BACKGROUND
currently next generation sequencing facilitates snp discovery and allows deeper analysis of genome variation  <cit> .
in plants, snp discovery has been performed either from rna-seq experiments  <cit>  or whole genome resequencing.
millions of polymorphisms have thus been discovered in arabidopsis  <cit> , rice  <cit> , soybean  <cit>  and maize  <cit> .
the tomato genome has recently been sequenced and the international tomato genome consortium has released a high-quality reference sequence  <cit> .
the available sequence covers 780 mb of the estimated 900 mb.
the annotation predicts  <dig>  gene models, among which  <dig>  were confirmed by rna-seq data.
an initial comparison of the genomes of the sequenced cultivated accession  and an accession of the closest wild relative, s. pimpinellifolium, revealed more than  <dig>  million snps representing a divergence of  <dig> %.
tomato is a model species for fruit development and composition and is also a vegetable of high economic importance.
it is grown all over the world, and its production has continuously increased over the last 50 years.
tomato originated in south america where all the wild species related to cultivated tomato grow in the andean region.
domestication probably started in peru or ecuador followed by diversification in mexico or alternatively domestication directly took place in mexico  <cit> .
tomato evolved following several bottlenecks that considerably reduced the molecular diversity of the cultivated accessions.
this hypothesis is supported by the very low polymorphism rate observed in cultivated species compared to wild relatives  <cit> , but also when analyzing diversity profiles of cherry-type tomato accessions , which are intermediate between wild and modern cultivated accessions  <cit> .
in contrast, tomato breeding has led to a wide range of phenotypic adaptations to different environments and different phenotypes for fruit shape, size and color  <cit> .
this was mainly due to introgressions from the related wild species and the discovery of major mutations  <cit> .
as a genetic model for fruit crops, tomato has been used in many qtl mapping and gene cloning studies.
due to the lack of molecular polymorphism, most of the gene and qtl mapping experiments were performed on inter-specific progeny involving a cultivated and a wild species  <cit> .
the use of wild relatives has allowed the discovery of several useful genes and qtls  <cit> .
since the first studies of tomato molecular diversity and gene mapping, molecular markers have evolved from rflp  <cit>  to aflp  <cit> , then ssr  <cit>  and later snp.
snps were first discovered through in silico mining of est  and amplicon sequencing of conserved ortholog sequences in different varieties  <cit> .
recently a large est sequencing effort allowed the building of an infinium array carrying ≈  <dig> snps .
in this article we present the polymorphisms detected from the resequencing of eight tomato accessions chosen to represent a large range of intraspecific variation.
while characterizing the diversity of  <dig> tomato accessions with  <dig> ssr and later  <dig> snps, we developed nested core collections representing a maximum of molecular and phenotypic variation  <cit> .
in order to discover snps and analyze the distribution of polymorphisms in the tomato genome, we have re-sequenced the whole genomes of eight lines corresponding to the smallest core collection composed of four cherry-type and four cultivated accessions.
the genome sequences were then aligned to the reference genome sequence and alignments were screened for snps.
the distribution and characteristics of the polymorphisms is presented.
a set of snps was cross validated with results from a genotyping array.
the distribution of polymorphisms between accessions and chromosomes is discussed in regard to the recent diversification of tomato.
RESULTS
we analysed two groups of accessions: a group of four cherry-type tomato accessions whose genomes consist in an admixture between the genomes of s. lycopersicum and s pimpinellifolium <cit>  and a group of four large-fruited lines typical of the cultivated accessions or breeding lines used  <dig> and  <dig>  the eight lines were chosen to maximise the molecular diversity detected with  <dig> ssr markers in a collection of  <dig> tomato accessions  <cit> .
following sanger sequencing of  <dig> amplicons in  <dig> accessions , we showed that 76% of the  <dig> snps identified in the collection were detected in at least one of these eight lines  <cit> .
furthermore, the  <dig> snps that were not polymorphic among the eight lines were only polymorphic in s. pimpinellifolium accessions.
we can thus predict that a large fraction of the snps present in any accession of the cultivated species were detected in this sample.
genome sequencing
genome sequencing of the eight tomato lines yielded  <dig> million reads, most of them being 101 bp paired-end reads.
after cleaning,  <dig> to 90% of the reads remained and were mapped to the high-quality genomic reference sequence of heinz  <dig>  <cit> .
a total of  <dig>  to  <dig> % of the reads mapped onto the genome, depending on the lines.
the reads covered  <dig> to 92% of the reference genome sequence.
the average sequence depth of coverage varied from  <dig> x to  <dig> x depending on the accession, with the average being  <dig> x .table  <dig>
total number of reads sequenced and mapped onto the heinz  <dig> reference genome after resequencing eight tomato accessions using illumina genome analyser
genome coverage was equivalent for all accessions and chromosomes except for one long region of chromosome  <dig> from the levovil accession, which corresponded to an introgression from a distant species .
the depth of coverage was also quite similar except for the peaks corresponding to regions with high homology with organelle genomes .
to avoid contamination with chloroplastic and mitochondrial dna reads, all the reads showing a depth higher than 128x were removed from subsequent analysis, as performed elsewhere  <cit> .figure  <dig>
genome view of the whole genome sequences  and zoom on chromosome  <dig>  of two lines .
the high peaks correspond to sequences with high homology with organelle genomes.
the chromosome  <dig> of levovil corresponds to the introgression from a wild related species .
polymorphisms in the eight lines
a total of  <dig> , <dig> unique snps and  <dig>  indels were detected when comparing each genome separately to the reference sequence, with the parameters defined in the materials and methods.
for detecting homozygous polymorphisms, we applied two filters: a minimum of  <dig> reads and a maximum of  <dig> reads had to be mapped at any position and a minimum allele frequency of  <dig>  was required.
if we increased the minimal depth to  <dig>  the number of snps dropped to  <dig> , <dig> but several polymorphisms previously detected by sanger sequencing were no longer detected, in particular in the three lines with a depth of coverage lower than 10x .
the total number of snps varied widely from one line to another, with a range of one to two million in the four s. l. cerasiforme accessions and from  <dig>  to  <dig>  in the four s. lycopersicum lines .
the total number of snps also varied widely between the different chromosomes .
chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> carried the highest number of snps  and very few snps were detected on chromosomes  <dig>   <dig>  and  <dig> .
the range of variation between the chromosomes reached 10-fold on average and 61-fold for the accession the most distant from the reference .figure  <dig>
distribution of the numbers of homozygous snps detected per chromosome and line for the four
s. l. cerasiforme
lines, cervil, plovdiv, la  <dig> and criollo  and the four
s. lycopersicum
large fruited lines, stupicke, ferum, levovil and la  <dig> .
the nucleotide diversity π  varied among the lines from  <dig> ×10- <dig> to  <dig> ×10- <dig>  in introns, these values ranged from  <dig> ×10- <dig>  to  <dig> ×10- <dig>  and in the coding sequences from  <dig> ×10- <dig> to  <dig> ×10- <dig> for the same lines .
it also varied from one chromosome to another, with chromosome  <dig> showing the lowest value  and chromosome  <dig> the highest .
the range of variation in π among the lines was higher than 100-fold for chromosome  <dig> while it was lower than 10-fold for chromosomes  <dig> and  <dig>  within the lines, the range varied from 8-fold for la  <dig> to 63-fold for cervil.figure  <dig>
distribution of the polymorphism rate  in intergenic regions, introns and coding sequences  in the four
s. l. cerasiforme
type lines and four
s. lycopersicum
large fruited lines.
the contribution of each line to the overall number of snps was also highly variable.
for instance, for the four s.l.
cerasiforme accessions, more than 75% of the snps detected in cervil were on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig>  while chromosomes  <dig> and  <dig> contributed to more than half of the snps of criollo.
the s. lycopersicum levovil accession presented an excess of snps on chromosome  <dig> , while this was evident on chromosome  <dig> for ferum  and on chromosome  <dig> for stupicke .
the distribution of the snps along each chromosome also showed high variation as illustrated in figure  <dig> and additional file  <dig> for every chromosome.
in general, snps were more frequent in the distal parts of chromosomes, which correspond to regions with higher recombination frequency  <cit>  and gene density  <cit> .
nevertheless some lines also exhibited large number of snps  in long regions covering the centromeric region such as on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig> for cervil, on chromosome  <dig>   <dig>   <dig> and  <dig> for plovdiv, on chromosome  <dig>   <dig>   <dig>   <dig> and  <dig> for la  <dig> and on chromosome  <dig>   <dig>   <dig> and  <dig> for criollo.
in the four large fruited lines, such patterns concerned only chromosome  <dig> for levovil,  <dig> for ferum and  <dig> for stupicke.
in these lines, a large number of regions were very poor in snps .
snp number did not appear to be related to the physical size of the chromosomes.
only  <dig>  snps were discovered on the longest chromosome , while more than  <dig>  were detected on chromosomes  <dig>   <dig> and  <dig>  covering each around 60 mb.figure  <dig>
distribution of the number of homozygous snp along the chromosomes  <dig>   <dig>  and  <dig> .
validation of snps with the infinium snp array
in order to validate the snps detected, we compared the genotypes obtained from the solcap snp array for  <dig> snps  <cit>  with the snps we detected for six of the eight lines.
we detected  <dig> snps  that matched perfectly.
among the  <dig> differences observed between our prediction and the solcap genotyping,  <dig> were different in every line and  <dig> just in one.
nevertheless 78% of the observed discrepancies were genotyped as heterozygous on the array and may thus correspond to a genotyping error on the array.
if we do not take into account the heterozygous snps and those that were identical in every line, the rate of discrepancy dropped to below 1%.
detection of indels
a total of  <dig>  unique indels were detected in the eight lines compared to the reference genome.
this number varied from  <dig>  to  <dig>  in cherry tomato lines and from  <dig>  to  <dig>  in s. lycopersicum lines .
their distribution across chromosomes was more homogeneous than for snps, although a few chromosomes with a high density compared to the average could be detected .
in most cases, the chromosomes carrying a high number of snps also exhibited a high number of indels.
the correlation between snp and indel numbers on the  <dig> chromosomes was higher than  <dig>  for all lines except for la  <dig> .
the frequency of indels varied on average from one per 14 kb for cervil to one per 270 kb for levovil.
at the chromosome level, these values ranged from one indel per  <dig>  kb to 717 kb.
the majority of indels corresponded to a unique base modification, but a maximum of 32 bp deletions and 25 bp insertions were detected.
the number of insertions was a little higher than the number of deletions  according to the lines.figure  <dig>
distribution of the number of indels detected per chromosome and line for the four s. l. cerasiforme lines  and the four s. lycopersicum lines .
heterozygous snps
tomato is an autogamous crop and the sequenced accessions were maintained by controlled self pollination.
we thus expected a very low rate of residual heterozygosity.
an snp was declared heterozygous when the frequency of both alleles was comprised between  <dig>  and  <dig> .
the total number of unique heterozygous snps was  <dig>  .
the distribution of heterozygous snps was much more homogeneous across lines and chromosomes than the distribution of homozygous snps .
the heterozygous snps corresponded to a variable fraction of the total snps .
a large part of the heterozygous snps  were assigned to chromosome  <dig>  which represents only  <dig> % of the reference genome and carries a large amount of repeated sequences.
we could hardly identify any chromosome fragment in any line which could represent residual heterozygosity covering several hundreds of kb.
this suggested that a large part of the heterozygous snps could result from mapping paralog sequences rather than revealing actual residual heterozygozity.
snp annotation
among the snps, 57% were in intergenic regions, 34% in upstream or downstream regions of a gene, 5% were intronic and  <dig> % in coding sequences.
the effect of each snp was classified according to snpeff v <dig> b software  <cit>  into four classes  “modifier”, for the snps located outside the genes, in non transcribed regions or in introns,  “low effect” for variants in coding regions which do not change the amino acid sequence,  “moderate” effect for variants which change the amino acid sequence and  “high effect” for variants which modify splice sites, stop or start codons .
table  <dig> shows the proportion of variants in each class.
more than 98% of the snps were classified as modifiers.
the fraction of moderate variants ranged from  <dig>  to  <dig> % according to the accessions and the low effect from  <dig>  to  <dig> %.
the high effect variants represented the smallest class, with  <dig> to  <dig> snps depending on the line.table  <dig>
distribution of the snp effect per type of effect in the four cherry-type  and four
s. lycopersicum
lines
s. l. cera
s. l. cera
s. l. cera
s. l. cera
s. lyc
s. lyc
s. lyc
s. lyc
high effect
total
937
701
648
572
273
240
220
184
moderate effect
total
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
low effect
total
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
modifier
total
<dig> ,654
<dig> ,629
<dig> ,290
<dig> ,706
<dig>
<dig>
<dig>
<dig>
snps were annotated using snpeff onto the sl <dig>  reference genome.
among the snps detected in coding sequences, 40% led to synonymous amino acid changes, 56% to non synonymous amino acid changes, with  <dig> % causing a start or stop loss or gain,  <dig> % a change in splice site,  <dig> % a stop in the coding sequence and  <dig> % a non synonymous start.
the percentage of indel with high effects  was higher than for snps  as an indel may rapidly cause a frame shift in the sequence .
the snps with a high effect impacted  <dig> genes.
go annotation of these genes revealed an excess of genes related to apoptosis and trna processing.
the snps with moderate  effects impacted  <dig>  genes, corresponding to several functions, with an excess of go categories related to stress responses.
the distribution into functional category of the genes subjected to high effect modifications were quite different for the eight lines, as illustrated in figure  <dig> for two distant lines.
the genes affected in the lines that are the closest to the reference sequence were mostly related to regulatory processes while in cervil, the most distant line, they were involved in all categories.figure  <dig>
distribution of the gene ontology of the genes for which snp with high effect were detected in cervil  and levovil  tomato lines.
copy number variant  identification
structural variations were detected in the genomes of the five lines with coverage higher than 10x by a global analysis of the read depth variation in 2000 bp-windows.
the comparison of read depth along the chromosomes revealed at least  <dig> regions where a significant variation in depth in at least one line suggested a cnv.
a maximum number of cnv was detected for cervil .
in contrast, la  <dig> showed an excess of regions with higher depth than the average .
on average,  <dig> of the  <dig> regions matched with a gene region, and in total  <dig> genes were impacted.
a significant excess of genes corresponding to cell death processes were detected.
discussion
several experiments have identified snps in tomato.
a few thousand snps have been detected in est sequences  <cit>  or through rna-seq experiments  <cit> .
the comparison of the reference sequence of the cultivated accession heinz  <dig> and the draft genome of s. pimpinellifolium accession la  <dig> allowed the discovery of more than  <dig>  million polymorphisms  <cit> .
in the present study, a genome wide analysis of eight tomato lines allowed the discovery of more than  <dig> million snps and almost  <dig>  indels heterogeneously distributed across the chromosomes and the lines, which could be utilized for subsequent genetic analysis and for tomato improvement.
data quality and conditions of snp discovery
the whole genome sequences of the eight lines were mapped onto the heinz  <dig> reference sequence for polymorphism discovery.
only 3-5% of the reads could not be mapped in spite of the stringent criteria.
this rate is much lower than the ratio of 20% of unmapped reads for s. pimpinellifolium <cit>  or 15% in rice  <cit> .
the low rate of unmapped reads resulted from  the high quality of the reference genome sequence and of the sequences produced,  the low percentage of repeated sequences in the tomato genome and  the low polymorphism level in the lines studied.
in contrast, a strong reduction of the genome coverage was observed for levovil on chromosome  <dig> in the region carrying the tomato mosaic virus resistance gene , introgressed from a distant species, s. peruvanium <cit> .
the lower coverage observed only for this chromosome suggested that this phenomenon is caused by the high divergence between the species, and not by copy number variation or indels with respect to the reference genome.
illumina sequencing allowed the detection of more than  <dig> million snps.
the error rate for illumina sequencing is low  and we applied a stringent selection criterion on read quality and retained only the snps that reached a minimum of 4x coverage per individual.
when we increased the threshold to a minimum coverage of 8x, the number of snps dropped to about  <dig> million , but several snps previously detected by sanger sequencing  <cit>  were no longer detected.
we thus preferred a less stringent threshold.
finally the cross validation with the snp array data gives a high level of confidence in the snps.
polymorphism detection is now possible in closely related accessions
most of the snps were detected in one of the cherry tomato lines.
cherry tomato genome was shown to consist in an admixture between the genomes of s. lycopersicum and s pimpinellifolium <cit> , resulting in regions with high polymorphism compared to the reference genome  and regions with low polymorphism.
the percentage of unique snps provided by the four s. lycopersicum were on average lower than 10% with the exception of chromosome  <dig>  for which stupicke provided 65% of the unique snps.
this is in agreement with the distances among the lines .
we assessed the number of common polymorphisms between lines in a pairwise approach including the snps detected in s. pimpinellifolium la  <dig> .
when comparing the two lines most distant from the reference genome, cervil and plovdiv ,  <dig>  snps were common to both lines, and thus  <dig>  and  <dig>  million snps were specific to each line.
if we compare these two lines to the s. pimpinellifolium genome, we detected  <dig>  and  <dig>  million snps common to the wild species, respectively.
thus each line carried around  <dig>  snp not detected when comparing la  <dig> and heinz  <dig>  this suggested that there is still a high number of snps to be discovered in s. pimpinellifolium and cherry-type accessions.table  <dig>
number of common snp  and indel  in all the pairs of comparisons
s. lyc
s. lyc
s. lyc
s. lyc
s. l. cera
s. l. cera
s. l. cera
s. l. cera
s. pim
accessions consist in four s. lycopersicum , four cherry-type  and one s. pimpinellifolium  accessions.
the first line and column indicate the number of snp and indel detected when compared to the reference genome  <cit> .
in cultivated tomato, the scarcity of polymorphisms at the molecular level hampered the construction of saturated intraspecific maps until snp discovery.
interestingly, even in the two lines that are the closest to the reference genome , one half to two-thirds of the snps remained specific to each line.
even the chromosomes with the lowest snp number exhibited more than  <dig>  snps.
it is thus now possible to build genetic maps of almost any cross and address genetic questions at the intraspecific level, which was not possible before the availability of resequencing approaches.
new rapid and low-cost techniques based on next-generation sequencing platforms have been proposed to identify snps among lines.
they consist either in a first genome reduction before sequencing or in low coverage whole genome resequencing such as genotyping by sequencing   <cit> .
in tomato, depending on the distance between the lines, genome reduction may lead to a low number of snps and gbs may be preferred in intraspecific crosses.
non random distribution of polymorphisms
the snps and indels appeared non-randomly distributed between different chromosomes, but also within each chromosome .
for instance, the overall number of snps detected on chromosome  <dig> was 10-fold lower than that on chromosome  <dig>  despite good coverage, a few regions appeared with a low snp density in every line, for example: a few mb in the middle of chromosomes  <dig> and  <dig> .
such snp “deserts” are also reported in other species  <cit>  and must be confirmed in a larger sample.
the snp numbers were not related to the length of chromosomes or to gene density.
some regions, particularly at the distal ends of the chromosomes, carried a large proportion of the polymorphisms .
the four s. lycopersicum lines also showed some regions poor in snps compared to the four cherry-type tomato lines, notably on chromosome  <dig>   <dig>   <dig> and  <dig>  the most striking feature is the occurrence of large regions covering more than 10 mb, present in one or two lines, and carrying large number of snps.
this kind of pattern appeared on chromosome  <dig> and  <dig> for cervil, on chromosome  <dig> for plovdiv, on chromosome  <dig> for levovil, on chromosome  <dig> for la  <dig> and ferum and on chromosome  <dig> for stupicke.
cervil and plovdiv presented the same profiles for chromosome  <dig> and  <dig>  with regions of low snp density spread over regions of higher snp density.
charles rick, a pioneer in tomato genetics, underlined the role of natural hybridization in tomato, particularly in south america where cultivated accessions may grow close to wild relatives  <cit> : this phenomenon could have resulted in large introgressions, as shown here, particularly in the cherry tomato accessions.figure  <dig>
single nucleotide polymorphisms  variation across the genome in the two groups of four cherry-type tomato lines  followed by the four cultivated lines .
the x-axis represents the physical distance along the chromosomes, in which each tick-mark is one megabase.
for each chromosome, the regions with extremely low snp frequencies  are shown in white, and the regions with the 20% highest density of the snps  are shown as red blocks.
since the early 20th century, tomato breeders have crossed cultivars with wild species in order to transfer resistance genes  <cit> .
this has resulted first in the introgression of large dna fragments of the wild species surrounding the resistance gene, inducing linkage drag.
subsequent backcrosses reduced the introgression size with more or less success  <cit> .
the introgression of disease resistance genes in many cultivars has strongly influenced the snp patterns.
the reference genome of heinz  <dig> carries several fragments introgressed from s. pimpinellifolium <cit> , notably the resistance genes against verticilllium  and fusarium .
other introgression events from s. pimpinellifolium in the heinz  <dig> genome have been reported, particularly a large one on chromosome  <dig>  <cit> .
among the resequenced lines, ferum carried the ve gene, ferum and criollo carried the i <dig> resistance gene, but it was not possible to relate the presence/absence of these genes with variations in polymorphism rate.
cervil carried the resistance gene to fusarium radicis on chromosome  <dig> .
chromosome  <dig> of levovil carried the tmv resistance gene introgressed from s. peruvianum .
this introgression from a distant species reduced the coverage depth in the region, but the number of snps detected with the mapped reads was higher than in the rest of the genome for this line.
for the other regions it is more difficult to identify any known introgressed gene.
these regions often cover the centromeric regions where the recombination rate is lower  <cit>  and thus an introgressed fragment may cover a large part of the chromosome.
our results confirmed the observations based on the snp array showing that variable polymorphism rates from one chromosome to another reveal the breeding history  <cit> .
structural modification
in s. pimpinellifolium,  <dig>  genome regions were lacking when compared to heinz  <dig> with large regions missing on chromosome  <dig> and  <dig>  <cit> .
we detected around  <dig>  cnv in the five lines with a coverage depth higher than 10x.
this number is much lower than in allogamous species like maize where structural variations are much more frequent  <cit> .
the frequency of cnv could be related to the snp frequency, except for la  <dig> which presented an excess of indels and cnv compared to its snp number.
snp annotation
annotation of snps and indels in the eight lines showed that less than 5% of the polymorphisms occurred in coding regions.
the  <dig>  unique polymorphisms with significant effects  affected  <dig>  genes.
non synonymous to synonymous ratios ranged from  <dig>  on average in the four cherry tomato lines to  <dig>  on average in the four cultivated lines.
these values are close to those detected in soybean , and in rice .
the nucleotide diversity decreased in the coding sequences in every line, as expected.
the four cultivated lines exhibited a lower overall diversity compared to the four cherry-type accessions, but also a lower ratio of snp between non coding and coding sequences, reflecting the purifying effect of breeding selection.
snps with large effects are often detected at higher frequencies in stress related genes as shown in maize  <cit>  or in arabidopsis thaliana <cit> .
an excess of genes related to cell death and regulator genes was also detected in the polymorphisms with high effects detected between s. lycopersicum and s. pimpinellifolium <cit> .
in the present study, we observed the same trend for the  <dig>  and  <dig> genes showing high effect snps and indels, as well as in the  <dig>  genes affected by cnvs.
a catalogue of variations useful for genetic studies
for years, we have studied the progeny of the cross between two of the studied lines, cervil and levovil.
we identified several qtls for fruit quality traits  <cit>  and fine mapped some of them  <cit> .
the availability of the reference genome allowed us to rapidly positionally clone a qtl controlling locule number  <cit> .
the availability of the annotated sequences of both lines considerably facilitates the identification of the genes and alleles underlying the qtls.
recently we constructed a multi allelic genetic intercross  population derived from the intercross of the eight lines.
with a broad genetic basis and higher recombination fraction than bi-parental populations, the magic population is particularly interesting for qtl identification  <cit> .
based on our resequencing effort, a set of snps regularly spaced along the chromosomes was identified in order to construct a genetic map of the population and for qtl mapping.
genome wide association is a complementary approach to identify qtls.
the admixture state of cherry tomato accessions is particularly adapted to such analysis  <cit> .
once a region carrying a qtl is identified using an snp array, the availability of the catalogue of snps present in that region and their annotation will be very useful for the identification of the putative snp responsible for the qtl.
beyond providing a highly valuable resource in terms of polymorphism, this catalogue allows a look at the past, revisiting and interpreting the breeding history of accessions and foreseeing the future through the use of high density mapping and detection of fine haplotypes and imputation of snps on large accessions panels.
CONCLUSIONS
next generation sequencing has provoked a revolution in plant research and genetics and offers a wide range of applications  <cit> .
in the present study, we used eight very diverse lines to detect more than  <dig> million snps, around  <dig>  indels and  <dig>  cnvs.
we showed that it was possible to detect thousands of snps even in closely related lines like heinz  <dig> and levovil, offering new perspectives for tomato breeding.
the distribution of snps was heterogeneous and revealed traces of ancient introgressions or breeding efforts.
these data are particularly useful for the identification of qtls and new alleles.
today several projects resequencing tomato accessions are underway  <cit> .
the number of snps available will thus rapidly increase, allowing the identification of new introgressions and regions of the genome under selection.

 currently next generation sequencing facilitates snp discovery and allows deeper analysis of genome variation. in plants, snp discovery has been performed either from rna-seq experiments or whole genome resequencing. millions of polymorphisms have thus been discovered in arabidopsis, rice, soybean and maize. the tomato genome has recently been sequenced and the international tomato genome consortium has released a high-quality reference sequence. the available sequence covers 780 mb of the estimated 900 mb. the annotation predicts gene models, among which were confirmed by rna-seq data. an initial comparison of the genomes of the sequenced cultivated accession and an accession of the closest wild relative, s. pimpinellifolium, revealed more than million snps representing a divergence of %. tomato is a model species for fruit development and composition and is also a vegetable of high economic importance. it is grown all over the world, and its production has continuously increased over the last 50 years. tomato originated in south america where all the wild species related to cultivated tomato grow in the andean region. domestication probably started in peru or ecuador followed by diversification in mexico or alternatively domestication directly took place in mexico. tomato evolved following several bottlenecks that considerably reduced the molecular diversity of the cultivated accessions. this hypothesis is supported by the very low polymorphism rate observed in cultivated species compared to wild relatives, but also when analyzing diversity profiles of cherry-type tomato accessions, which are intermediate between wild and modern cultivated accessions. in contrast, tomato breeding has led to a wide range of phenotypic adaptations to different environments and different phenotypes for fruit shape, size and color. this was mainly due to introgressions from the related wild species and the discovery of major mutations. as a genetic model for fruit crops, tomato has been used in many qtl mapping and gene cloning studies. due to the lack of molecular polymorphism, most of the gene and qtl mapping experiments were performed on inter-specific progeny involving a cultivated and a wild species. the use of wild relatives has allowed the discovery of several useful genes and qtls. since the first studies of tomato molecular diversity and gene mapping, molecular markers have evolved from rflp to aflp, then ssr and later snp. snps were first discovered through in silico mining of est and amplicon sequencing of conserved ortholog sequences in different varieties. recently a large est sequencing effort allowed the building of an infinium array carrying ≈  snps. in this article we present the polymorphisms detected from the resequencing of eight tomato accessions chosen to represent a large range of intraspecific variation. while characterizing the diversity of tomato accessions with ssr and later snps, we developed nested core collections representing a maximum of molecular and phenotypic variation. in order to discover snps and analyze the distribution of polymorphisms in the tomato genome, we have re-sequenced the whole genomes of eight lines corresponding to the smallest core collection composed of four cherry-type and four cultivated accessions. the genome sequences were then aligned to the reference genome sequence and alignments were screened for snps. the distribution and characteristics of the polymorphisms is presented. a set of snps was cross validated with results from a genotyping array. the distribution of polymorphisms between accessions and chromosomes is discussed in regard to the recent diversification of tomato.  we analysed two groups of accessions: a group of four cherry-type tomato accessions whose genomes consist in an admixture between the genomes of s. lycopersicum and s pimpinellifolium and a group of four large-fruited lines typical of the cultivated accessions or breeding lines used and the eight lines were chosen to maximise the molecular diversity detected with ssr markers in a collection of tomato accessions. following sanger sequencing of amplicons in accessions, we showed that 76% of the snps identified in the collection were detected in at least one of these eight lines. furthermore, the snps that were not polymorphic among the eight lines were only polymorphic in s. pimpinellifolium accessions. we can thus predict that a large fraction of the snps present in any accession of the cultivated species were detected in this sample. genome sequencing genome sequencing of the eight tomato lines yielded million reads, most of them being 101 bp paired-end reads. after cleaning, to 90% of the reads remained and were mapped to the high-quality genomic reference sequence of heinz. a total of to % of the reads mapped onto the genome, depending on the lines. the reads covered to 92% of the reference genome sequence. the average sequence depth of coverage varied from x to x depending on the accession, with the average being x.table total number of reads sequenced and mapped onto the heinz reference genome after resequencing eight tomato accessions using illumina genome analyser genome coverage was equivalent for all accessions and chromosomes except for one long region of chromosome from the levovil accession, which corresponded to an introgression from a distant species. the depth of coverage was also quite similar except for the peaks corresponding to regions with high homology with organelle genomes. to avoid contamination with chloroplastic and mitochondrial dna reads, all the reads showing a depth higher than 128x were removed from subsequent analysis, as performed elsewhere.figure genome view of the whole genome sequences and zoom on chromosome of two lines. the high peaks correspond to sequences with high homology with organelle genomes. the chromosome of levovil corresponds to the introgression from a wild related species. polymorphisms in the eight lines a total of, unique snps and indels were detected when comparing each genome separately to the reference sequence, with the parameters defined in the materials and methods. for detecting homozygous polymorphisms, we applied two filters: a minimum of reads and a maximum of reads had to be mapped at any position and a minimum allele frequency of was required. if we increased the minimal depth to the number of snps dropped to, but several polymorphisms previously detected by sanger sequencing were no longer detected, in particular in the three lines with a depth of coverage lower than 10x. the total number of snps varied widely from one line to another, with a range of one to two million in the four s. l. cerasiforme accessions and from to in the four s. lycopersicum lines. the total number of snps also varied widely between the different chromosomes. chromosomes and carried the highest number of snps and very few snps were detected on chromosomes and. the range of variation between the chromosomes reached 10-fold on average and 61-fold for the accession the most distant from the reference.figure distribution of the numbers of homozygous snps detected per chromosome and line for the four s. l. cerasiforme lines, cervil, plovdiv, la and criollo and the four s. lycopersicum large fruited lines, stupicke, ferum, levovil and la. the nucleotide diversity π varied among the lines from ×10- to ×10- in introns, these values ranged from ×10- to ×10- and in the coding sequences from ×10- to ×10- for the same lines. it also varied from one chromosome to another, with chromosome showing the lowest value and chromosome the highest. the range of variation in π among the lines was higher than 100-fold for chromosome while it was lower than 10-fold for chromosomes and within the lines, the range varied from 8-fold for la to 63-fold for cervil.figure distribution of the polymorphism rate in intergenic regions, introns and coding sequences in the four s. l. cerasiforme type lines and four s. lycopersicum large fruited lines. the contribution of each line to the overall number of snps was also highly variable. for instance, for the four s.l. cerasiforme accessions, more than 75% of the snps detected in cervil were on chromosomes and while chromosomes and contributed to more than half of the snps of criollo. the s. lycopersicum levovil accession presented an excess of snps on chromosome, while this was evident on chromosome for ferum and on chromosome for stupicke. the distribution of the snps along each chromosome also showed high variation as illustrated in figure  and additional file for every chromosome. in general, snps were more frequent in the distal parts of chromosomes, which correspond to regions with higher recombination frequency and gene density. nevertheless some lines also exhibited large number of snps in long regions covering the centromeric region such as on chromosomes and for cervil, on chromosome and for plovdiv, on chromosome and for la and on chromosome and for criollo. in the four large fruited lines, such patterns concerned only chromosome for levovil, for ferum and for stupicke. in these lines, a large number of regions were very poor in snps. snp number did not appear to be related to the physical size of the chromosomes. only snps were discovered on the longest chromosome, while more than were detected on chromosomes and covering each around 60 mb.figure distribution of the number of homozygous snp along the chromosomes and. validation of snps with the infinium snp array in order to validate the snps detected, we compared the genotypes obtained from the solcap snp array for snps with the snps we detected for six of the eight lines. we detected snps that matched perfectly. among the differences observed between our prediction and the solcap genotyping, were different in every line and just in one. nevertheless 78% of the observed discrepancies were genotyped as heterozygous on the array and may thus correspond to a genotyping error on the array. if we do not take into account the heterozygous snps and those that were identical in every line, the rate of discrepancy dropped to below 1%. detection of indels a total of unique indels were detected in the eight lines compared to the reference genome. this number varied from to in cherry tomato lines and from to in s. lycopersicum lines. their distribution across chromosomes was more homogeneous than for snps, although a few chromosomes with a high density compared to the average could be detected. in most cases, the chromosomes carrying a high number of snps also exhibited a high number of indels. the correlation between snp and indel numbers on the chromosomes was higher than for all lines except for la. the frequency of indels varied on average from one per 14 kb for cervil to one per 270 kb for levovil. at the chromosome level, these values ranged from one indel per  kb to 717 kb. the majority of indels corresponded to a unique base modification, but a maximum of 32 bp deletions and 25 bp insertions were detected. the number of insertions was a little higher than the number of deletions according to the lines.figure distribution of the number of indels detected per chromosome and line for the four s. l. cerasiforme lines and the four s. lycopersicum lines. heterozygous snps tomato is an autogamous crop and the sequenced accessions were maintained by controlled self pollination. we thus expected a very low rate of residual heterozygosity. an snp was declared heterozygous when the frequency of both alleles was comprised between and. the total number of unique heterozygous snps was. the distribution of heterozygous snps was much more homogeneous across lines and chromosomes than the distribution of homozygous snps. the heterozygous snps corresponded to a variable fraction of the total snps. a large part of the heterozygous snps were assigned to chromosome which represents only % of the reference genome and carries a large amount of repeated sequences. we could hardly identify any chromosome fragment in any line which could represent residual heterozygosity covering several hundreds of kb. this suggested that a large part of the heterozygous snps could result from mapping paralog sequences rather than revealing actual residual heterozygozity. snp annotation among the snps, 57% were in intergenic regions, 34% in upstream or downstream regions of a gene, 5% were intronic and % in coding sequences. the effect of each snp was classified according to snpeff v b software into four classes “modifier”, for the snps located outside the genes, in non transcribed regions or in introns, “low effect” for variants in coding regions which do not change the amino acid sequence, “moderate” effect for variants which change the amino acid sequence and “high effect” for variants which modify splice sites, stop or start codons. table  shows the proportion of variants in each class. more than 98% of the snps were classified as modifiers. the fraction of moderate variants ranged from to % according to the accessions and the low effect from to %. the high effect variants represented the smallest class, with to snps depending on the line.table distribution of the snp effect per type of effect in the four cherry-type and four s. lycopersicum lines s. l. cera s. l. cera s. l. cera s. l. cera s. lyc s. lyc s. lyc s. lyc high effect total 937 701 648 572 273 240 220 184 moderate effect total low effect total modifier total,654,629,290,706 snps were annotated using snpeff onto the sl reference genome. among the snps detected in coding sequences, 40% led to synonymous amino acid changes, 56% to non synonymous amino acid changes, with % causing a start or stop loss or gain, % a change in splice site, % a stop in the coding sequence and % a non synonymous start. the percentage of indel with high effects was higher than for snps as an indel may rapidly cause a frame shift in the sequence. the snps with a high effect impacted genes. go annotation of these genes revealed an excess of genes related to apoptosis and trna processing. the snps with moderate effects impacted genes, corresponding to several functions, with an excess of go categories related to stress responses. the distribution into functional category of the genes subjected to high effect modifications were quite different for the eight lines, as illustrated in figure  for two distant lines. the genes affected in the lines that are the closest to the reference sequence were mostly related to regulatory processes while in cervil, the most distant line, they were involved in all categories.figure distribution of the gene ontology of the genes for which snp with high effect were detected in cervil and levovil tomato lines. copy number variant identification structural variations were detected in the genomes of the five lines with coverage higher than 10x by a global analysis of the read depth variation in 2000 bp-windows. the comparison of read depth along the chromosomes revealed at least regions where a significant variation in depth in at least one line suggested a cnv. a maximum number of cnv was detected for cervil. in contrast, la showed an excess of regions with higher depth than the average. on average, of the regions matched with a gene region, and in total genes were impacted. a significant excess of genes corresponding to cell death processes were detected. discussion several experiments have identified snps in tomato. a few thousand snps have been detected in est sequences or through rna-seq experiments. the comparison of the reference sequence of the cultivated accession heinz and the draft genome of s. pimpinellifolium accession la allowed the discovery of more than million polymorphisms. in the present study, a genome wide analysis of eight tomato lines allowed the discovery of more than million snps and almost indels heterogeneously distributed across the chromosomes and the lines, which could be utilized for subsequent genetic analysis and for tomato improvement. data quality and conditions of snp discovery the whole genome sequences of the eight lines were mapped onto the heinz reference sequence for polymorphism discovery. only 3-5% of the reads could not be mapped in spite of the stringent criteria. this rate is much lower than the ratio of 20% of unmapped reads for s. pimpinellifolium or 15% in rice. the low rate of unmapped reads resulted from the high quality of the reference genome sequence and of the sequences produced, the low percentage of repeated sequences in the tomato genome and the low polymorphism level in the lines studied. in contrast, a strong reduction of the genome coverage was observed for levovil on chromosome in the region carrying the tomato mosaic virus resistance gene, introgressed from a distant species, s. peruvanium. the lower coverage observed only for this chromosome suggested that this phenomenon is caused by the high divergence between the species, and not by copy number variation or indels with respect to the reference genome. illumina sequencing allowed the detection of more than million snps. the error rate for illumina sequencing is low and we applied a stringent selection criterion on read quality and retained only the snps that reached a minimum of 4x coverage per individual. when we increased the threshold to a minimum coverage of 8x, the number of snps dropped to about million, but several snps previously detected by sanger sequencing were no longer detected. we thus preferred a less stringent threshold. finally the cross validation with the snp array data gives a high level of confidence in the snps. polymorphism detection is now possible in closely related accessions most of the snps were detected in one of the cherry tomato lines. cherry tomato genome was shown to consist in an admixture between the genomes of s. lycopersicum and s pimpinellifolium, resulting in regions with high polymorphism compared to the reference genome and regions with low polymorphism. the percentage of unique snps provided by the four s. lycopersicum were on average lower than 10% with the exception of chromosome for which stupicke provided 65% of the unique snps. this is in agreement with the distances among the lines. we assessed the number of common polymorphisms between lines in a pairwise approach including the snps detected in s. pimpinellifolium la. when comparing the two lines most distant from the reference genome, cervil and plovdiv, snps were common to both lines, and thus and million snps were specific to each line. if we compare these two lines to the s. pimpinellifolium genome, we detected and million snps common to the wild species, respectively. thus each line carried around snp not detected when comparing la and heinz this suggested that there is still a high number of snps to be discovered in s. pimpinellifolium and cherry-type accessions.table number of common snp and indel in all the pairs of comparisons s. lyc s. lyc s. lyc s. lyc s. l. cera s. l. cera s. l. cera s. l. cera s. pim accessions consist in four s. lycopersicum, four cherry-type and one s. pimpinellifolium accessions. the first line and column indicate the number of snp and indel detected when compared to the reference genome. in cultivated tomato, the scarcity of polymorphisms at the molecular level hampered the construction of saturated intraspecific maps until snp discovery. interestingly, even in the two lines that are the closest to the reference genome, one half to two-thirds of the snps remained specific to each line. even the chromosomes with the lowest snp number exhibited more than snps. it is thus now possible to build genetic maps of almost any cross and address genetic questions at the intraspecific level, which was not possible before the availability of resequencing approaches. new rapid and low-cost techniques based on next-generation sequencing platforms have been proposed to identify snps among lines. they consist either in a first genome reduction before sequencing or in low coverage whole genome resequencing such as genotyping by sequencing. in tomato, depending on the distance between the lines, genome reduction may lead to a low number of snps and gbs may be preferred in intraspecific crosses. non random distribution of polymorphisms the snps and indels appeared non-randomly distributed between different chromosomes, but also within each chromosome. for instance, the overall number of snps detected on chromosome was 10-fold lower than that on chromosome despite good coverage, a few regions appeared with a low snp density in every line, for example: a few mb in the middle of chromosomes and. such snp “deserts” are also reported in other species and must be confirmed in a larger sample. the snp numbers were not related to the length of chromosomes or to gene density. some regions, particularly at the distal ends of the chromosomes, carried a large proportion of the polymorphisms. the four s. lycopersicum lines also showed some regions poor in snps compared to the four cherry-type tomato lines, notably on chromosome and the most striking feature is the occurrence of large regions covering more than 10 mb, present in one or two lines, and carrying large number of snps. this kind of pattern appeared on chromosome and for cervil, on chromosome for plovdiv, on chromosome for levovil, on chromosome for la and ferum and on chromosome for stupicke. cervil and plovdiv presented the same profiles for chromosome and with regions of low snp density spread over regions of higher snp density. charles rick, a pioneer in tomato genetics, underlined the role of natural hybridization in tomato, particularly in south america where cultivated accessions may grow close to wild relatives: this phenomenon could have resulted in large introgressions, as shown here, particularly in the cherry tomato accessions.figure single nucleotide polymorphisms variation across the genome in the two groups of four cherry-type tomato lines followed by the four cultivated lines. the x-axis represents the physical distance along the chromosomes, in which each tick-mark is one megabase. for each chromosome, the regions with extremely low snp frequencies are shown in white, and the regions with the 20% highest density of the snps are shown as red blocks. since the early 20th century, tomato breeders have crossed cultivars with wild species in order to transfer resistance genes. this has resulted first in the introgression of large dna fragments of the wild species surrounding the resistance gene, inducing linkage drag. subsequent backcrosses reduced the introgression size with more or less success. the introgression of disease resistance genes in many cultivars has strongly influenced the snp patterns. the reference genome of heinz carries several fragments introgressed from s. pimpinellifolium, notably the resistance genes against verticilllium and fusarium. other introgression events from s. pimpinellifolium in the heinz genome have been reported, particularly a large one on chromosome. among the resequenced lines, ferum carried the ve gene, ferum and criollo carried the i resistance gene, but it was not possible to relate the presence/absence of these genes with variations in polymorphism rate. cervil carried the resistance gene to fusarium radicis on chromosome. chromosome of levovil carried the tmv resistance gene introgressed from s. peruvianum. this introgression from a distant species reduced the coverage depth in the region, but the number of snps detected with the mapped reads was higher than in the rest of the genome for this line. for the other regions it is more difficult to identify any known introgressed gene. these regions often cover the centromeric regions where the recombination rate is lower and thus an introgressed fragment may cover a large part of the chromosome. our results confirmed the observations based on the snp array showing that variable polymorphism rates from one chromosome to another reveal the breeding history. structural modification in s. pimpinellifolium, genome regions were lacking when compared to heinz with large regions missing on chromosome and. we detected around cnv in the five lines with a coverage depth higher than 10x. this number is much lower than in allogamous species like maize where structural variations are much more frequent. the frequency of cnv could be related to the snp frequency, except for la which presented an excess of indels and cnv compared to its snp number. snp annotation annotation of snps and indels in the eight lines showed that less than 5% of the polymorphisms occurred in coding regions. the unique polymorphisms with significant effects affected genes. non synonymous to synonymous ratios ranged from on average in the four cherry tomato lines to on average in the four cultivated lines. these values are close to those detected in soybean, and in rice. the nucleotide diversity decreased in the coding sequences in every line, as expected. the four cultivated lines exhibited a lower overall diversity compared to the four cherry-type accessions, but also a lower ratio of snp between non coding and coding sequences, reflecting the purifying effect of breeding selection. snps with large effects are often detected at higher frequencies in stress related genes as shown in maize or in arabidopsis thaliana. an excess of genes related to cell death and regulator genes was also detected in the polymorphisms with high effects detected between s. lycopersicum and s. pimpinellifolium. in the present study, we observed the same trend for the and genes showing high effect snps and indels, as well as in the genes affected by cnvs. a catalogue of variations useful for genetic studies for years, we have studied the progeny of the cross between two of the studied lines, cervil and levovil. we identified several qtls for fruit quality traits and fine mapped some of them. the availability of the reference genome allowed us to rapidly positionally clone a qtl controlling locule number. the availability of the annotated sequences of both lines considerably facilitates the identification of the genes and alleles underlying the qtls. recently we constructed a multi allelic genetic intercross population derived from the intercross of the eight lines. with a broad genetic basis and higher recombination fraction than bi-parental populations, the magic population is particularly interesting for qtl identification. based on our resequencing effort, a set of snps regularly spaced along the chromosomes was identified in order to construct a genetic map of the population and for qtl mapping. genome wide association is a complementary approach to identify qtls. the admixture state of cherry tomato accessions is particularly adapted to such analysis. once a region carrying a qtl is identified using an snp array, the availability of the catalogue of snps present in that region and their annotation will be very useful for the identification of the putative snp responsible for the qtl. beyond providing a highly valuable resource in terms of polymorphism, this catalogue allows a look at the past, revisiting and interpreting the breeding history of accessions and foreseeing the future through the use of high density mapping and detection of fine haplotypes and imputation of snps on large accessions panels.  next generation sequencing has provoked a revolution in plant research and genetics and offers a wide range of applications. in the present study, we used eight very diverse lines to detect more than million snps, around indels and cnvs. we showed that it was possible to detect thousands of snps even in closely related lines like heinz and levovil, offering new perspectives for tomato breeding. the distribution of snps was heterogeneous and revealed traces of ancient introgressions or breeding efforts. these data are particularly useful for the identification of qtls and new alleles. today several projects resequencing tomato accessions are underway. the number of snps available will thus rapidly increase, allowing the identification of new introgressions and regions of the genome under selection. 
here we describe the whole-genome resequencing of eight accessions chosen to represent a large range of intra-specific variability and the identification and annotation of novel polymorphisms. substantial genetic differences exist between the sequenced tomato accessions and the reference sequence. the distribution of snps and indels across and within chromosomes was highly heterogeneous revealing introgressions from wild species and the mosaic structure of the genomes of the cherry tomato accessions. the completion of the tomato genome sequence and recent advances in dna sequencing technology allow for in-depth characterization of genetic variation present in the tomato genome. comparison of the sequences with the reference genome yielded more than million single nucleotide polymorphisms. like many self-pollinated crops, cultivated tomato accessions show a low molecular but high phenotypic diversity. this study represents the first whole genome resequencing experiment in cultivated tomato.
 one of the goals of genomics is to identify the genetic loci responsible for variation in phenotypic traits. the completion of the tomato genome sequence and recent advances in dna sequencing technology allow for in-depth characterization of genetic variation present in the tomato genome. like many self-pollinated crops, cultivated tomato accessions show a low molecular but high phenotypic diversity. here we describe the whole-genome resequencing of eight accessions chosen to represent a large range of intra-specific variability and the identification and annotation of novel polymorphisms.  the eight genomes were sequenced using the gaii illumina platform. comparison of the sequences with the reference genome yielded more than million single nucleotide polymorphisms. this number varied from to million according to the accessions. almost indels were detected. the distribution of snps and indels across and within chromosomes was highly heterogeneous revealing introgressions from wild species and the mosaic structure of the genomes of the cherry tomato accessions. in-depth annotation of the polymorphisms identified more than unique non-synonymous snps. in addition putative copy-number variations were identified.  this study represents the first whole genome resequencing experiment in cultivated tomato. substantial genetic differences exist between the sequenced tomato accessions and the reference sequence. the heterogeneous distribution of the polymorphisms may be related to introgressions that occurred during domestication or breeding. the annotated snps, indels and cnvs identified in this resequencing study will serve as useful genetic tools, and as candidate polymorphisms in the search for phenotype-altering dna variations. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tomatogenomesequencesingle nucleotide polymorphismintrogressionissue-copyright-statement© biomed central ltd 2013 