BACKGROUND
changes in the copy number of dna sequences have been identified as one of the main mechanisms producing genetic variability in eukaryotes  <cit> .
these changes may include the duplication of genomes or chromosomes, duplication of genome segments including genes, and even small duplications both in intergenic and in coding regions  <cit> .
examples of phenotypic effects due to this type of genome variation are increasingly found both in plant species and in animal systems, including humans, where a number of genetic diseases have been linked to copy number variation of specific genes  <cit> .
the availability of genome sequences of an increasing number of individuals and species, or specific populations within them, has multiplied the examples of this feature of genomic variability.
genome sequences are being obtained at an increased rate with the systematic application of massive sequencing techniques.
in particular, the genome sequences of different plant species are being published.
these include species used in genetic or molecular analysis, such as arabidopsis thaliana and brachypodium dystachion, of interest for their evolutionary relation with other plants, or crop plants of interest in agriculture.
in this respect one of the questions that can be addressed at this moment through resequencing of the appropriate varieties is the genomic variability of plant species that occurs at both interspecific and intraspecific levels.
these data may help to understand how plant species adapt to changing environments including domestication.
cucurbits are interesting systems to study changes in genome structure for a number of reasons.
they form a family of species that has been the object of genetic analysis for a long time, they occupy a particular place in the phylogenetic classification of plant species, and they are an important crop worldwide and a component of the diet of many european and asiatic countries since historical times.
they have relatively compact genomes  and do not seem to have undergone whole genome duplications besides the ancestral one in the origin of flowering plants  <cit> .
it has also been shown that they have a surprisingly low number of genes coding for proteins with similarity to pathogen resistance-related genes  <cit> .
the genome sequences of three important cucurbit crops, cucumber, melon, and watermelon, have already been published  <cit> .
the analysis of the high intra-specific genetic variation and morphologic diversity of cucumis melo has led to tentatively classify the subspecific variability of this species into several cultivar groups that can be further broadly grouped into two subspecies, melo and agrestis <cit> .
the published melon reference genome belongs to the double-haploid line dhl <dig>  derived from a cross between two phylogenetically distant melon cultivars: pi  <dig> , cultivar group conomon, subspecies agrestis, and the t <dig>  line, cultivar group inodorus, of the subspecies melo <cit> .
resequencing data from both dhl <dig> parentals as well as from the reference line itself have already been produced using illumina technology  <cit> .
here, results are presented on genome variability of melon after resequencing two additional melon accessions corresponding to a cultivated and an old-relative melon variety.
the variation in copy number of different genes has been calculated and it appears there are hotspots of variability in a number of clusters corresponding to genes related to pathogen resistance, indicating species adaptation to changing biotic environments through a genomic strategy.
RESULTS
genome resequencing of two cucumis melo varieties
to study gene content variations that could be primarily responsible for at least part of the wide phenotypic diversity of melon, two distant melon varieties were chosen for further genome sequencing.
one of these cultivars was c- <dig>  an accession from cabo verde previously classified as c. melo ssp.
agrestis based on the character of the pubescence type of the female flower hypantium  <cit> .
this accession has tiny seeds and very small round fruits  with smooth, dark green, black-spotted skin, and non-aromatic flesh .
the second variety was c- <dig>  a cultivar of the subspecies melo whose seeds had been collected from a local market in rawa .
the fruits of this accession have yellow-orange, smooth skin, very aromatic flesh, and are of medium size  .
the adscription of c- <dig> and c- <dig> to the agrestis and melo groups, respectively, was confirmed building a phylogenetic tree to position those cultivars relative to five other melon varieties .
genomic dna from young leaves of both cultivars was used to produce two paired-end libraries of average insert size ca.
500 bp, subsequently sequenced using the illumina genome analyser iix technology.
around  <dig> million pairs of cleaned, high-quality reads of maximum length 152 bp were obtained for each cultivar .
similar amounts of illumina reads previously obtained from dhl <dig> and its two parental lines  <cit>  were also included in the analysis .
in all, between  <dig>  gb  and  <dig>  gb  of total sequence length were produced, representing ×13-× <dig> genomic coverage, assuming a melon genome size of 454 mb  <cit> .
these paired-end data sets were then aligned to the melon reference genome to produce bam files as described in the methods section.
a
b
araw, unfiltered data.
b75% of the total length is contained in all reads ≥ n <dig>
detection and functional characterization of pav genes
in order to detect genes putatively absent from a genomic region in one cultivar, compared to the corresponding region in the reference genome , the genomic coordinates of all  <dig>  melon annotated genes were used to search the alignment files for regions of very low coverage in at least one of the four melon cultivars analyzed .
the mapping patterns at the candidate loci were visually inspected to produce a final list of  <dig>  putative pav genes .
this list can be found in additional file 2: table s <dig>  together with the information on gene absence distribution for each melon cultivar and gene functional annotations.
compared to the reference genome, a total of  <dig> genes  were absent in only one cultivar, while  <dig> genes  were absent in two cultivars,  <dig>  in three cultivars, and only  <dig> genes were missing in more than three cultivars.
in all,  <dig>  pav events were detected, distributed in all cultivars analyzed, including  <dig> in dhl <dig>  which probably represents mapping artifacts as no pav would be expected when mapping dhl <dig> reads against the dhl <dig> reference genome itself.
only two genes were absent from dhl <dig> but present in both parental genomes, which clearly indicates false positives for the detection of pavs.
in addition, not all melon varieties had the same content of pav genes.
in cultivar c- <dig> there were  <dig> pav events , while c- <dig>  t <dig> and pi  <dig> had, respectively,  <dig> ,  <dig> , and  <dig>  events.
however, it must be taken into account that both t <dig> and pi  <dig> are the parental lines of dhl <dig> and so it is expected that the number of pav events would be significantly lower than those found when comparing two not so closely related melon cultivars, as each one contributes to around 50% of the reference genome.
in all, the average number of absent genes per cultivar was  <dig>  and, therefore, ca.
<dig> %  of all genes were absent, on average, in the analyzed cultivars as compared to dhl <dig>  these figures are comparable with values published for arabidopsis and rice  <cit> .
the functional annotation of the candidate pav genes was obtained from the melonomics webpage  <cit> .
also, a blast2go analysis was performed to gather further information on genes with unknown function.
additionally, functions were tentatively assigned to ten of these genes using phylogeny-based information from the melon phylome  <cit> , as for the general annotation of the genome  <cit> .
using this combined approach, and excluding all genes with only generic information available, such as 'ankyrin repeat-containing', 'coiled-coil', 'oxidoreductase activity' and 'catalytic activity', functional information was finally assigned to  <dig> pav genes  .
however, upon analyzing the functional annotations, it was found that  <dig> of these genes were annotated as encoding transposon-related sequences.
the melon reference genome has been annotated after masking mobile elements  <cit> .
the inspection of these  <dig> sequences showed that  <dig> were true transposons that somehow had not been masked during the sequencing assembly; the rest did show homology with proteins in our transposon databases but with e-values lower than 1e- <dig>  these sequences probably represent degenerate forms of old mobile elements, transposon fragments that remain in the genome after an excision event, or they are genes that have some degree of similarity to transposon sequences.
the  <dig> putative pav genes with functional annotations other than transposon-related sequences were classified according to their ontology .
more than 80% of the annotated genes could be grouped into nine categories: amino-acid, carbohydrate and lipid metabolism, biotic or abiotic stress response, terpenoid biosynthesis, transcription/translation processes, transport, and two groups containing generic p <dig> and protein/dna- binding proteins that could not be further classified due to lack of more specific information.
interestingly, the most populated functional category was that of stress response proteins , with a group of  <dig> tir-nbs-lrr/nb-arc disease-resistance proteins by far the largest subclass of pav genes having an associated function.
it is known that structural genome variants in plants are prone to affect stress response genes, particularly those for disease resistance  <cit> .
these genes have been found to be underrepresented in the genomes of cucurbits when compared to those of other plant species.
previous studies have shown that, although whole-genome duplication events seem to be absent from the lineages leading to c. melo, several gene families have expanded specifically in the cucumis genus compared with other cucurbits, or in c. melo when compared with cucumis sativus l.   <cit> .
the list of expanded families includes defense response, lipid metabolism, cell wall structure, translation and transport gene families which, as we report above, are also enriched in pav genes.
crossing the lists of pav and expanded families to search for coincidences, a total of  <dig> % of genes in families that have expanded specifically in the cucumis genus and  <dig> % in c. melo were found to be pav.
these values are  <dig> - <dig>  times higher than the abundance of pav genes,  <dig> %, in the melon gene complement.
presence of pav genes in multi-copy gene families and gene clusters
it has been previously suggested that with genes belonging to multi-copy gene families, and especially those arranged in clusters, there is a greater chance of presence/absence polymorphism, probably due to unequal crossover between homologous genes.
this seems to be particularly the case for disease resistance genes: there is evidence that clusters of these genes may well be hotspots for the generation of pav  <cit> .
in melon, previous studies have shown that resistance genes are frequently found in clusters in the genome; most notably, half of the  <dig> predicted melon nbs-lrr genes groups within nine clusters  <cit> .
a region spanning 760 kb of linkage group v  and containing the vat gene locus is of particular interest.
it contains  <dig> nbs-lrr resistance genes, the highest concentration of r genes found in the melon genome.
a microsynteny comparison of part of this region and its homolog in the cucumber genome has already shown the existence of pav polymorphisms affecting several r genes  <cit> .
the presence of pav genes in multi-copy gene families and gene clusters was further investigated to see whether presence/absence variants were associated to these families with higher frequency than randomly expected.
to this end, the melon gene complement was first grouped in families of homologous genes as explained in the methods section.
as a result,  <dig> % of all genes were classified as belonging to single-member families or singletons, while  <dig>  genes were members of  <dig>  multi-copy gene families.
by comparing the pav and multi-copy gene lists, it was found that  <dig> pav genes  can be classified as multi-copy while  <dig>  non-pav genes  belong to multi-copy families .
therefore, there was a significantly higher proportion  of multi-copy genes in the pav subset of the melon gene complement.
as only  <dig> % of singleton genes were classified as pav compared to  <dig> % pavs in the multi-copy fraction, genes present in the genome in more than one copy are five times more likely to be affected by pav than singleton genes.
most of the multi-copy gene families contain only two  or three  genes.
these small families represent 41% of all multi-copy genes but only 29% of all multi-copy pav genes.
on the other hand,  <dig> families of 7– <dig> gene members contain 15% of all multi-copy genes but 22% of all multi-copy pav genes.
therefore, pav genes appear to have a slightly higher tendency to be present in more populated gene families in the melon genome.
to see whether the physical location of analogous genes could also be related to the occurrence of presence/absence polymorphisms, the presence of pav genes in clusters of genes with a significant degree of similarity was investigated.
by classifying multi-copy genes in clusters , a total of  <dig>  genes, of which  <dig> were pavs, grouped in  <dig> clusters .
therefore, the proportion of pav genes clustered in the melon genome  is significantly higher than the  <dig> % of non-pav clustered genes .
also, as only 4% of non-clustered genes are pav compared to the 8% of clustered genes, it seems clear that the chance of genes present in the genome in tandem arrays being affected by pav is double that of isolated genes.
of all  <dig> clusters, 77% are just tandems of two genes, and the longest cluster found had  <dig> genes.
significantly, 14% of all clustered genes, but 28% of all pavs in tandem, were found in the  <dig> clusters of five or more genes, indicating that pavs tend to be present in longer clusters.
validation of putative pav genes
the prediction of pav genes has been based solely on the analysis of the alignment files, with no specific experimental support.
since many pav genes belong to medium-large multigene families, there is more than a negligible possibility of mapping artifacts mistakenly taken for putative pavs.
therefore, it was decided to determine which fraction of putative pav genes was truly absent from the analyzed cultivars.
the validation of pavs was mostly carried out in a  <dig>  mb region that spans the 760 kb region described above, due to its high level of pav polymorphisms and multi-copy gene content .
since all reads were mapped against the dhl <dig> genome, it was important to confirm that no chimeras had been produced during the assembly of this region on the reference genome.
the sequence of a 250 kb fragment with the melo3c004258- <dig> genes, previously obtained from three overlapping bacs, was used to validate the corresponding sequence in the assembled reference genome  <cit> .
to validate the melo3c004290- <dig> gene fragment, a tiling path of  <dig> bac clones spanning 713 kb and comprising the melon genes melo3c004287-melo3c <dig> was obtained as described in the methods section .
the bac lengths were estimated by averaging 3– <dig> pulsed-field electrophoretic runs for each clone .
the estimated lengths of eight clones differed by 10 kb on average  when compared with the expected values obtained by mapping the bac-end sequences to the reference genome assembly.
this result is acceptable bearing in mind both the expected error in the bac length estimations based on pulsed-field electrophoresis and, more importantly, the abundance of stretches of ns on the reference genome.
their sizes had been adjusted on the basis of pair-end mapping information during the assembly process and, therefore, can be expected to differ to some extent from the actual lengths of the sequences they represent.
in particular, two clones spanning the melo3c04318- <dig> region differed from their expected lengths by only 7 kb, which indicates that the 150 kb and 90 kb fragments absent in that region in the two agrestis cultivars  are most probably present in the reference dhl <dig> sequence.
two overlapping clones spanning the melo3c04287- <dig> genes were found to be 50–60 kb larger than their expected sizes.
as both clones share 36 kb of sequence, this suggests that about 50 kb are missing from the reference genome sequence somewhere in the shared region, which has only one annotated gene .
in all, the existence of a contig of bacs whose sequence ends map adequately to the reference genome validates the overall sequence structure of, at least, the melo3c04295- <dig> region from dhl <dig>  although minor artifacts cannot be discarded.
in a complementary approach, individual putative pav regions were confirmed by pcr using primers flanking the candidate regions and genomic dna from the studied varieties, and bac or genomic dna from the reference genome.
a total of  <dig> genes were analyzed,  <dig> belonging to the  <dig>  mb region of linkage group v. the results confirmed  <dig>  of the putative pav genes while six genes that had been considered partially absent were found in the respective cultivars .
a
b
c
a
d
acoordinates on the scaffold/contig that contains the gene.
bcv: c- <dig>  iq: c- <dig>  pi: pi  <dig>  ps: t <dig>
cdhl <dig> sequence fragment that contains the gene and is absent relative to the reference sequence.
dc: pav confirmed; e: exon deletion; i: intron deletion; si: sequence insertion relative to the reference.
sequencing of the amplified pcr bands showed that the absence of a genomic fragment was often accompanied by the presence of sequences that were absent from the reference genome, from just a few to several thousand nucleotides.
in two instances these new sequences were found to show homology with transposons, indicating the role that mobile elements may play in the generation of pav polymorphisms  <cit> .
analysis of pavs in a 1 mb region of linkage group v
as mentioned above, the greatest density of resistance genes in the melon genome was found in a 760 kb fragment on linkage group v. if a cluster of lipoxygenase genes and one phytochelatin gene in the immediate vicinity of this fragment are also considered, the resulting  <dig>  mb region contains  <dig> annotated genes  of which  <dig>  are most probably involved in different aspects of biotic or abiotic stress responses .
as well as  <dig> nbs-lrr-tir and seven cc-nbs-lrr genes having homology with resistance genes, there are nine lipoxygenase genes, which may have roles in pest resistance and response to wounding, and one phytochelatin gene, that might be involved in resistance to metal poisoning  <cit> .
these resistance genes are grouped in several clusters and 47% of them show pav, not only in the analyzed melon lines but also when compared with the syntenic regions in two other cucurbits with sequenced genomes: cucumber, where only eight tir-nbs-lrr, one cc-nbs-lrr, and no phytochelatins are found, and watermelon, with only five tir-nbs-lrr, one cc-nbs-lrr, and no phytochelatins .
the highest concentration of pav events detected in the melon genome was found in this region, with nearly 30% of all the genes partially or totally absent from at least one of the analyzed melon cultivars.
in the vicinity of these genes, besides  <dig> pav in stress response genes, there are also pav genes in a cluster of gdsl lipases and a cluster of terpene cyclases, and eight additional pav genes of unknown function.
this region contains the longest continuous gene-containing pav sequence found in melon: a fragment of 150 kb containing the genes melo3c04318- <dig> and completely absent from the c- <dig> cultivar, while a 90 kb fragment containing the genes 4319– <dig> was absent from pi  <dig>  the agrestis parental of dhl <dig>  and another fragment of 86 kb containing five resistance genes was absent from c- <dig> .
annotation of the reference melon genome has shown that the number of putative disease resistance genes of the nbs-lrr family in melon and cucumber is lower than in other plant species  <cit> .
although the expansion of the lipoxygenase gene family has been suggested as a mechanism to compensate for this reduction in cucumber  <cit> , no such expansion has been observed in melon.
however, our results show that a high degree of pav polymorphism affects several families of disease resistance genes.
it must also be taken into account that only absent genes relative to the dhl <dig> reference genome could be detected using our approach, so additional pav events may be found if the resequencing reads are mapped against the genome of other cultivars.
the 'pan-genome' concept has already been proposed to reflect the fact that single genome sequences do not seem to account for the full set of genes in plant species  <cit> .
it is then possible that the full set of active melon r-genes cannot be obtained by analyzing the annotated genes from just one cultivar.
in fact, different combinations of present and absent r-genes in a particular cultivar  might well confer various levels of resistance to different pathogens or strains.
as an example, the vat gen, an economically important gene conferring resistance to aphid colonization, has been cloned in pi  <dig> and shown to be a cc-nbs-lrr gene syntenic to those found on the cluster of seven cc-nbs-lrr genes in the  <dig>  mb region of dhl <dig>  <cit> .
two alleles were found at this locus with different resistance specificities.
however, it has been reported that the cloning of candidates in other melon accessions is particularly difficult, probably due to the existence of several sources of variability, including the variable complexity of the cluster of resistance genes depending on the genotype  <cit> .
related results have been found in other plant species.
for example, nematode resistance mediated by the soybean rhg <dig> qtl has been found to be conferred by copy number variation: resistant plants contain ten tandem copies of a 31 kb segment that spans three dissimilar genes, all of which contribute to the resistance  <cit> .
CONCLUSIONS
this article presents the quantification of pavs in the genome of melon by comparing the published reference genome to four additional melon cultivars.
we found that multi-copy melon gene families are particularly affected by pav polymorphism.
this is in agreement with results in other species and it may respond to the need for multiple copies of a gene for new functions required for the evolution of the species.
on further analyses, gene clusters appeared as hotspots of pavs in the melon genome, with significant clustering in cases of biotic stress response-related genes.
in particular, melon has two large clusters of sequences coding for nbs-lrr proteins where important resistance loci such as fom <dig> or vat map.
the analysis of a  <dig>  mb region containing the vat locus revealed the highest density of pav events as well as the longest continuous gene-containing pav sequence in melon.
cucurbit genomes have the peculiarity that some gene families related to pathogen resistance are smaller than in most other plant species.
disease resistance genes are essential for the survival of the species and, therefore, they have also been the object of high selective pressure during domestication and breeding.
this work reports the finding of high sequence variability involving genome fragments containing pathogen resistance genes in the melon genome.
the variability of these clusters may well be the reason why, in some cases, it has been difficult to identify specific resistance genes in these regions.
the results also show the importance of sequencing different cultivars of a particular cultivated plant species to characterize its genome variability, the understanding of its evolutionary history, and to help define breeding strategies.

 changes in the copy number of dna sequences have been identified as one of the main mechanisms producing genetic variability in eukaryotes. these changes may include the duplication of genomes or chromosomes, duplication of genome segments including genes, and even small duplications both in intergenic and in coding regions. examples of phenotypic effects due to this type of genome variation are increasingly found both in plant species and in animal systems, including humans, where a number of genetic diseases have been linked to copy number variation of specific genes. the availability of genome sequences of an increasing number of individuals and species, or specific populations within them, has multiplied the examples of this feature of genomic variability. genome sequences are being obtained at an increased rate with the systematic application of massive sequencing techniques. in particular, the genome sequences of different plant species are being published. these include species used in genetic or molecular analysis, such as arabidopsis thaliana and brachypodium dystachion, of interest for their evolutionary relation with other plants, or crop plants of interest in agriculture. in this respect one of the questions that can be addressed at this moment through resequencing of the appropriate varieties is the genomic variability of plant species that occurs at both interspecific and intraspecific levels. these data may help to understand how plant species adapt to changing environments including domestication. cucurbits are interesting systems to study changes in genome structure for a number of reasons. they form a family of species that has been the object of genetic analysis for a long time, they occupy a particular place in the phylogenetic classification of plant species, and they are an important crop worldwide and a component of the diet of many european and asiatic countries since historical times. they have relatively compact genomes and do not seem to have undergone whole genome duplications besides the ancestral one in the origin of flowering plants. it has also been shown that they have a surprisingly low number of genes coding for proteins with similarity to pathogen resistance-related genes. the genome sequences of three important cucurbit crops, cucumber, melon, and watermelon, have already been published. the analysis of the high intra-specific genetic variation and morphologic diversity of cucumis melo has led to tentatively classify the subspecific variability of this species into several cultivar groups that can be further broadly grouped into two subspecies, melo and agrestis. the published melon reference genome belongs to the double-haploid line dhl derived from a cross between two phylogenetically distant melon cultivars: pi, cultivar group conomon, subspecies agrestis, and the t line, cultivar group inodorus, of the subspecies melo. resequencing data from both dhl parentals as well as from the reference line itself have already been produced using illumina technology. here, results are presented on genome variability of melon after resequencing two additional melon accessions corresponding to a cultivated and an old-relative melon variety. the variation in copy number of different genes has been calculated and it appears there are hotspots of variability in a number of clusters corresponding to genes related to pathogen resistance, indicating species adaptation to changing biotic environments through a genomic strategy.  genome resequencing of two cucumis melo varieties to study gene content variations that could be primarily responsible for at least part of the wide phenotypic diversity of melon, two distant melon varieties were chosen for further genome sequencing. one of these cultivars was c- an accession from cabo verde previously classified as c. melo ssp. agrestis based on the character of the pubescence type of the female flower hypantium. this accession has tiny seeds and very small round fruits with smooth, dark green, black-spotted skin, and non-aromatic flesh. the second variety was c- a cultivar of the subspecies melo whose seeds had been collected from a local market in rawa. the fruits of this accession have yellow-orange, smooth skin, very aromatic flesh, and are of medium size. the adscription of c- and c- to the agrestis and melo groups, respectively, was confirmed building a phylogenetic tree to position those cultivars relative to five other melon varieties. genomic dna from young leaves of both cultivars was used to produce two paired-end libraries of average insert size ca. 500 bp, subsequently sequenced using the illumina genome analyser iix technology. around million pairs of cleaned, high-quality reads of maximum length 152 bp were obtained for each cultivar. similar amounts of illumina reads previously obtained from dhl and its two parental lines were also included in the analysis. in all, between gb and gb of total sequence length were produced, representing ×13-× genomic coverage, assuming a melon genome size of 454 mb. these paired-end data sets were then aligned to the melon reference genome to produce bam files as described in the methods section. a b araw, unfiltered data. b75% of the total length is contained in all reads ≥ n detection and functional characterization of pav genes in order to detect genes putatively absent from a genomic region in one cultivar, compared to the corresponding region in the reference genome, the genomic coordinates of all melon annotated genes were used to search the alignment files for regions of very low coverage in at least one of the four melon cultivars analyzed. the mapping patterns at the candidate loci were visually inspected to produce a final list of putative pav genes. this list can be found in additional file 2: table s together with the information on gene absence distribution for each melon cultivar and gene functional annotations. compared to the reference genome, a total of genes were absent in only one cultivar, while genes were absent in two cultivars, in three cultivars, and only genes were missing in more than three cultivars. in all, pav events were detected, distributed in all cultivars analyzed, including in dhl which probably represents mapping artifacts as no pav would be expected when mapping dhl reads against the dhl reference genome itself. only two genes were absent from dhl but present in both parental genomes, which clearly indicates false positives for the detection of pavs. in addition, not all melon varieties had the same content of pav genes. in cultivar c- there were pav events, while c- t and pi had, respectively,,, and events. however, it must be taken into account that both t and pi are the parental lines of dhl and so it is expected that the number of pav events would be significantly lower than those found when comparing two not so closely related melon cultivars, as each one contributes to around 50% of the reference genome. in all, the average number of absent genes per cultivar was and, therefore, ca. % of all genes were absent, on average, in the analyzed cultivars as compared to dhl these figures are comparable with values published for arabidopsis and rice. the functional annotation of the candidate pav genes was obtained from the melonomics webpage. also, a blast2go analysis was performed to gather further information on genes with unknown function. additionally, functions were tentatively assigned to ten of these genes using phylogeny-based information from the melon phylome, as for the general annotation of the genome. using this combined approach, and excluding all genes with only generic information available, such as'ankyrin repeat-containing','coiled-coil','oxidoreductase activity' and'catalytic activity', functional information was finally assigned to pav genes. however, upon analyzing the functional annotations, it was found that of these genes were annotated as encoding transposon-related sequences. the melon reference genome has been annotated after masking mobile elements. the inspection of these sequences showed that were true transposons that somehow had not been masked during the sequencing assembly; the rest did show homology with proteins in our transposon databases but with e-values lower than 1e- these sequences probably represent degenerate forms of old mobile elements, transposon fragments that remain in the genome after an excision event, or they are genes that have some degree of similarity to transposon sequences. the putative pav genes with functional annotations other than transposon-related sequences were classified according to their ontology. more than 80% of the annotated genes could be grouped into nine categories: amino-acid, carbohydrate and lipid metabolism, biotic or abiotic stress response, terpenoid biosynthesis, transcription/translation processes, transport, and two groups containing generic p and protein/dna- binding proteins that could not be further classified due to lack of more specific information. interestingly, the most populated functional category was that of stress response proteins, with a group of tir-nbs-lrr/nb-arc disease-resistance proteins by far the largest subclass of pav genes having an associated function. it is known that structural genome variants in plants are prone to affect stress response genes, particularly those for disease resistance. these genes have been found to be underrepresented in the genomes of cucurbits when compared to those of other plant species. previous studies have shown that, although whole-genome duplication events seem to be absent from the lineages leading to c. melo, several gene families have expanded specifically in the cucumis genus compared with other cucurbits, or in c. melo when compared with cucumis sativus l.. the list of expanded families includes defense response, lipid metabolism, cell wall structure, translation and transport gene families which, as we report above, are also enriched in pav genes. crossing the lists of pav and expanded families to search for coincidences, a total of % of genes in families that have expanded specifically in the cucumis genus and % in c. melo were found to be pav. these values are - times higher than the abundance of pav genes, %, in the melon gene complement. presence of pav genes in multi-copy gene families and gene clusters it has been previously suggested that with genes belonging to multi-copy gene families, and especially those arranged in clusters, there is a greater chance of presence/absence polymorphism, probably due to unequal crossover between homologous genes. this seems to be particularly the case for disease resistance genes: there is evidence that clusters of these genes may well be hotspots for the generation of pav. in melon, previous studies have shown that resistance genes are frequently found in clusters in the genome; most notably, half of the predicted melon nbs-lrr genes groups within nine clusters. a region spanning 760 kb of linkage group v and containing the vat gene locus is of particular interest. it contains nbs-lrr resistance genes, the highest concentration of r genes found in the melon genome. a microsynteny comparison of part of this region and its homolog in the cucumber genome has already shown the existence of pav polymorphisms affecting several r genes. the presence of pav genes in multi-copy gene families and gene clusters was further investigated to see whether presence/absence variants were associated to these families with higher frequency than randomly expected. to this end, the melon gene complement was first grouped in families of homologous genes as explained in the methods section. as a result, % of all genes were classified as belonging to single-member families or singletons, while genes were members of multi-copy gene families. by comparing the pav and multi-copy gene lists, it was found that pav genes can be classified as multi-copy while non-pav genes belong to multi-copy families. therefore, there was a significantly higher proportion of multi-copy genes in the pav subset of the melon gene complement. as only % of singleton genes were classified as pav compared to % pavs in the multi-copy fraction, genes present in the genome in more than one copy are five times more likely to be affected by pav than singleton genes. most of the multi-copy gene families contain only two or three genes. these small families represent 41% of all multi-copy genes but only 29% of all multi-copy pav genes. on the other hand, families of 7– gene members contain 15% of all multi-copy genes but 22% of all multi-copy pav genes. therefore, pav genes appear to have a slightly higher tendency to be present in more populated gene families in the melon genome. to see whether the physical location of analogous genes could also be related to the occurrence of presence/absence polymorphisms, the presence of pav genes in clusters of genes with a significant degree of similarity was investigated. by classifying multi-copy genes in clusters, a total of genes, of which were pavs, grouped in clusters. therefore, the proportion of pav genes clustered in the melon genome is significantly higher than the % of non-pav clustered genes. also, as only 4% of non-clustered genes are pav compared to the 8% of clustered genes, it seems clear that the chance of genes present in the genome in tandem arrays being affected by pav is double that of isolated genes. of all clusters, 77% are just tandems of two genes, and the longest cluster found had genes. significantly, 14% of all clustered genes, but 28% of all pavs in tandem, were found in the clusters of five or more genes, indicating that pavs tend to be present in longer clusters. validation of putative pav genes the prediction of pav genes has been based solely on the analysis of the alignment files, with no specific experimental support. since many pav genes belong to medium-large multigene families, there is more than a negligible possibility of mapping artifacts mistakenly taken for putative pavs. therefore, it was decided to determine which fraction of putative pav genes was truly absent from the analyzed cultivars. the validation of pavs was mostly carried out in a  mb region that spans the 760 kb region described above, due to its high level of pav polymorphisms and multi-copy gene content. since all reads were mapped against the dhl genome, it was important to confirm that no chimeras had been produced during the assembly of this region on the reference genome. the sequence of a 250 kb fragment with the melo3c004258- genes, previously obtained from three overlapping bacs, was used to validate the corresponding sequence in the assembled reference genome. to validate the melo3c004290- gene fragment, a tiling path of bac clones spanning 713 kb and comprising the melon genes melo3c004287-melo3c was obtained as described in the methods section. the bac lengths were estimated by averaging 3– pulsed-field electrophoretic runs for each clone. the estimated lengths of eight clones differed by 10 kb on average when compared with the expected values obtained by mapping the bac-end sequences to the reference genome assembly. this result is acceptable bearing in mind both the expected error in the bac length estimations based on pulsed-field electrophoresis and, more importantly, the abundance of stretches of ns on the reference genome. their sizes had been adjusted on the basis of pair-end mapping information during the assembly process and, therefore, can be expected to differ to some extent from the actual lengths of the sequences they represent. in particular, two clones spanning the melo3c04318- region differed from their expected lengths by only 7 kb, which indicates that the 150 kb and 90 kb fragments absent in that region in the two agrestis cultivars are most probably present in the reference dhl sequence. two overlapping clones spanning the melo3c04287- genes were found to be 50–60 kb larger than their expected sizes. as both clones share 36 kb of sequence, this suggests that about 50 kb are missing from the reference genome sequence somewhere in the shared region, which has only one annotated gene. in all, the existence of a contig of bacs whose sequence ends map adequately to the reference genome validates the overall sequence structure of, at least, the melo3c04295- region from dhl although minor artifacts cannot be discarded. in a complementary approach, individual putative pav regions were confirmed by pcr using primers flanking the candidate regions and genomic dna from the studied varieties, and bac or genomic dna from the reference genome. a total of genes were analyzed, belonging to the  mb region of linkage group v. the results confirmed of the putative pav genes while six genes that had been considered partially absent were found in the respective cultivars. a b c a d acoordinates on the scaffold/contig that contains the gene. bcv: c- iq: c- pi: pi ps: t cdhl sequence fragment that contains the gene and is absent relative to the reference sequence. dc: pav confirmed; e: exon deletion; i: intron deletion; si: sequence insertion relative to the reference. sequencing of the amplified pcr bands showed that the absence of a genomic fragment was often accompanied by the presence of sequences that were absent from the reference genome, from just a few to several thousand nucleotides. in two instances these new sequences were found to show homology with transposons, indicating the role that mobile elements may play in the generation of pav polymorphisms. analysis of pavs in a 1 mb region of linkage group v as mentioned above, the greatest density of resistance genes in the melon genome was found in a 760 kb fragment on linkage group v. if a cluster of lipoxygenase genes and one phytochelatin gene in the immediate vicinity of this fragment are also considered, the resulting  mb region contains annotated genes of which are most probably involved in different aspects of biotic or abiotic stress responses. as well as nbs-lrr-tir and seven cc-nbs-lrr genes having homology with resistance genes, there are nine lipoxygenase genes, which may have roles in pest resistance and response to wounding, and one phytochelatin gene, that might be involved in resistance to metal poisoning. these resistance genes are grouped in several clusters and 47% of them show pav, not only in the analyzed melon lines but also when compared with the syntenic regions in two other cucurbits with sequenced genomes: cucumber, where only eight tir-nbs-lrr, one cc-nbs-lrr, and no phytochelatins are found, and watermelon, with only five tir-nbs-lrr, one cc-nbs-lrr, and no phytochelatins. the highest concentration of pav events detected in the melon genome was found in this region, with nearly 30% of all the genes partially or totally absent from at least one of the analyzed melon cultivars. in the vicinity of these genes, besides pav in stress response genes, there are also pav genes in a cluster of gdsl lipases and a cluster of terpene cyclases, and eight additional pav genes of unknown function. this region contains the longest continuous gene-containing pav sequence found in melon: a fragment of 150 kb containing the genes melo3c04318- and completely absent from the c- cultivar, while a 90 kb fragment containing the genes 4319– was absent from pi the agrestis parental of dhl and another fragment of 86 kb containing five resistance genes was absent from c-. annotation of the reference melon genome has shown that the number of putative disease resistance genes of the nbs-lrr family in melon and cucumber is lower than in other plant species. although the expansion of the lipoxygenase gene family has been suggested as a mechanism to compensate for this reduction in cucumber, no such expansion has been observed in melon. however, our results show that a high degree of pav polymorphism affects several families of disease resistance genes. it must also be taken into account that only absent genes relative to the dhl reference genome could be detected using our approach, so additional pav events may be found if the resequencing reads are mapped against the genome of other cultivars. the'pan-genome' concept has already been proposed to reflect the fact that single genome sequences do not seem to account for the full set of genes in plant species. it is then possible that the full set of active melon r-genes cannot be obtained by analyzing the annotated genes from just one cultivar. in fact, different combinations of present and absent r-genes in a particular cultivar might well confer various levels of resistance to different pathogens or strains. as an example, the vat gen, an economically important gene conferring resistance to aphid colonization, has been cloned in pi and shown to be a cc-nbs-lrr gene syntenic to those found on the cluster of seven cc-nbs-lrr genes in the  mb region of dhl. two alleles were found at this locus with different resistance specificities. however, it has been reported that the cloning of candidates in other melon accessions is particularly difficult, probably due to the existence of several sources of variability, including the variable complexity of the cluster of resistance genes depending on the genotype. related results have been found in other plant species. for example, nematode resistance mediated by the soybean rhg qtl has been found to be conferred by copy number variation: resistant plants contain ten tandem copies of a 31 kb segment that spans three dissimilar genes, all of which contribute to the resistance.  this article presents the quantification of pavs in the genome of melon by comparing the published reference genome to four additional melon cultivars. we found that multi-copy melon gene families are particularly affected by pav polymorphism. this is in agreement with results in other species and it may respond to the need for multiple copies of a gene for new functions required for the evolution of the species. on further analyses, gene clusters appeared as hotspots of pavs in the melon genome, with significant clustering in cases of biotic stress response-related genes. in particular, melon has two large clusters of sequences coding for nbs-lrr proteins where important resistance loci such as fom or vat map. the analysis of a  mb region containing the vat locus revealed the highest density of pav events as well as the longest continuous gene-containing pav sequence in melon. cucurbit genomes have the peculiarity that some gene families related to pathogen resistance are smaller than in most other plant species. disease resistance genes are essential for the survival of the species and, therefore, they have also been the object of high selective pressure during domestication and breeding. this work reports the finding of high sequence variability involving genome fragments containing pathogen resistance genes in the melon genome. the variability of these clusters may well be the reason why, in some cases, it has been difficult to identify specific resistance genes in these regions. the results also show the importance of sequencing different cultivars of a particular cultivated plant species to characterize its genome variability, the understanding of its evolutionary history, and to help define breeding strategies. 
data on gene presence/absence variation in melon was obtained by resequencing a cultivated accession and an old-relative melon variety, and using previously obtained resequencing data from three other melon cultivars, among them dhl on which the current draft melon genome sequence is based. the highest concentration of pav events detected in the melon genome was found in a  mb region of linkage group v, which also shows the highest density of melon stress-response genes. a total of pav events were detected, involving % of the predicted melon gene complement. the most populated functional category among the pav genes of known function was that of stress response proteins. changes in the copy number of dna sequences are one of the main mechanisms generating genome variability in eukaryotes. our results suggest that genes from multi-copy families are five times more likely to be affected by pav than singleton genes. in addition, these results show the limitations of single reference genome sequences as the only basis for characterization and cloning of resistance genes. also, the chance of genes present in the genome in tandem arrays being affected by pav is double that of isolated genes, with pav genes tending to be in longer clusters. the increasing availability of genome sequences through the application of next-generation massive sequencing technologies has allowed the study of genomic polymorphisms at both the interspecific and intraspecific levels, thus helping to understand how species adapt to changing environments through genome variability. as cucurbits are known to possess a significantly lower number of defense-related genes compared to other plant species, pav variation may play an important role in generating new pathogen resistances at the subspecies level. the first genome-wide report of pav variation among several melon cultivars is presented here. multi-copy and clustered genes, especially those with putative stress-response functions, were found to be particularly affected by pav polymorphisms. in particular, this region contains the longest continuous gene-containing pav sequence so far identified in melon.
 changes in the copy number of dna sequences are one of the main mechanisms generating genome variability in eukaryotes. these changes are often related to phenotypic effects such as genetic disorders or novel pathogen resistance. the increasing availability of genome sequences through the application of next-generation massive sequencing technologies has allowed the study of genomic polymorphisms at both the interspecific and intraspecific levels, thus helping to understand how species adapt to changing environments through genome variability.  data on gene presence/absence variation in melon was obtained by resequencing a cultivated accession and an old-relative melon variety, and using previously obtained resequencing data from three other melon cultivars, among them dhl on which the current draft melon genome sequence is based. a total of pav events were detected, involving % of the predicted melon gene complement. in all, an average % of genes were absent from each analyzed cultivar as compared to the dhl reference genome. the most populated functional category among the pav genes of known function was that of stress response proteins. our results suggest that genes from multi-copy families are five times more likely to be affected by pav than singleton genes. also, the chance of genes present in the genome in tandem arrays being affected by pav is double that of isolated genes, with pav genes tending to be in longer clusters. the highest concentration of pav events detected in the melon genome was found in a  mb region of linkage group v, which also shows the highest density of melon stress-response genes. in particular, this region contains the longest continuous gene-containing pav sequence so far identified in melon.  the first genome-wide report of pav variation among several melon cultivars is presented here. multi-copy and clustered genes, especially those with putative stress-response functions, were found to be particularly affected by pav polymorphisms. as cucurbits are known to possess a significantly lower number of defense-related genes compared to other plant species, pav variation may play an important role in generating new pathogen resistances at the subspecies level. in addition, these results show the limitations of single reference genome sequences as the only basis for characterization and cloning of resistance genes. 