of unknown function. differences in these receptors within and between closely related species of mice are likely to underpin a range of behavioural responses. to investigate these differences, we interrogated the vr gene repertoire from inbred strains of mice using massively parallel sequencing.  approximately half of the vr genes that we investigated could be successfully resolved, and those that were unambiguously mapped resulted in an extremely accurate dataset. collectively vrs have over twice the coding sequence variation of the genome average; but we identify striking non-random distribution of these variants within and between genes, clusters, clades and functional classes of vrs. we show that functional vr gene repertoires differ considerably between different mus subspecies and species, suggesting these receptors may play a role in mediating behavioural adaptations. finally, we provide evidence that widely-used, highly inbred laboratory-derived strains have a greatly reduced, but not entirely redundant capacity for differential pheromone-mediated behaviours.  together our results suggest that the unusually variable vr repertoires of mice have a significant role in encoding differences in olfactory-mediated responses and behaviours. our dataset has expanded over nine fold the known number of mouse vr alleles, and will enable mechanistic analyses into the genetics of innate behavioural differences in mice. vomeronasalreceptorolfactionpheromonebehaviourgenome sequencingsingle nucleotide polymorphismmouse 