BACKGROUND
studies of genetic variation have long focused on snps but over the last decade, novel insights into the genomic bases of phenotypic variation have come from analysing other types of polymorphisms including structural variations .
genes with variable copy numbers have long been known in humans  but screening the entire genome for quantitatively variable dna segments is more recent .
dna copy number variations  are a class of sv which may vary in size and are being intensively studied in the human as they are associated with a variety of phenotypes, mostly diseases  <cit> .
although, there is no strict consensus regarding the definition of cnvs, a few common features can be found.
cnvs are typically described as dna fragments exceeding 1 kb  <cit>  with a minimum sequence identity of 90%  <cit>  and most authors indicate that cnvs should not include transposons nor arise from transposon activity  <cit> .
we refer to presence/absence variation  when zero copies are present in an individual genome  <cit> , representing a subset of cnvs.
cnvs can either be inherited from the previous generations or appear de novo in an individual.
the per-locus rate for genomic rearrangements was estimated between 2 × 10e− <dig> and  <dig>  × 10e− <dig> in humans  <cit> .
mechanisms that have been identified as resulting in cnvs  include non-allelic homologous recombination  <cit> , non-homologous end joining  <cit> , break induced replication  <cit> , single strand annealing  <cit> , breakage-fusion-bridge  <cit>  and all replication slippages of the dna-polymerase during dna replication and reparation  <cit> .
despite this variety of mechanisms, the majority of cnvs are linked to the occurrence of low-copy-repeats  that are dna segments larger than  <dig> kb, dispersed throughout the genome and share a sequence identity higher than 95%.
lcrs have been reported to favor genomic rearrangements in flanking regions and described as being responsible for cnv ‘hotspots’  <cit> .
gene cnvs in human have been linked to various phenotypes such as the ability to digest starch  <cit>  and to the predisposition to diseases such breast cancer  <cit> , among others.
in plants, a sv affecting three genes was linked to pathogen resistance in soybean  <cit>  and a gene duplication was shown to cause higher grain yield in rice  <cit> .
studying cnv at the genome level can even reveal evolutionary footprints, as within the primate lineage  <cit> , which implies that inherited cnvs are subject to the same evolutionary forces as all other dna polymorphisms.
taken together, these examples illustrate the importance of cnvs for phenotypic variation and the evolution.
however, few studies have investigated cnvs and associated genes at the genome-scale in plants except for a few model species including maize  <cit> , arabidopsis  <cit> , soybean  <cit> , barley  <cit> , rice  <cit>  and poplar  <cit> .
a widely used approach for the discovery and analysis of cnvs at the genome scale involves comparative hybridizations on arrays of bacs or oligonucleotide sequences  <cit> .
the method is known as acgh .
oligonucleotide arrays afford rapid synthesis  <cit>  and design flexibility, which can be advantageous to overcome the more variable signal of oligonucleotide probes compared to bacs, e.g., by using a minimum of three probes per genomic region to reduce variance  <cit> .
as the technology has developed, statistical approaches to detect probe sets with abnormal intensities have also been implemented, most often looking for breakpoints in intensity signal between neighboring probes .
a disadvantage of these methods is that they require a high quality reference genome for the species under investigation which is often lacking for non-model organisms.
to date, gene cnvs have not been investigated in genomes as large as those encountered in most conifers .
in this report, we developed an acgh for white spruce  and investigated north-american spruce species.
white spruce and black spruce  are largely sympatric and span from the atlantic to the pacific coasts in canada.
picea glauca can hybridize with picea engelmanni  to produce a hybrid known as interior spruce which is widely distributed in the rocky mountains  <cit> .
genome draft assemblies have been obtained for the diploid genomes of white spruce  <cit>  and norway spruce  <cit>  but are highly fragmented.
however, considerable genome resources are available for white spruce including a gene catalog  <cit> , sequence capture datasets  <cit> , and a gene expression database  <cit> .
in this report, we took advantage of this genomic knowledge to pursue three objectives: 1) to develop a cgh array for spruce species targeting thousands of genes and a custom analysis pipeline centered on gene sequences; 2) to determine in spruce the frequency and characteristics of cnvs and genes involved in by analysing pedigreed families; and 3) to compare results from white spruce, black spruce and interior spruce.
we used our cgh array in offspring-parent comparisons and were able to detect thousands of cnvs within and across spruce families.
our findings indicated a role for cnvs in adaptation and suggested that the hybrid genome of interior spruce is less dynamic in regards to cnvs.
RESULTS
detection of cnv genes using cgh array and control for false discovery rate
a two-step approach was used to identify suitable acgh probes for assessing relative gene copy number variations in comparisons between test and reference genomes .
we designed probes starting from a genomic assembly of the white spruce gene space which was developed from sequence-capture data and represented ~ <dig>  distinct genes  <cit>  in the white spruce gene catalogue  <cit> .
first, a set of  <dig> million sense and antisense probes tilled along the gene space assembly was designed by genotypic inc.  and an array was produced with the entire set of  <dig> million probes .
second, preliminary comparative hybridizations were used to select a reduced set of genes meeting probe coverage and sequence length criteria .
a final set ~ <dig>  effective probes was used to design an array targeting  <dig>  gene sequences of 500 bp or more  with a minimum of six probes  evenly distributed along the gene sequence; the maximum number of probes reached  <dig> for a single  <dig>  bp gene sequence .fig.
<dig> overview of the experimental approach deployed to investigate the occurrence of cnvs within spruce pedigrees.
a) full-sibs are produced between two mature individuals by means of controlled cross-pollination in nursery.
b) the ‘female’ parent genomic dna is employed as the reference genome while the genomic dna of descendants are used as test genomes.
c) distribution of probes number per gene.
d) distribution of probes and corrected intensity ratios along one gene sequence  in cnv when comparing one descendant to the # <dig> parent
the 180 k probe acgh design was effective in discovering inherited cnvs in four different spruce families by comparing the dna of full-sib individuals  to one parent .
here, two families were from white spruce  and the two other families were from black spruce  and interior spruce , respectively; we analysed  <dig> individuals per family in a total of  <dig> test-reference hybridizations.
a log ratio of hybridization fluorescence intensities was calculated for each probe following the test-reference comparisons by using r package  and in-house python programs taking into account the dye and gc gene content biases.
the vast majority of probes gave a log intensity ratio close to  <dig>  as expected when the reference and test genomes have the same number of copies.
this expected number at a given locus is two since spruces are diploid but a higher numbers of copies cannot be dismissed.
over all comparisons, the number of positive and negative ratios were very similar.fig.
<dig> hybridizations and cnv detection results.
a) distribution of the log <dig> ratios for all probes over all families.
b) distributions of genes found in cnv among spruce families
several measures were taken to develop a reliable approach to identify genes displaying cnvs.
the experimental design involved repeatedly assessing intensity ratios for a given gene by using several individuals from a pedigree and  <dig> probes per gene in average.
self-self hybridizations in which a single genome was hybridized in duplicate on the same array but with different dyes were used in addition to the test-reference comparisons.
self-self hybridizations were carried out in all three species  and served to define robust cnv detection parameters, i.e., two major criteria for cnv detection, which resulted in a false discovery rate < < 1% in all comparisons over all species.
first, an individual probe criterion was defined as: the absolute value of the log <dig> ratio must be superior to  <dig>  | or |log2|).
second, a gene level criterion was defined based on cross-probe repeatability such that a minimum of 83% of probes per gene must meet the log ratio criterion which translates in an average of  <dig> significant probes per gene declared as representing a cnv.
inherited cnvs were found for many different genes but with low frequencies
we detected cnvs affecting a total of  <dig> distinct genes when considering all comparisons and species .
as the dna was extracted from a sample comprising several needles , hence large number of cells, the cnvs most likely affected the entire tested organism and were mostly representative of a variation inherited from the previous generation.
cnvs with an absolute log ratio superior to  <dig> made up only  <dig> % to  <dig> % of the gene cnvs and represented the upper limit above which large copy number differences are undistinguishable from presence/absence variations  given the maximum intensities that can be reached using acgh.
the number of individuals scored positively for any given gene cnv ranged from  <dig> to  <dig> per family .
this led us to classify gene cnvs as common  and infrequent , the latter accounted for 64% of gene cnvs on average .
the average number of gene cnvs detected within an individual was  <dig>  over all families.
two individuals in each of the white spruce families and one individual in the black spruce family accounted for nearly half of the infrequent gene cnvs in each family and represented outliers within those families .table  <dig> numbers of gene cnvs within each family and percentages of frequent, infrequent and presence/absence variations
familya
afamilies are named according to the main text
fig.
<dig> numbers of gene cnvs within each individual for the four pedigrees.
despite some variation, the hybrid descendants presented fewer cnv genes than the pure species descendants
cnvs were shared among species and less abundant in hybrid
the number of cnvs detected represented on average 10% of the tested genes but varied between families, i.e.,  <dig> to  <dig> in white spruce,  <dig> in black spruce and only  <dig> in interior spruce.
the reduced number of gene cnvs in the hybrid  was found both at the individual level and the family level compared to the other spruce families .
this difference was statistically tested and the number of gene cnvs was significantly lower in the interior spruce family than in white spruce and black spruce families  while the difference between the three latter families was not significant .
we examined the nature of the differences between species by considering the overlap in the gene cnv sets between the pedigrees.
the different families shared from  <dig> to  <dig> gene cnvs, including a core set of  <dig> gene cnvs found in all spruces .
the number of shared gene cnvs among families was in accordance with the relative divergence between the spruce species.
the two white spruce families had the most shared gene cnvs , white spruce and the interior spruce families had fewer  and the black spruce family shared the smallest numbers of gene cnvs with other pedigrees .
gene cnvs are enriched for adaptation-related functions and narrow expression profiles
the potential biological implications of gene cnvs were evaluated using enrichment tests for terms in gene ontology annotations.
in white spruce, the comparison of gene cnvs to the entire set of tested genes on the cgh array revealed a significant enrichment in a number of functions related to  <dig> biological processes  most significantly including the ‘response to stress’ term.
using the recently updated pfam annotations of the white spruce gene catalog  <cit> , enrichment was found for oxygenase, synthetase, hydrolase and cytochrome-p <dig> activities.
in black spruce, go-term enrichment was found in eight biological processes including most significantly ‘response to stress’ and ‘defense response’ .table  <dig> enrichment test in go annotations for white and black spruce species, results from fatigo in babelomics  <dig>
picea glauca
picea mariana
the different numbers of gene cnvs detected between species did not translate into functional annotation differences.
the core set of  <dig> gene cnvs found in all of the families is significantly enriched in functions related to defense response.
the hybrid family sharing gene cnvs with the white spruce families may be expected by inheritance of genetic material from the white spruce species but did not explain gene cnvs also shared with black spruce.
hence, such cnvs sharing between all families is more likely indicative of ancestral genetic variations that remain variable in several north-american picea species.
in addition to defense responses-related functions, all spruce species and the hybrid were enriched in the stress responses while annotations of the  <dig> genes specifically found in black spruce were not significantly enriched in any biological process compared to white spruce specific genes.
on the other hand, the  <dig> genes specifically found in the hybrid were not enriched in functions related to defense nor stress responses but enriched in functions related to responses to hormonal, organic and chemical substances.
expression levels for  <dig> % of the gene cnvs were previously assessed in various organs at different stages of development in white spruce for a total of eight tissues  <cit> .
gene cnvs appeared expressed in wide range of tissues and organs, including vegetative buds, foliage, primary and secondary xylem, phelloderm, roots, megagametophyte and embryogenic cells with no significant enrichment for any of them.
however, the set of gene cnvs is significantly enriched in genes expressed in only one or two tissues, with no or little variation between tissues.
cnvs were distributed across the entire genome
a dense genetic map has been continuously developed since  <dig> for white spruce , which has allowed to locate the position of  <dig> genes, observe a high synteny between spruce species and dissect the genomic architecture for several traits of adaptive and economic interest in spruce species  <cit> .
a total of  <dig>  of the cnv genes were located on this spruce genetic map.
the cnvs were distributed over the entire genome; each linkage group included from  <dig>  to  <dig>  gene cnvs and had an average of  <dig>  cnvs .
the average distances between two mapped gene cnvs were  <dig> ,  <dig> ,  <dig>  and  <dig>  cm for the ws <dig>  ws <dig>  bs and is families respectively, and was inversely proportional to the number of mapped gene cnvs in each family.
no significant clustering of gene cnvs was observed within linkage groups although a total of  <dig> cases of two or more genes within a 1 cm region were found across the three spruce species .fig.
<dig> distribution over the genome and frequency of genes found in cnv also positioned upon the spruce genetic map  <cit> .
each point represents the position of a gene found in cnv in one cgh comparison; the piling is indicative of the number of individuals presenting the cnv
discussion
we analysed thousands of gene cnvs and found that they are less abundant in a hybrid compared to two pure spruce species.
the biological functions of the gene cnvs indicate a role in stress and defense responses.
we discuss the development of cnv detection in wild species and the insights gained from our findings into genome biology, evolution and adaptation.
detecting gene cnvs within pedigrees
we developed gene-based cnv detection procedures to analyse wild species, even those with large genomes.
a low threshold was used for cnv detection  compared to other recent studies .
this apparently low value was permitted because of an array design that included a relatively large number of probes which presented such significant signal for the same gene.
previous reports have recommended a minimum of three probes per genomic segment in order to effectively estimate the relative number of copies when using oligonucleotide cgh .
when analysing genes, some authors set a requirement that all of the three to four probes tested be significant for positive scoring of cnvs .
we used many more probes per gene sequence  which provided a strong support for the estimated ratios and robust detection parameters with very low fdr.
in these conditions, the number of cnvs detected was inflated substantially when we lowered the proportion of significant probes  and was only slightly affected when we lowered the significance threshold for intensity ratios.
we did not detect a bias towards negative or positive log ratios in the overall data set nor when comparing individual offspring to a parental reference genome.
this result appeared to be at odds with those from other organisms showing that losses often dominate over gains  <cit> ; however, the detection of more losses than gains could be due to a reference genome bias, at least in some cases  <cit> .
this type of bias is not expected in our experiment as we used parent-offspring comparisons which reduced sequence divergences between the test and reference genomes, both being similarly distant from the genome used for probe design.
it has also been argued that a higher number of positive cnvs  may be expected because genomes are likely more tolerant to duplications than deletion possibly resulting in loss of function  <cit> .
our approach, in which we sampled multiple offspring, was designed to uncover and identify inherited pre-existing gene cnvs shared across a pedigree rather than de novo cnvs arising from recent structural variations.
however, many of the cnvs in spruces  were found in only one individual within a pedigree, as observed in other plants  <cit> .
these infrequent cnvs may represent three different cases: i) random effects due to the small sample size , which is supported by the detection of these same cnvs in at least one other family ; ii) false-positives, despite the very small number of cnvs in self-self hybridizations; iii) de novo structural variations which occurred in or during reproduction of the previous generation.
in the subset of cnvs found only once over the entire dataset  the proportion of negative and positive log ratios was balanced, suggesting that there was no prevalence of de novo copy gains or losses.
impact and distribution of cnvs across the genome
the proportion of genes positively scored for cnvs was  <dig> % in white spruce  which is similar or slightly higher in magnitude than found in whole genome analyses in model species and crop plants ;  <cit>  ;  <cit>  ;  <cit>  ;  <cit>  ,  <cit>  ).
the high proportion found in spruce can be either attributed to the detection of relatively short cnv regions enabled by the high probe density in our array design, or the occurrence of more than one gene located within the same long dna sequence in cnv.
in that case, some cnvs may represent only one and the same dna segment that has been duplicated or lost.
this could not be verified for most of the cnvs as their genomic positions were unknown.
hence, the rate of cnvs may not precisely reflect the rate of actual structural variations but rather provided an estimate of the order of magnitude.
in total, 9% of the genes surveyed  using acgh and 6% of the genes found in cnv had previously been positioned on a genetic linkage map in picea glauca  <cit> .
this lower proportion of mapped cnvs than expected by chance alone is not surprising because the map was mainly developed using snp from large high-throughput genotyping arrays  <cit>  which likely selected against genes that display cnvs.
localizations on the genetic map showed that cnv genes were widely distributed at a low density over the entire genome  with a minimum of  <dig> cnv genes per linkage group and average distance of 27 cm between successive cnvs within species.
in addition,  <dig> cases of  <dig> or  <dig> cnv genes located within 1-cm of each other suggested the occurrence of structural variations involving chromosomal segments with more than one gene.
even though the number of mapped cnvs was too small to precisely assess the frequency of such events, it would represent an estimated rate of svs close to  <dig> %.
multi-gene svs have already been described in soybean  <cit>  and a rate of 5–10% was reported in maize/teosinte by using acgh  <cit> .
in light of these reports, more svs affecting more than one gene in spruce could be expected.
however, conifer genes are often separated by very long non-coding regions  such that multi-gene svs may be less common in this phylum.
furthermore, the discovery of cnvs in close proximity to each other raises the question of their origin.
the first hypothesis may be that these genes are among the few genes actually separated by relatively small non-coding sequences.
another explanation would be that these genes are widely separated but structural variations that are typically even wider in conifers, hence allowing to encompass several coding sequences.
finally, one possible explanation is that these genes may otherwise originate from different events of sv within the same genomic region; such hotspots for structural variations have been found in other model organisms .
further characterization of these genetically linked cnvs genes and a finer dissection of structural variations could be achieved by analysing larger populations , hence allowing to directly map these genetic variations and infer the occurrence of one or more events of structural variation.
numbers of shared cnvs agrees with the estimated time of species divergence
the proportion of shared gene cnvs across the different picea species was in accordance with their phylogenetic distance, i.e., more gene cnvs were shared among more closely related families and species.
the highest overlap was found between the two white spruce families, suggesting that the  <dig> shared gene cnvs are not limited to one parental lineage but more likely to be spread at a significant level in the population.
the second highest overlaps were between interior spruce and white spruce families despite the lower number of gene cnvs in the hybrid.
engelmann and white spruces, the parental species of the hybrid interior spruce, diverged only  <dig>  myears ago, a relatively short timeframe compared to the  <dig>  myears separating the white/engelmann spruce lineage from the black spruce lineage  <cit> .
in line with their relative times of divergence, engelmann and white spruce can hybridize while black spruce is reproductively isolated from both of the other species  <cit>  and also shared the smallest number of cnvs with the other spruce families.
evolutionary forces have also been linked to cnv diversity in other plants.
in a study comparing domesticated and wild barely accessions for instance, it was shown that strong directional selection associated with breeding in barley reduced cnv diversity  <cit> .
hence, cnv diversity in barley mirrored the behaviour of other dna polymorphisms indicating that evolutionary forces likely have similar impacts on cnvs than on other types of dna polymorphism.
similarly, cnv diversity assessed using a human acgh allowed to detect gene cnvs shared among primates that likely resulted from gene duplication in common ancestors  <cit> .
therefore, gene cnvs shared between the different spruce species likely represent ancestral polymorphisms that have not been eliminated by genetic drift or directional selection during the course of evolution, or alternatively, have been maintained by balancing or divergent selection.
role of cnvs in the evolution of forest trees
a wide range of gene cnvs have been linked to quantitative phenotypic variation in model organisms  <cit> .
in forest trees, quantitative genetic variations have been linked to adaptation and is characteristic of key adaptive traits such as growth phenology and cold tolerance.
genetic variation in adaptive traits has been studied in many conifers including picea spp.
and was often observed to follow gradual changes over wide ranging geographic areas and environmental conditions as opposed to drastic changes linked to specific conditions .
here we examine the potential impact of cnvs on adaptation in trees and predict that they make a qualitatively distinct contribution compared to variations in coding sequences resulting in amino acid substitutions.
the two types of mutation have different impacts at the molecular level and are expected to have different evolutionary consequences.
changes in amino acid sequences may alter protein function and therefore are more often likely to be deleterious and purged, or to be recessive and only impact phenotype in homozygotes  <cit> .
cnvs alter gene dosage and may affect expression which is akin to cis- acting regulatory variation.
as such cnvs are more likely to alter phenotype in heterozygotes and thus be more directly visible to selection  <cit> .
gene dosage effects caused by cnvs may also be detrimental, for example by creating an imbalance in multi-protein complexes  <cit> , but this is likely to be restricted to certain classes of genes and be more prevalent in house-keeping functions.
the relative effects of cnvs and amino acid changes will also depend on the function and expression of the genes product.
here, cnv genes were enriched for biological functions related to stress response, defense response and metabolic processes, which was also observed in barley and maize  <cit>  and is in line with a role in adaptation.
the cnv genes were also enriched for narrow transcript accumulation profiles in white spruce  <cit> , suggesting more specialized functions rather than house-keeping roles which reinforces the potential for cnvs to contribute to adaptive phenotypes.
considering these observations, cnvs affecting genes have the potential to make a unique contribution to adaptation in trees as a result of their qualitatively distinct impact on evolution and the types of genes affected.
we further suggest that cnvs could be especially important for short term adaptability and thus in responding to the acceleration of environmental change.
fewer cnvs in the hybrid species
significantly fewer cnv genes were found for the interior spruce family , a hybrid resulting from the natural introgression of white and engelmann spruce.
we examined whether the difference could be due to technical reasons such as is producing fewer successful hybridizations as the probe set was designed with pure white spruce sequences.
however, the data indicated that the numbers of successfully tested genes were nearly identical in all three species including the more distantly related black spruce.
this is consistent with the expectation that the procedure we used was able to tolerate up to three mismatches per probe  and the average degree of polymorphism found among sequences of white, black and engelmann spruces.
the occurrence of cnvs is underpinned by mechanisms that are likely to be conserved across all living organisms  <cit>  and are predominantly associated with low-copy-repeats , such as unequal interchromosomal recombinations, among others.
lcrs are conserved  dna segments that occur a few times throughout the genome  <cit> .
interior spruce has a diploid genome that originally came from contributions of white spruce and engelmann spruce.
as a result, a higher level of heterozygosity is observed in the individuals of interior spruce compared to individuals of pure species.
such dual origin is expected to have introduced a higher level of diversity in lcrs found in hybrid than that found in individuals of pure species.
hence, a lower sequence identity between lcr sequences of homologous chromosomes is expected in the hybrid genome which would have entangled the mechanisms leading to gene cnvs.
as a consequence, the interior spruce individuals has less numerous cnvs than white spruce or black spruce individuals; these cnvs may result from other types of breakage-reparation mechanisms that are not based upon sequence identity.
these results suggest that hybrids have systemically reduced copy number variability indicative of a less dynamic genome which in turn would limit genome evolvability.
future studies investigating other cases of introgression, either from the wild or from artificial cross-breeding, would support or dismiss this hypothesis.
CONCLUSIONS
investigations of non-model species have broadened the scope of genomic studies and contribute to the understanding of ecological diversity.
more affordable sequencing and genotyping techniques as well as the advent of next-generation sequencing have paved the way to these developments.
this report illustrates how techniques of transcriptomics and gene-space characterization in a non-model system can in turn be used to design cgh arrays and study cnvs on a genome scale.
approaches using whole genome sequencing data from model organisms to detect usually small cnvs have been developed; however different detection methods and algorithms have produced different results from the same datasets  <cit> .
more recently, continuous development of technologies to sequence and characterize larger dna segments have allowed to investigate larger structural variations in model organisms using third generation pacbio sequencing  <cit>  or genome mapping on nanochannel arrays  <cit> .
these technologies can be applied to non-model organisms, although the 20-gb conifer genomes would still remain challenging.
an advantage of the approach outlined here was the very low fdr which indicated robust results.
although it is time and resource consuming, testing several offspring from the same parents was also helpful as it confirmed the discovery of cnvs by repeated identification in related individuals.
this approach also throws light onto inherited gene cnvs which are of interest for investigating the role of cnvs in adaptation and evolution as well as establishing a framework to study their relation to phenotype, e.g., through qtl mapping.
as gene cnvs could result in dosage effects on phenotypic variation, they may play a key role in quantitative trait variations such as growth and phenology in plants.
here, we reported a lower number of cnvs within a hybrid pedigree than in families from pure species, suggesting that hybrids may systemically have a lower diversity in cnvs.
considering the gene annotations related to stress and defense responses, this reduced genetic diversity may act on phenotypic variability and evolution of hybrids in response to natural selection.
future investigations to assess the impact of cnvs on the evolution of species could focus on cnvs at the population level, looking for signatures of selection across geographical ranges and varying environmental conditions.
applied to a variety of species, such analyses may reveal new insights into adaptation and speciation.

 studies of genetic variation have long focused on snps but over the last decade, novel insights into the genomic bases of phenotypic variation have come from analysing other types of polymorphisms including structural variations. genes with variable copy numbers have long been known in humans but screening the entire genome for quantitatively variable dna segments is more recent. dna copy number variations are a class of sv which may vary in size and are being intensively studied in the human as they are associated with a variety of phenotypes, mostly diseases. although, there is no strict consensus regarding the definition of cnvs, a few common features can be found. cnvs are typically described as dna fragments exceeding 1 kb with a minimum sequence identity of 90% and most authors indicate that cnvs should not include transposons nor arise from transposon activity. we refer to presence/absence variation when zero copies are present in an individual genome, representing a subset of cnvs. cnvs can either be inherited from the previous generations or appear de novo in an individual. the per-locus rate for genomic rearrangements was estimated between 2 × 10e− and  × 10e− in humans. mechanisms that have been identified as resulting in cnvs include non-allelic homologous recombination, non-homologous end joining, break induced replication, single strand annealing, breakage-fusion-bridge and all replication slippages of the dna-polymerase during dna replication and reparation. despite this variety of mechanisms, the majority of cnvs are linked to the occurrence of low-copy-repeats that are dna segments larger than kb, dispersed throughout the genome and share a sequence identity higher than 95%. lcrs have been reported to favor genomic rearrangements in flanking regions and described as being responsible for cnv ‘hotspots’. gene cnvs in human have been linked to various phenotypes such as the ability to digest starch and to the predisposition to diseases such breast cancer, among others. in plants, a sv affecting three genes was linked to pathogen resistance in soybean and a gene duplication was shown to cause higher grain yield in rice. studying cnv at the genome level can even reveal evolutionary footprints, as within the primate lineage, which implies that inherited cnvs are subject to the same evolutionary forces as all other dna polymorphisms. taken together, these examples illustrate the importance of cnvs for phenotypic variation and the evolution. however, few studies have investigated cnvs and associated genes at the genome-scale in plants except for a few model species including maize, arabidopsis, soybean, barley, rice and poplar. a widely used approach for the discovery and analysis of cnvs at the genome scale involves comparative hybridizations on arrays of bacs or oligonucleotide sequences. the method is known as acgh. oligonucleotide arrays afford rapid synthesis and design flexibility, which can be advantageous to overcome the more variable signal of oligonucleotide probes compared to bacs, e.g., by using a minimum of three probes per genomic region to reduce variance. as the technology has developed, statistical approaches to detect probe sets with abnormal intensities have also been implemented, most often looking for breakpoints in intensity signal between neighboring probes. a disadvantage of these methods is that they require a high quality reference genome for the species under investigation which is often lacking for non-model organisms. to date, gene cnvs have not been investigated in genomes as large as those encountered in most conifers. in this report, we developed an acgh for white spruce and investigated north-american spruce species. white spruce and black spruce are largely sympatric and span from the atlantic to the pacific coasts in canada. picea glauca can hybridize with picea engelmanni to produce a hybrid known as interior spruce which is widely distributed in the rocky mountains. genome draft assemblies have been obtained for the diploid genomes of white spruce and norway spruce but are highly fragmented. however, considerable genome resources are available for white spruce including a gene catalog, sequence capture datasets, and a gene expression database. in this report, we took advantage of this genomic knowledge to pursue three objectives: 1) to develop a cgh array for spruce species targeting thousands of genes and a custom analysis pipeline centered on gene sequences; 2) to determine in spruce the frequency and characteristics of cnvs and genes involved in by analysing pedigreed families; and 3) to compare results from white spruce, black spruce and interior spruce. we used our cgh array in offspring-parent comparisons and were able to detect thousands of cnvs within and across spruce families. our findings indicated a role for cnvs in adaptation and suggested that the hybrid genome of interior spruce is less dynamic in regards to cnvs.  detection of cnv genes using cgh array and control for false discovery rate a two-step approach was used to identify suitable acgh probes for assessing relative gene copy number variations in comparisons between test and reference genomes. we designed probes starting from a genomic assembly of the white spruce gene space which was developed from sequence-capture data and represented ~ distinct genes in the white spruce gene catalogue. first, a set of million sense and antisense probes tilled along the gene space assembly was designed by genotypic inc. and an array was produced with the entire set of million probes. second, preliminary comparative hybridizations were used to select a reduced set of genes meeting probe coverage and sequence length criteria. a final set ~ effective probes was used to design an array targeting gene sequences of 500 bp or more with a minimum of six probes evenly distributed along the gene sequence; the maximum number of probes reached for a single  bp gene sequence.fig. overview of the experimental approach deployed to investigate the occurrence of cnvs within spruce pedigrees. a) full-sibs are produced between two mature individuals by means of controlled cross-pollination in nursery. b) the ‘female’ parent genomic dna is employed as the reference genome while the genomic dna of descendants are used as test genomes. c) distribution of probes number per gene. d) distribution of probes and corrected intensity ratios along one gene sequence in cnv when comparing one descendant to the # parent the 180 k probe acgh design was effective in discovering inherited cnvs in four different spruce families by comparing the dna of full-sib individuals to one parent. here, two families were from white spruce and the two other families were from black spruce and interior spruce, respectively; we analysed individuals per family in a total of test-reference hybridizations. a log ratio of hybridization fluorescence intensities was calculated for each probe following the test-reference comparisons by using r package and in-house python programs taking into account the dye and gc gene content biases. the vast majority of probes gave a log intensity ratio close to as expected when the reference and test genomes have the same number of copies. this expected number at a given locus is two since spruces are diploid but a higher numbers of copies cannot be dismissed. over all comparisons, the number of positive and negative ratios were very similar.fig. hybridizations and cnv detection results. a) distribution of the log ratios for all probes over all families. b) distributions of genes found in cnv among spruce families several measures were taken to develop a reliable approach to identify genes displaying cnvs. the experimental design involved repeatedly assessing intensity ratios for a given gene by using several individuals from a pedigree and probes per gene in average. self-self hybridizations in which a single genome was hybridized in duplicate on the same array but with different dyes were used in addition to the test-reference comparisons. self-self hybridizations were carried out in all three species and served to define robust cnv detection parameters, i.e., two major criteria for cnv detection, which resulted in a false discovery rate < < 1% in all comparisons over all species. first, an individual probe criterion was defined as: the absolute value of the log ratio must be superior to | or |log2|). second, a gene level criterion was defined based on cross-probe repeatability such that a minimum of 83% of probes per gene must meet the log ratio criterion which translates in an average of significant probes per gene declared as representing a cnv. inherited cnvs were found for many different genes but with low frequencies we detected cnvs affecting a total of distinct genes when considering all comparisons and species. as the dna was extracted from a sample comprising several needles, hence large number of cells, the cnvs most likely affected the entire tested organism and were mostly representative of a variation inherited from the previous generation. cnvs with an absolute log ratio superior to made up only % to % of the gene cnvs and represented the upper limit above which large copy number differences are undistinguishable from presence/absence variations given the maximum intensities that can be reached using acgh. the number of individuals scored positively for any given gene cnv ranged from to per family. this led us to classify gene cnvs as common and infrequent, the latter accounted for 64% of gene cnvs on average. the average number of gene cnvs detected within an individual was over all families. two individuals in each of the white spruce families and one individual in the black spruce family accounted for nearly half of the infrequent gene cnvs in each family and represented outliers within those families.table numbers of gene cnvs within each family and percentages of frequent, infrequent and presence/absence variations familya afamilies are named according to the main text fig. numbers of gene cnvs within each individual for the four pedigrees. despite some variation, the hybrid descendants presented fewer cnv genes than the pure species descendants cnvs were shared among species and less abundant in hybrid the number of cnvs detected represented on average 10% of the tested genes but varied between families, i.e., to in white spruce, in black spruce and only in interior spruce. the reduced number of gene cnvs in the hybrid was found both at the individual level and the family level compared to the other spruce families. this difference was statistically tested and the number of gene cnvs was significantly lower in the interior spruce family than in white spruce and black spruce families while the difference between the three latter families was not significant. we examined the nature of the differences between species by considering the overlap in the gene cnv sets between the pedigrees. the different families shared from to gene cnvs, including a core set of gene cnvs found in all spruces. the number of shared gene cnvs among families was in accordance with the relative divergence between the spruce species. the two white spruce families had the most shared gene cnvs, white spruce and the interior spruce families had fewer and the black spruce family shared the smallest numbers of gene cnvs with other pedigrees. gene cnvs are enriched for adaptation-related functions and narrow expression profiles the potential biological implications of gene cnvs were evaluated using enrichment tests for terms in gene ontology annotations. in white spruce, the comparison of gene cnvs to the entire set of tested genes on the cgh array revealed a significant enrichment in a number of functions related to biological processes most significantly including the ‘response to stress’ term. using the recently updated pfam annotations of the white spruce gene catalog, enrichment was found for oxygenase, synthetase, hydrolase and cytochrome-p activities. in black spruce, go-term enrichment was found in eight biological processes including most significantly ‘response to stress’ and ‘defense response’.table enrichment test in go annotations for white and black spruce species, results from fatigo in babelomics picea glauca picea mariana the different numbers of gene cnvs detected between species did not translate into functional annotation differences. the core set of gene cnvs found in all of the families is significantly enriched in functions related to defense response. the hybrid family sharing gene cnvs with the white spruce families may be expected by inheritance of genetic material from the white spruce species but did not explain gene cnvs also shared with black spruce. hence, such cnvs sharing between all families is more likely indicative of ancestral genetic variations that remain variable in several north-american picea species. in addition to defense responses-related functions, all spruce species and the hybrid were enriched in the stress responses while annotations of the genes specifically found in black spruce were not significantly enriched in any biological process compared to white spruce specific genes. on the other hand, the genes specifically found in the hybrid were not enriched in functions related to defense nor stress responses but enriched in functions related to responses to hormonal, organic and chemical substances. expression levels for % of the gene cnvs were previously assessed in various organs at different stages of development in white spruce for a total of eight tissues. gene cnvs appeared expressed in wide range of tissues and organs, including vegetative buds, foliage, primary and secondary xylem, phelloderm, roots, megagametophyte and embryogenic cells with no significant enrichment for any of them. however, the set of gene cnvs is significantly enriched in genes expressed in only one or two tissues, with no or little variation between tissues. cnvs were distributed across the entire genome a dense genetic map has been continuously developed since for white spruce, which has allowed to locate the position of genes, observe a high synteny between spruce species and dissect the genomic architecture for several traits of adaptive and economic interest in spruce species. a total of of the cnv genes were located on this spruce genetic map. the cnvs were distributed over the entire genome; each linkage group included from to gene cnvs and had an average of cnvs. the average distances between two mapped gene cnvs were,, and  cm for the ws ws bs and is families respectively, and was inversely proportional to the number of mapped gene cnvs in each family. no significant clustering of gene cnvs was observed within linkage groups although a total of cases of two or more genes within a 1 cm region were found across the three spruce species.fig. distribution over the genome and frequency of genes found in cnv also positioned upon the spruce genetic map. each point represents the position of a gene found in cnv in one cgh comparison; the piling is indicative of the number of individuals presenting the cnv discussion we analysed thousands of gene cnvs and found that they are less abundant in a hybrid compared to two pure spruce species. the biological functions of the gene cnvs indicate a role in stress and defense responses. we discuss the development of cnv detection in wild species and the insights gained from our findings into genome biology, evolution and adaptation. detecting gene cnvs within pedigrees we developed gene-based cnv detection procedures to analyse wild species, even those with large genomes. a low threshold was used for cnv detection compared to other recent studies. this apparently low value was permitted because of an array design that included a relatively large number of probes which presented such significant signal for the same gene. previous reports have recommended a minimum of three probes per genomic segment in order to effectively estimate the relative number of copies when using oligonucleotide cgh. when analysing genes, some authors set a requirement that all of the three to four probes tested be significant for positive scoring of cnvs. we used many more probes per gene sequence which provided a strong support for the estimated ratios and robust detection parameters with very low fdr. in these conditions, the number of cnvs detected was inflated substantially when we lowered the proportion of significant probes and was only slightly affected when we lowered the significance threshold for intensity ratios. we did not detect a bias towards negative or positive log ratios in the overall data set nor when comparing individual offspring to a parental reference genome. this result appeared to be at odds with those from other organisms showing that losses often dominate over gains; however, the detection of more losses than gains could be due to a reference genome bias, at least in some cases. this type of bias is not expected in our experiment as we used parent-offspring comparisons which reduced sequence divergences between the test and reference genomes, both being similarly distant from the genome used for probe design. it has also been argued that a higher number of positive cnvs may be expected because genomes are likely more tolerant to duplications than deletion possibly resulting in loss of function. our approach, in which we sampled multiple offspring, was designed to uncover and identify inherited pre-existing gene cnvs shared across a pedigree rather than de novo cnvs arising from recent structural variations. however, many of the cnvs in spruces were found in only one individual within a pedigree, as observed in other plants. these infrequent cnvs may represent three different cases: i) random effects due to the small sample size, which is supported by the detection of these same cnvs in at least one other family; ii) false-positives, despite the very small number of cnvs in self-self hybridizations; iii) de novo structural variations which occurred in or during reproduction of the previous generation. in the subset of cnvs found only once over the entire dataset the proportion of negative and positive log ratios was balanced, suggesting that there was no prevalence of de novo copy gains or losses. impact and distribution of cnvs across the genome the proportion of genes positively scored for cnvs was % in white spruce which is similar or slightly higher in magnitude than found in whole genome analyses in model species and crop plants;;;;, ). the high proportion found in spruce can be either attributed to the detection of relatively short cnv regions enabled by the high probe density in our array design, or the occurrence of more than one gene located within the same long dna sequence in cnv. in that case, some cnvs may represent only one and the same dna segment that has been duplicated or lost. this could not be verified for most of the cnvs as their genomic positions were unknown. hence, the rate of cnvs may not precisely reflect the rate of actual structural variations but rather provided an estimate of the order of magnitude. in total, 9% of the genes surveyed using acgh and 6% of the genes found in cnv had previously been positioned on a genetic linkage map in picea glauca. this lower proportion of mapped cnvs than expected by chance alone is not surprising because the map was mainly developed using snp from large high-throughput genotyping arrays which likely selected against genes that display cnvs. localizations on the genetic map showed that cnv genes were widely distributed at a low density over the entire genome with a minimum of cnv genes per linkage group and average distance of 27 cm between successive cnvs within species. in addition, cases of or cnv genes located within 1-cm of each other suggested the occurrence of structural variations involving chromosomal segments with more than one gene. even though the number of mapped cnvs was too small to precisely assess the frequency of such events, it would represent an estimated rate of svs close to %. multi-gene svs have already been described in soybean and a rate of 5–10% was reported in maize/teosinte by using acgh. in light of these reports, more svs affecting more than one gene in spruce could be expected. however, conifer genes are often separated by very long non-coding regions such that multi-gene svs may be less common in this phylum. furthermore, the discovery of cnvs in close proximity to each other raises the question of their origin. the first hypothesis may be that these genes are among the few genes actually separated by relatively small non-coding sequences. another explanation would be that these genes are widely separated but structural variations that are typically even wider in conifers, hence allowing to encompass several coding sequences. finally, one possible explanation is that these genes may otherwise originate from different events of sv within the same genomic region; such hotspots for structural variations have been found in other model organisms. further characterization of these genetically linked cnvs genes and a finer dissection of structural variations could be achieved by analysing larger populations, hence allowing to directly map these genetic variations and infer the occurrence of one or more events of structural variation. numbers of shared cnvs agrees with the estimated time of species divergence the proportion of shared gene cnvs across the different picea species was in accordance with their phylogenetic distance, i.e., more gene cnvs were shared among more closely related families and species. the highest overlap was found between the two white spruce families, suggesting that the shared gene cnvs are not limited to one parental lineage but more likely to be spread at a significant level in the population. the second highest overlaps were between interior spruce and white spruce families despite the lower number of gene cnvs in the hybrid. engelmann and white spruces, the parental species of the hybrid interior spruce, diverged only myears ago, a relatively short timeframe compared to the myears separating the white/engelmann spruce lineage from the black spruce lineage. in line with their relative times of divergence, engelmann and white spruce can hybridize while black spruce is reproductively isolated from both of the other species and also shared the smallest number of cnvs with the other spruce families. evolutionary forces have also been linked to cnv diversity in other plants. in a study comparing domesticated and wild barely accessions for instance, it was shown that strong directional selection associated with breeding in barley reduced cnv diversity. hence, cnv diversity in barley mirrored the behaviour of other dna polymorphisms indicating that evolutionary forces likely have similar impacts on cnvs than on other types of dna polymorphism. similarly, cnv diversity assessed using a human acgh allowed to detect gene cnvs shared among primates that likely resulted from gene duplication in common ancestors. therefore, gene cnvs shared between the different spruce species likely represent ancestral polymorphisms that have not been eliminated by genetic drift or directional selection during the course of evolution, or alternatively, have been maintained by balancing or divergent selection. role of cnvs in the evolution of forest trees a wide range of gene cnvs have been linked to quantitative phenotypic variation in model organisms. in forest trees, quantitative genetic variations have been linked to adaptation and is characteristic of key adaptive traits such as growth phenology and cold tolerance. genetic variation in adaptive traits has been studied in many conifers including picea spp. and was often observed to follow gradual changes over wide ranging geographic areas and environmental conditions as opposed to drastic changes linked to specific conditions. here we examine the potential impact of cnvs on adaptation in trees and predict that they make a qualitatively distinct contribution compared to variations in coding sequences resulting in amino acid substitutions. the two types of mutation have different impacts at the molecular level and are expected to have different evolutionary consequences. changes in amino acid sequences may alter protein function and therefore are more often likely to be deleterious and purged, or to be recessive and only impact phenotype in homozygotes. cnvs alter gene dosage and may affect expression which is akin to cis- acting regulatory variation. as such cnvs are more likely to alter phenotype in heterozygotes and thus be more directly visible to selection. gene dosage effects caused by cnvs may also be detrimental, for example by creating an imbalance in multi-protein complexes, but this is likely to be restricted to certain classes of genes and be more prevalent in house-keeping functions. the relative effects of cnvs and amino acid changes will also depend on the function and expression of the genes product. here, cnv genes were enriched for biological functions related to stress response, defense response and metabolic processes, which was also observed in barley and maize and is in line with a role in adaptation. the cnv genes were also enriched for narrow transcript accumulation profiles in white spruce, suggesting more specialized functions rather than house-keeping roles which reinforces the potential for cnvs to contribute to adaptive phenotypes. considering these observations, cnvs affecting genes have the potential to make a unique contribution to adaptation in trees as a result of their qualitatively distinct impact on evolution and the types of genes affected. we further suggest that cnvs could be especially important for short term adaptability and thus in responding to the acceleration of environmental change. fewer cnvs in the hybrid species significantly fewer cnv genes were found for the interior spruce family, a hybrid resulting from the natural introgression of white and engelmann spruce. we examined whether the difference could be due to technical reasons such as is producing fewer successful hybridizations as the probe set was designed with pure white spruce sequences. however, the data indicated that the numbers of successfully tested genes were nearly identical in all three species including the more distantly related black spruce. this is consistent with the expectation that the procedure we used was able to tolerate up to three mismatches per probe and the average degree of polymorphism found among sequences of white, black and engelmann spruces. the occurrence of cnvs is underpinned by mechanisms that are likely to be conserved across all living organisms and are predominantly associated with low-copy-repeats, such as unequal interchromosomal recombinations, among others. lcrs are conserved dna segments that occur a few times throughout the genome. interior spruce has a diploid genome that originally came from contributions of white spruce and engelmann spruce. as a result, a higher level of heterozygosity is observed in the individuals of interior spruce compared to individuals of pure species. such dual origin is expected to have introduced a higher level of diversity in lcrs found in hybrid than that found in individuals of pure species. hence, a lower sequence identity between lcr sequences of homologous chromosomes is expected in the hybrid genome which would have entangled the mechanisms leading to gene cnvs. as a consequence, the interior spruce individuals has less numerous cnvs than white spruce or black spruce individuals; these cnvs may result from other types of breakage-reparation mechanisms that are not based upon sequence identity. these results suggest that hybrids have systemically reduced copy number variability indicative of a less dynamic genome which in turn would limit genome evolvability. future studies investigating other cases of introgression, either from the wild or from artificial cross-breeding, would support or dismiss this hypothesis.  investigations of non-model species have broadened the scope of genomic studies and contribute to the understanding of ecological diversity. more affordable sequencing and genotyping techniques as well as the advent of next-generation sequencing have paved the way to these developments. this report illustrates how techniques of transcriptomics and gene-space characterization in a non-model system can in turn be used to design cgh arrays and study cnvs on a genome scale. approaches using whole genome sequencing data from model organisms to detect usually small cnvs have been developed; however different detection methods and algorithms have produced different results from the same datasets. more recently, continuous development of technologies to sequence and characterize larger dna segments have allowed to investigate larger structural variations in model organisms using third generation pacbio sequencing or genome mapping on nanochannel arrays. these technologies can be applied to non-model organisms, although the 20-gb conifer genomes would still remain challenging. an advantage of the approach outlined here was the very low fdr which indicated robust results. although it is time and resource consuming, testing several offspring from the same parents was also helpful as it confirmed the discovery of cnvs by repeated identification in related individuals. this approach also throws light onto inherited gene cnvs which are of interest for investigating the role of cnvs in adaptation and evolution as well as establishing a framework to study their relation to phenotype, e.g., through qtl mapping. as gene cnvs could result in dosage effects on phenotypic variation, they may play a key role in quantitative trait variations such as growth and phenology in plants. here, we reported a lower number of cnvs within a hybrid pedigree than in families from pure species, suggesting that hybrids may systemically have a lower diversity in cnvs. considering the gene annotations related to stress and defense responses, this reduced genetic diversity may act on phenotypic variability and evolution of hybrids in response to natural selection. future investigations to assess the impact of cnvs on the evolution of species could focus on cnvs at the population level, looking for signatures of selection across geographical ranges and varying environmental conditions. applied to a variety of species, such analyses may reveal new insights into adaptation and speciation. 
gene cnvs were assessed in three full-sib families from species with gb genomes, i.e., white and black spruce, and interior spruce - a natural hybrid. we propose that cnvs make a qualitatively distinct contribution to adaptation which could be important for short term change. the hybrid family presented significantly fewer cnvs, suggesting that the admixture of two species within one genome reduces the occurrence of cnvs. a genome-scale understanding of cnvs is available in a few plants but none are wild species, leaving a knowledge gap regarding their genome biology and evolutionary role. we developed a reliable cnv detection method for species lacking contiguous reference genome. the approach we developed is of particular interest in non-model species lacking a reference genome. we discovered hundreds of gene cnvs in each species, in total, which were enriched in functions related to stress and defense responses and narrow expression profiles, indicating a potential role in adaptation. copy number variationcomparative genomic hybridizationnon-model speciesgenomic structural variationgenome architecturespecies hybridizationpicea glaucapicea marianapicea engelmanniconifersissue-copyright-statement© the author 2017
 copy number variations have been linked to different phenotypes in human, including many diseases. a genome-scale understanding of cnvs is available in a few plants but none are wild species, leaving a knowledge gap regarding their genome biology and evolutionary role. we developed a reliable cnv detection method for species lacking contiguous reference genome. we selected multiple probes within gene sequences and developed comparative genome hybridization on arrays. gene cnvs were assessed in three full-sib families from species with gb genomes, i.e., white and black spruce, and interior spruce - a natural hybrid.  we discovered hundreds of gene cnvs in each species, in total, which were enriched in functions related to stress and defense responses and narrow expression profiles, indicating a potential role in adaptation. the number of shared cnvs was in accordance with the degree of relatedness between individuals and species. the genetically mapped subset of these genes showed a wide distribution across the genome, implying numerous structural variations. the hybrid family presented significantly fewer cnvs, suggesting that the admixture of two species within one genome reduces the occurrence of cnvs.  the approach we developed is of particular interest in non-model species lacking a reference genome. our findings point to a role for cnvs in adaptation. their reduced abundance in the hybrid may limit genetic variability and evolvability of hybrids. we propose that cnvs make a qualitatively distinct contribution to adaptation which could be important for short term change. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords copy number variationcomparative genomic hybridizationnon-model speciesgenomic structural variationgenome architecturespecies hybridizationpicea glaucapicea marianapicea engelmanniconifersissue-copyright-statement© the author 2017 