BACKGROUND
solanum pimpinellifolium has high breeding potential for biotic and abiotic stress tolerance and fruit quality traits such as fruit weight, internal and external color, firmness, fruit shape, lycopene and high soluble solids content.
as a result, this wild species has been frequently used as a donor in tomato breeding programs .
in addition, the s. pimpinellifolium genome is reported to harbor more favorable and fewer unfavorable alleles for breeding improved cultivars compared to other wild tomato species including s. cheesmanii, s. chmielewskii and s. habrochaites  <cit> .
s. pimpinellifolium is more closely related to cultivated tomato  than the other wild tomato species.
thus, utilization of s. pimpinellifolium in breeding minimizes the consequences of linkage drag  <cit> .
efficient introgression of favorable alleles from s. pimpinellifolium into s. lycopersicum by both positive and background selection requires high-throughput marker identification and genotyping as well as identification of the qtls  that control the traits of interest  <cit> .
cultivated tomato has extensive genomic resources for molecular breeding.
the genome has been completely sequenced and a large amount of data about genome-wide intraspecific and interspecific allelic variation are available.
more than  <dig> interspecific tomato linkage maps were constructed using ssr , cosii  and snp  markers.
nine of these maps were constructed using mapping populations derived from crosses between s. pimpinellifolium and s. lycopersicum for mapping of fruit quality traits and disease tolerance .
most of these low resolution linkage maps were constructed with rflp , cosii and ssr markers.
four recently constructed linkage maps contain snp markers designed from the solanaceae genomics network  <cit>  and tomato mapping resource databases .
the maps introduced by salinan et al.
<cit>  and capel et al.
<cit> , respectively, contained  <dig> and  <dig> snp markers genotyped in s. pimpinellifolium and s. lycopersicum using the high-resolution melting  method.
another map with  <dig> markers was constructed with snps discovered by restriction site associated dna sequencing  technology and veracode snp makers  <cit> .
the traitgenetics expimp <dig> linkage map had the highest number of interspecific snp markers   <cit> .
two additional studies have described development of snp markers by sequencing.
in the work of causse et al.
<cit> , eight  s. lycopersicum accessions were sequenced to identify  <dig>  unique, non-synonymous snps and  <dig>  putative copy-number variations .
the other study used sequencing data available from the ncbi database for nine tomato accessions   <cit>  is a practical and inexpensive method for high-throughput snp discovery and genotyping.
the gbs approach uses next-generation sequencing  technologies for multiplex sequencing of restriction site-associated dna  <cit> .
alignment of the sequence reads generated from a population enables simultaneous snp discovery and genotyping .
to date, the gbs approach has been commonly used for snp discovery and genotyping in crop species such as wheat  <cit> , rice  <cit>  and maize  <cit> .
however, the rad-seq method, which is similar to gbs has been used in tomato  <cit> .
this technique was used to discover  <dig> snps in an f2: <dig> population derived from a cross between s. pimpinellifolium  and s. lycopersicum  to identify qtls for late blight resistance  <cit> ).
the primary goal of the current study was to use the gbs approach for high-throughput identification of interspecific snps in the s. pimpinellifolium  and s. lycopersicum cv.
tueza genomes.
the second aim of this work was the identification of qtls for fruit quality traits using the identified markers.
to achieve these aims, an ibl  population  comprising  <dig> individuals was developed using an advanced backcross  qtl strategy.
the donor parent was the wild tomato species s. pimpinellifolium  and the recurrent parent was an elite fresh market tomato, s. lycopersicum cv.
tueza.
ibl populations are used to introgress favorable alleles from wild species into the cultivated genome in tomato breeding.
although unbalanced populations such as ibls are not suitable for linkage map construction due to unequal representation of the parental genomes, they are valuable genetic resources for qtl mapping because ibl populations contain small introgressions from the wild species in the cultivated tomato genome.
such small introgressions and the higher occurrence of crossing-over events as compared to f <dig> and bc populations improve the resolution of qtl mapping in ibl populations  <cit> .
fruit quality traits such as fruit weight, external and internal color, and firmness affect consumer preference and define the market value of tomato cultivars.
thus, tomato breeders are interested in the improvement of tomato cultivars for these traits and several studies were performed to identify qtls controlling them .
the most comprehensive qtl mapping study for tomato fruit quality traits was performed by doganlar et al.
<cit> .
in this study,  <dig> qtls were identified for  <dig> fruit quality traits using an ibl population derived from the cross between s. pimpinellifolium  and a processing tomato cultivar .
despite the high number of loci identified for fruit quality traits, most qtl analyses were performed using low resolution genetic linkage maps and transient populations, with the notable exceptions of work with ibls  <cit>  and rils  <cit> .
in the present study, qtls were identified for  <dig> fruit quality traits  in an ibl population using interspecific snp loci discovered through a gbs approach.
this is the first time that gbs methodology was implemented in tomato for generation of a high resolution physical map for qtl mapping of fruit quality traits.
the results of the present study demonstrate the efficiency of gbs for snp discovery and qtl mapping in tomato.
methods
plant materials
an interspecific ibl  population derived from the cross s. lycopersicum cv.
tueza x s. pimpinellifolium  was used as plant material in the study.
tueza is a cultivated fresh market tomato line with large , red, slightly flattened round fruits.
la <dig> is a wild type tomato with small, red, round fruits.
the ibl population and parents were grown by multi tohum seed company .
a total of  <dig> plants per genotype were grown in double rows with 140 cm between wide rows and 50 cm between narrow rows.
within rows, plants were spaced at 40 cm intervals.
for basal fertilization, 500 kg 15:15: <dig>  fertilizer and 50 t of composted manure were applied per ha.
drip irrigation was used with fertigation  at each irrigation using 1–2– <dig> fertilizer until first fruit set, 2–1- <dig> fertilizer until first fruit ripening and 1–1- <dig> fertilizer after first fruit ripening.
total genomic dna was isolated from the leaf tissue bulked from  <dig> plants of the parental accessions and  <dig> individuals of the ibl population using a ctab method  <cit> .
genomic dna was quantified using qubit™ quantitation assay .
dna integrity was checked on a 1% agarose gel.
genotyping by sequencing
genomic dna from the  <dig> individuals of the ibl population and parental accessions were subjected to gbs analysis by the university of wisconsin biotechnology center .
sequencing library preparation protocol, including restriction enzyme digestion, use of barcode adapters, sample pooling and amplification, was performed as described by elshire et al.
<cit> .
the 95-plex library was sequenced with the illumina hiseq next-generation sequencing platform .
gbs data analysis and snp identification
gbs data analysis was performed using the gbs discovery pipeline of tassel version  <dig>  software  <cit> .
the fastq and sample key files  generated from raw sequence reads by the casava  <dig> .
<dig> software package  were used as input for processing in the pipeline.
before the analysis, 64-base reads were generated by trimming reads having the barcodes for each genotype followed by an apeki cut site using the fastqtotagcountplugin of the pipeline.
reads with unidentified bases  were excluded from analysis.
the barcoded sequence reads were collapsed into unique sequence tags with counts using the fastqtotagcountplugin with default parameters with the exception that minimum number of times a tag must be present was set to  <dig>  tag count files that contained the sequence tags that passed the minimum count threshold of  <dig> were merged into a master file using the mergemultipletagcountplugin.
the master tags in fastq format generated by tagcounttofastqplugin were aligned to the tomato s. lycopersicum reference genome using the bowtie <dig> plugin with default parameters  <cit> .
samconverterplugin generated the “tags on physical map”  file which contained information about the physical positions of the master tags which had the best unique alignments with the reference genome.
in addition to the topm file, the “tags by taxa”  file that contained tag counts of each barcode generated by fastqtotbtplugin was used for snp calling according to the parameters of the tagstosnpbyalignmentplugin .
snps were recorded in a hapmap file for each chromosome.
mergeduplicatesnpsplugin was used to merge the duplicate snps.
snps were filtered based on minimum taxon coverage , minimum site coverage , linkage disequilibrium with neighboring snps , minimum r <dig> value for the ld filter :  <dig> , and minimum bonferroni-corrected p-value for the ld filter :  <dig> .
a physical map of the identified snps was drawn using mapchart software  <cit> .
phenotypic evaluation
tomato fruits at the normal market stage were evaluated for  <dig> qualitative fruit quality traits: fruit weight, dry matter weight, external color, internal color, locule number, wall thickness, firmness, fruit shape, stem scar, total soluble solids content and ph.
phenotyping was performed as described in the first such work using s. pimpinellifolium  <cit>  and most of the studies thereafter  <cit> .
fruit weight  was determined by bulking the fruit from  <dig> plants and calculating the mean weight of  <dig> representative tomato fruits.
fruits from  <dig> plants per genotype were bulked and characterized for external and internal color, fruit firmness, shape, stem scar, locule number, wall thickness and total soluble solids content.
external  and internal fruit colour  were visually determined for each individual using a scale from  <dig> to  <dig> .
a total of 100 g tomato fruits were dried and weighed to calculate fruit dry matter.
fruit firmness  was determined by hand squeezing using a scale of  <dig> to  <dig> .
ratio of fruit length to fruit width represented fruit shape  with a scale from  <dig> to  <dig> .
stem scar diameter  represented stem scar size .
locule number  was counted in transversely-cut fruits.
fruit wall thickness   was visually determined using a scale from  <dig> to  <dig>   <cit> .
total soluble solids content of the tomato fruits was measured using a refractometer.
the ph of juice from the fruits was measured with a ph meter.
qtl mapping
qgene version  <dig>   <cit>  was used for qtl analysis.
the cim  qtl analysis method uses both interval mapping and multiple regression analysis and was performed with automatic forward cofactor selection and a scan interval of  <dig>  mb.
a total of  <dig>  random permutations were performed with parameters  to calculate the genome-wide lod threshold  <cit> .
correlation analysis between traits was performed using pasw software  <cit> .
RESULTS
gbs
a total of  <dig> , <dig> reads were generated by sequencing of the 95-plex library.
from these reads,  <dig>  sequence tags were generated.
a majority of the sequence tags  were uniquely aligned to the tomato genome.
the remaining tags were either aligned to multiple positions  or could not be located  to the tomato genome assembly.
the  <dig>  sequence tags uniquely aligned to the genome were used for genome-wide high-throughput snp discovery.
sequences of the merged gbs tags in sam  file format can be accessed at sra  database under accession number srp <dig>
snp identification
tag alignment to the reference genome revealed  <dig>  unique snp loci  between the s. lycopersicum and s. pimpinellifolium genomes.
the snp loci were found on all  <dig> chromosomes of tomato .
the physical map constructed with the identified snp loci had sufficient coverage for genome analysis such as qtl mapping and contained snps that were evenly distributed along the chromosomes with  <dig> gaps in size of more than 2 mb .
after filtration based on the proportion of missing data  and parameters such as minimum taxon coverage , minimum site coverage , linkage disequilibrium with neighboring snps , minimum r <dig> value for the ld filter :  <dig> , minimum bonferroni-corrected p-value for the ld filter :  <dig> , a total of  <dig>  snp loci were retained.
while the average distance between adjacent loci was  <dig>  kb for the pre-filtration snps, frequency was reduced to one snp per  <dig>  kb after filtering .
chromosome t <dig> had the highest frequency of filtered snps with an average distance of  <dig>  kb between adjacent markers .fig.
<dig> physical map of the  <dig>  snps retained after the filtering process.
detailed information about the physical map and snp locations is available in the additional file 2
anumber and frequency of snps physically mapped in tomato genome
bnumber and frequency of snps retained after the filtration process
the majority of the snps  identified in this study were transition mutations  as expected.
the most frequently observed substitution types, a/g and c/t transitions, had similar frequencies:  <dig>  and  <dig> %, respectively.
c/g transversion was the least common substitution type  .
the observed transition/transversion ratio was  <dig> .
phenotypic variation
a total of  <dig> ibl individuals and parental accessions were characterized for  <dig> fruit quality traits including fruit weight, dry matter weight, external and internal color, locule number, wall thickness, firmness, fruit shape, stem scar, soluble solids content and ph.
the parents of the ibl population had extreme phenotypes for fruit weight, wall thickness, stem scar and soluble solids content traits .
all of the traits segregated in the ibl population.
fruit weight, wall thickness, firmness and stem scar traits displayed the highest variation in the population with coefficients of variation  ranging from  <dig>  to  <dig> %, .
with the exception of soluble solids content and ph, the remaining traits  had considerable variation in the population .
the ph and soluble solids contents displayed the lowest variation with  <dig> and  <dig> % cv, respectively .
all traits except external color, locule number and fruit shape displayed normal and continuous distributions .table  <dig> statistics for fruit quality traits measured in ibl population and parents; s. lycopersicum cv.
tueza and s. pimpinellifolium cv.
la1589
correlation analysis demonstrated significant associations between some traits.
fruit weight was correlated to all traits except dry matter weight, internal color, fruit shape and ph.
fruit weight was positively correlated to locule number  and wall thickness  and negatively weakly correlated to external color  and soluble solids content .
dry matter weight was moderately correlated to soluble solids content .
external color was weakly correlated to internal color  and locule number .
locule number was moderately correlated to stem scar  .
qtl mapping
the  <dig>  genome-wide snp loci  that were retained after filtering were used in qtl mapping of the fruit quality traits.
cim analysis was performed and a logarithm of odds  threshold  generated by  <dig>  permutations was used to identify qtls for each trait.
for fruit weight, the lod threshold was  <dig>   and three qtls  were identified on chromosomes t <dig>  t <dig> and t <dig> .
the percentage of phenotypic variation  explained by the qtls varied from  <dig> to 26% .
the qtl on chromosome t <dig>  had the highest pve, 26%.
three qtls were identified for dry matter weight on chromosome t <dig> based on a lod threshold of  <dig> .
the pves of these loci were  <dig>   <dig> and 14% for dw <dig> , dw <dig>  and dw <dig> , respectively .table  <dig> qtls identified for  <dig> fruit quality traits
effectc
s. pimpinellifolium
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. lycopersicum
s. lycopersicum
s. pimpinellifolium
s. pimpinellifolium
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. pimpinellifolium
s. pimpinellifolium
s. pimpinellifolium
s. lycopersicum
s. lycopersicum
s. lycopersicum
s. lycopersicum
apeak position of qtl
bpercentage of phenotypic variation explained by identified qtl
cparental allele associated with increased trait value
a total of  <dig> qtls were identified for external and internal color in tomato based on lod thresholds of  <dig>  and  <dig>  respectively .
for the external color, two qtls were identified on chromosomes t <dig> and t <dig> with a total pve of 21%.
a total of nine qtls were identified for internal color on chromosomes t <dig>  t <dig>  t <dig>  t <dig>  t <dig>  t <dig>  and t <dig>  the percentage of phenotypic variation  explained by the loci varied from  <dig> to 24% .
two qtls were identified for locule number on chromosomes t <dig> and t <dig> with a lod threshold of  <dig>  .
pves of the qtls were 30% for ln <dig>  and 13% for ln <dig>  .
lod threshold for wall thickness was  <dig>  and two qtls were identified on chromosomes t <dig> and t <dig>  pves of the qtls were  <dig> and 13%, for wall <dig>  and wall <dig> , respectively.
a total of four qtls with lod scores higher than the threshold  were identified for firmness on chromosomes t <dig>  t <dig> and t <dig>  pves of the qtls varied from  <dig> to 24% .
for fruit shape, four qtls were detected on chromosomes t <dig>  t <dig> and t <dig>  .
pves of the qtls for the trait ranged from  <dig> to 26%.
the qtl on chromosome t <dig> had the highest pve  .
two qtls were determined for stem scar  on chromosomes t <dig> and t <dig> .
pves of the qtls were  <dig> and 3% for sc <dig>  and sc <dig> , respectively .
lod thresholds for soluble solids content and ph traits were  <dig>  and  <dig> , respectively .
for soluble solids content, four qtls were identified on chromosomes t <dig>  t <dig>  t <dig> and t <dig>  pves of the qtls varied from  <dig> to 34%.
the qtl on chromosome t <dig> had the highest pve  .
for ph, two qtls were identified on chromosomes t <dig> and t <dig>  the qtl on chromosome t <dig> had a major allelic effect with a pve of 47%.
the pve of the qtl on chromosome t <dig> was 14% .
colocalization of the qtls indicates that a given qtl has an effect on more than one trait.
in the present study, a few colocalized qtls were detected.
qtls on chromosomes t <dig> and t <dig> for locule number colocalized with qtls for fruit weight and fruit shape, respectively.
qtls on chromosomes t <dig> and t <dig> for wall thickness colocalized with qtls for soluble solids content and fruit shape, respectively.
qtls for ph and external color colocalized on chromosome t <dig> .
qtl mapping revealed several s. pimpinellifolium alleles potentially useful for improving fruit traits.
a total of  <dig> qtls for traits such as fruit weight, external and internal color, firmness, soluble solids content and stem scar had favourable alleles provided by the s. pimpinellifolium parent.
s. pimpinellifolium had the highest breeding potential for internal color with six qtls on chromosomes t <dig>  t <dig>  t <dig> and t <dig> having s. pimpinellifolium alleles that improved the trait .
all the qtls for external color and firmness on chromosomes t <dig>  t <dig>  t <dig> and t <dig> had favourable s. pimpinellifolium alleles with total pves of  <dig> and 75%, respectively.
for soluble solids content, s. pimpinellifolium alleles improved the trait for two qtls, with pves of 23% and 34% .
also single qtls on chromosomes t <dig> and t <dig> for fruit weight and stem scar, respectively, had wild alleles which improved the traits = with pves of 15% and 3%, .
discussion
snp identification by gbs analysis
the analysis of gbs data in conjunction with a well-established reference genome is a relatively straightforward route for snp calling and marker ordering along chromosomes  <cit> .
in this work, most of the sequence tags  could be uniquely aligned to the tomato reference genome.
this was expected because complete genome assemblies of tomato are available.
in the present study, although  <dig>  snps were discovered by comparison of the s. lycopersicum and s. pimpinellifolium genome sequences, most of the snps  were excluded due to a high proportion of missing data.
nevertheless, a sufficient number of validated snp markers  remained and were found to be useful for qtl mapping.
in comparison with previous work, this study discovered fewer validated snps than are available in the solanaceae genomics network database  and those reported by chen et al.
<cit>  .
the current number of snps is also much lower than discovered by causse et al.
<cit>   and kim et al.
<cit>  ; however, these snps were not validated.
the average frequency of snps identified in this study was  <dig> snp per  <dig>  kb, much higher than reported for the s. lycopersicum × s. pimpinellifolium linkage maps of salinan et al.
<cit>  , capel et al.
<cit>   and chen et al.
<cit>  .
thus, the present research demonstrated that the gbs approach was efficient in constructing a snp-based physical map of sufficient resolution for qtl mapping in tomato.
phenotypic variation
the ibl population and parental genotypes were evaluated for  <dig> fruit quality traits in order to identify associated qtls.
sizable variation for all traits except soluble solids content and ph, and normal continuous distribution of all but three traits  were observed in the ibl population.
external color and locule number tended to skew toward more intense red color and higher locule numbers due to the unbalanced nature of the ibl population which favors the recurrent parent genotype.
the parents of the ibl population had extreme alleles for fruit weight, wall thickness, stem scar and soluble solids content traits .
although the parental alleles for soluble solids content were extreme, low variation was observed in the ibl population for the trait.
this finding implies an unbalanced introgression of s. pimpinellifolium alleles for soluble solids content into the s. lycopersicum genome.
the present study demonstrated correlations between fruit quality traits, however, most of the significant correlations were weak.
correlations between fruit weight and all traits except fruit shape, internal color, dry matter weight and ph demonstrated that fruit weight was associated with fruit quality traits such as locule number, wall thickness, firmness and stem scar.
fruit weight had a high positive correlation with locule number.
this is expected because increased locule number has a direct effect on fruit size and weight.
negative correlations of fruit weight with external color and soluble solids content indicate that intensity of external color decreases with increased fruit size due to decreased lycopene content and that sucrose content is negatively correlated with fruit volume.
this negative correlation was also reported by chen et al.
<cit> , doganlar et al.
<cit> , sun et al.
<cit>  and fulton et al.
<cit> .
correlation results between fruit weight and quality traits were consistent with the results of lippman and tanksley  <cit> , okmen et al.
<cit>  and fulton et al.
<cit> .
a direct effect of soluble solids content on dry matter weight was observed in the ibl population.
the positive correlation between internal color and external color was expected and consistent with previous reports .
these correlations can also be attributed to the pleiotropic effects of genes on different fruit quality traits.
qtl mapping
fruit quality parameters are important agronomic traits that increase the market value of both fresh market and processing tomatoes.
thus, there are many reports on qtl identification for fruit quality traits.
all previous qtl mapping studies were performed using low density linkage maps constructed with pcr-based markers  and rflp probes.
various parental lines and mapping populations such as bc2f <dig>  ibl and ril were used in these previous studies.
this is the first study in which qtls for fruit quality traits were identified by constructing a high density snp-based physical map using a recently developed ibl population that carries introgressions from the s. pimpinellifolium genome.
the physical map of snp markers was useful for qtl mapping as ibls are unbalanced populations which are not suitable for linkage map construction.
fruit weight is the focus of many studies because increased fruit weight has direct effects on tomato yield .
fruit size is also an important trait that directs consumer preferences.
medium and large tomatoes are usually preferred by consumers  <cit> .
in this work, three qtls were identified on chromosomes t <dig>  t <dig> and t <dig> for fruit weight.
previous studies identified three major and two minor qtls on chromosomes t <dig>  t <dig>  t <dig>  t <dig> and t <dig>  although qtl locations varied among these studies, all studies identified a qtl with major effect on chromosome t <dig> corresponding to a cloned gene that controls fruit weight   <cit> .
in the present study, the fruit weight qtl on chromosome t <dig> explained a variance of 15% for the trait, a value which is relatively low when compared with the same qtl in other studies .
differences in qtl magnitudes of effect and locations are most likely due to differences in population type used in the studies.
the present work is most similar to the work of doganlar et al.
<cit>  which also studied an ibl population, which used a processing tomato as the recurrent parent.
pve of the qtl on chromosome t <dig> was the same as that reported by doganlar et al.
<cit>   due to the similarity of the genetic structures of the populations  used in the two studies.
identification of previously undetected qtls on chromosomes t <dig> and t <dig> in the present work can be attributed to variation in the genetic backgrounds of the two mapping populations which is due to the use of different recurrent parents.
because dried tomatoes have a high economic value, fruit dry matter weight can be as important as fruit weight.
a previous qtl mapping study performed by saliba-colombani et al.
<cit>  identified qtls  on chromosomes t <dig>  t <dig> and t <dig> in a ril population developed from the cross between a cherry tomato cultivar and s. lycopersicum.
in other work, qtls were identified on chromosomes t <dig>  t <dig>  t <dig> and t <dig> using  <dig> introgression lines carrying s. chmielewski introgressions in a s. lycopersicum genetic background  <cit> .
in the present study, none of the above mentioned qtls were detected.
this result can be due to insufficient variation for dry matter weight between the parents and the moderate coefficient of variation detected for the trait in the mapping population.
pves of identified qtls ranged from  <dig> to 19%, suggesting that in contrast to fruit weight, dry matter weight is not controlled by major effect qtls.
in the present study, while a total of nine qtls were identified for internal color, only two loci were identified for external color.
the low number of qtls identified for external color might be due to the unbalanced segregation of the trait in the ibl population.
previous work detected qtls for external color on chromosomes t <dig>  t <dig>  t <dig>  t <dig>  t <dig>  t <dig>  t <dig> and t <dig> .
although a qtl was also identified on chromosome t <dig> in this work, the physical position of the closest marker  to the locus on the same chromosome by okmen et al.
<cit>  reveals that the two qtls are not identical.
for internal color, previous studies identified qtls on chromosomes t <dig>  t <dig>  t <dig>  t <dig>  t <dig>  t <dig> and t <dig> with pves that ranged between  <dig> to 30%  <cit> .
in the present work, qtls for internal color were identified on chromosomes t <dig>  t <dig> and t <dig>  the physical positions of the markers  linked to the three qtls indicated that they do not overlap with the qtls identified in previous work.
previous studies showed that locule number is controlled by six qtls on chromosomes t <dig>  t <dig>  t <dig>  t <dig>  t <dig> and t <dig>  <cit> .
in addition, a major gene for locule number was mapped at the  <dig>  mb position on chromosome t <dig>  <cit> .
the major qtl  containing this single gene  was also identified in the present study .
in addition to this major qtl, a new qtl with minor effect was identified on chromosome t <dig>
wall thickness and firmness are important fruit quality traits that define the shelf life of tomatoes.
qtls with minor effects on wall thickness were reported on chromosomes t <dig>  t <dig>  t <dig> and t <dig>  <cit> , however, these loci do not overlap with those reported in the present work.
previously, qtls for firmness were identified on chromosomes t <dig>  t <dig>  t <dig>  t <dig>  t <dig>  t <dig> and t <dig>  <cit> .
in addition to these previously identified qtls, four new qtls were identified for firmness trait in this work.
fruit shape and stem scar are appearance traits analysed in this study.
globular fruits with small stem scar are favoured in the market.
more than  <dig> qtls for fruit shape were identified in previous studies  <cit> .
in addition to these qtls, a total of four new qtls were identified in this work with minor effects on fruit shape.
for stem scar, seven qtls were previously identified in tomato  <cit> .
one of the two qtls identified in this study for the stem scar was previously reported at  <dig>  mb position on chromosome t <dig> with a low pve of 8%  <cit> .
soluble solids content and ph are important traits for fresh market tomatoes as they help define flavor  <cit> .
a total of five qtls were detected on chromosomes t <dig>  t <dig>  t <dig> and t <dig> in previous studies for soluble solids content  <cit>  the present report demonstrated that different qtls  control soluble solids content in fresh market tomatoes.
for ph, a total of six qtls were identified in tomato on chromosomes t <dig>  t <dig>  t <dig>  t <dig>  t <dig> and t <dig> in previous studies  <cit> .
while the position of the previously identified qtl on chromosome t <dig>  <cit>  was at 86 mb, the major effect qtl  identified on the same chromosome in this work was positioned at  <dig>  mb.
thus, the qtl identified in this study is close to the qtl previously identified by chen et al.
<cit> .
these two qtls might actually overlap because the snp-based map of the present study has much higher resolution than the linkage map of chen et al.
<cit> .
some qtls colocalized as expected.
for example, qtls for locule number coincided with those for fruit weight and fruit shape because increased locule number results in larger tomatoes.
however, colocalization of a qtl for ph with one for external color and colocalization of loci for wall thickness and soluble solids content were unexpected.
these unexpected colocalizations might be due to linkage of the genes that control the traits  <cit> .
this present study confirmed the high breeding potential of s. pimpinellifolium by detecting useful alleles for breeding of fruit quality traits such as fruit weight, external and internal color, firmness, soluble solids content and stem scar.
the findings were expected for external color and soluble solids content because s. pimpinellifolium had higher values than cultivated tomato.
in contrast, although s. pimpinellifolium had lower values than s. lycopersicum for fruit weight, internal color and stem scar, favorable s. pimpinellifolium alleles were detected for these traits.
this result was consistent with the work of top et al.
<cit> .
in that study, although s. pimpinellifolium had lower values than cultivated tomato for fruit weight and firmness, some individuals from an ibl  population derived from the cross s. lycopersicum and s. pimpinellifolium  showed higher values than s. lycopersicum .
CONCLUSIONS
the present research demonstrated that gbs was an efficient technique for construction of a snp-based physical map with sufficient resolution for mapping fruit quality qtls in tomato.
the identified snps were well distributed in the tomato genome.
this study also revealed valuable s. pimpinellifolium alleles for most of the traits.
thus, in this work, valuable findings were obtained for unlocking the genetic potential of this wild species for tomato breeding.
additional files
additional file 1: table s <dig>  tassel software parameters used for snp calling and filtering.
table s <dig>  types of substitutions represented by the identified snp loci.
table s <dig>  significant  correlations between tomato fruit traits.
correlations with p value >  <dig>  were considered to be non-significant .
fw = fruit weight, dw = dry matter weight, exc = external color, inc = internal color, ln = locule number, wall = wall thickness, firm = firmness, fs = fruit shape, scar = stem scar, ssc = soluble solids content.
table s <dig>  lod thresholds of  <dig> fruit quality traits calculated by  <dig>  random permutations with parameters .
table s <dig>  fruit quality qtls that colocalized.
figure s <dig>  frequency distributions for the fruit traits in the ibl population.
fw = fruit weight, dw = dry matter weight, exc = external color, inc = internal color, ln = locule number, wall = wall thickness, firm = firmness, fs = fruit shape, scar = stem scar, ssc = soluble solids content.
all the traits except external color, locule number and fruit shape showed normal and continuous distribution
additional file 2: additional excel file containing physical maps of identified and filtered snps and the linkage map.
abbreviations
abadvanced backcross
cimcomposite interval mapping
cosiiconserved ortholog set ii
cvcoefficients of variation
dwdry matter weight
excexternal color
firmfirmness
fsfruit shape
fwfruit weigh
gbsgenotyping by sequencing
hrmhigh-resolution melting
iblinbred backcross lines
incinternal color
lnlocule number
ngsnext-generation sequencing
pvepercentage of phenotypic variation explained by the qtl
qtlquantitative trait loci
rflprestriction fragment length polymorphism
scarstem scar
snpsingle nucleotide polymorphism
sscsoluble solids content
ssrsimple sequence sepeats
tbttags by taxa
topmtags on physical map
wallwall thickness

 solanum pimpinellifolium has high breeding potential for biotic and abiotic stress tolerance and fruit quality traits such as fruit weight, internal and external color, firmness, fruit shape, lycopene and high soluble solids content. as a result, this wild species has been frequently used as a donor in tomato breeding programs. in addition, the s. pimpinellifolium genome is reported to harbor more favorable and fewer unfavorable alleles for breeding improved cultivars compared to other wild tomato species including s. cheesmanii, s. chmielewskii and s. habrochaites. s. pimpinellifolium is more closely related to cultivated tomato than the other wild tomato species. thus, utilization of s. pimpinellifolium in breeding minimizes the consequences of linkage drag. efficient introgression of favorable alleles from s. pimpinellifolium into s. lycopersicum by both positive and background selection requires high-throughput marker identification and genotyping as well as identification of the qtls that control the traits of interest. cultivated tomato has extensive genomic resources for molecular breeding. the genome has been completely sequenced and a large amount of data about genome-wide intraspecific and interspecific allelic variation are available. more than interspecific tomato linkage maps were constructed using ssr, cosii and snp markers. nine of these maps were constructed using mapping populations derived from crosses between s. pimpinellifolium and s. lycopersicum for mapping of fruit quality traits and disease tolerance. most of these low resolution linkage maps were constructed with rflp, cosii and ssr markers. four recently constructed linkage maps contain snp markers designed from the solanaceae genomics network and tomato mapping resource databases. the maps introduced by salinan et al. and capel et al., respectively, contained and snp markers genotyped in s. pimpinellifolium and s. lycopersicum using the high-resolution melting method. another map with markers was constructed with snps discovered by restriction site associated dna sequencing technology and veracode snp makers. the traitgenetics expimp linkage map had the highest number of interspecific snp markers. two additional studies have described development of snp markers by sequencing. in the work of causse et al., eight s. lycopersicum accessions were sequenced to identify unique, non-synonymous snps and putative copy-number variations. the other study used sequencing data available from the ncbi database for nine tomato accessions is a practical and inexpensive method for high-throughput snp discovery and genotyping. the gbs approach uses next-generation sequencing technologies for multiplex sequencing of restriction site-associated dna. alignment of the sequence reads generated from a population enables simultaneous snp discovery and genotyping. to date, the gbs approach has been commonly used for snp discovery and genotyping in crop species such as wheat, rice and maize. however, the rad-seq method, which is similar to gbs has been used in tomato. this technique was used to discover snps in an f2: population derived from a cross between s. pimpinellifolium and s. lycopersicum to identify qtls for late blight resistance ). the primary goal of the current study was to use the gbs approach for high-throughput identification of interspecific snps in the s. pimpinellifolium and s. lycopersicum cv. tueza genomes. the second aim of this work was the identification of qtls for fruit quality traits using the identified markers. to achieve these aims, an ibl population comprising individuals was developed using an advanced backcross qtl strategy. the donor parent was the wild tomato species s. pimpinellifolium and the recurrent parent was an elite fresh market tomato, s. lycopersicum cv. tueza. ibl populations are used to introgress favorable alleles from wild species into the cultivated genome in tomato breeding. although unbalanced populations such as ibls are not suitable for linkage map construction due to unequal representation of the parental genomes, they are valuable genetic resources for qtl mapping because ibl populations contain small introgressions from the wild species in the cultivated tomato genome. such small introgressions and the higher occurrence of crossing-over events as compared to f and bc populations improve the resolution of qtl mapping in ibl populations. fruit quality traits such as fruit weight, external and internal color, and firmness affect consumer preference and define the market value of tomato cultivars. thus, tomato breeders are interested in the improvement of tomato cultivars for these traits and several studies were performed to identify qtls controlling them. the most comprehensive qtl mapping study for tomato fruit quality traits was performed by doganlar et al.. in this study, qtls were identified for fruit quality traits using an ibl population derived from the cross between s. pimpinellifolium and a processing tomato cultivar. despite the high number of loci identified for fruit quality traits, most qtl analyses were performed using low resolution genetic linkage maps and transient populations, with the notable exceptions of work with ibls and rils. in the present study, qtls were identified for fruit quality traits in an ibl population using interspecific snp loci discovered through a gbs approach. this is the first time that gbs methodology was implemented in tomato for generation of a high resolution physical map for qtl mapping of fruit quality traits. the results of the present study demonstrate the efficiency of gbs for snp discovery and qtl mapping in tomato. methods plant materials an interspecific ibl population derived from the cross s. lycopersicum cv. tueza x s. pimpinellifolium was used as plant material in the study. tueza is a cultivated fresh market tomato line with large, red, slightly flattened round fruits. la is a wild type tomato with small, red, round fruits. the ibl population and parents were grown by multi tohum seed company. a total of plants per genotype were grown in double rows with 140 cm between wide rows and 50 cm between narrow rows. within rows, plants were spaced at 40 cm intervals. for basal fertilization, 500 kg 15:15: fertilizer and 50 t of composted manure were applied per ha. drip irrigation was used with fertigation at each irrigation using 1–2– fertilizer until first fruit set, 2–1- fertilizer until first fruit ripening and 1–1- fertilizer after first fruit ripening. total genomic dna was isolated from the leaf tissue bulked from plants of the parental accessions and individuals of the ibl population using a ctab method. genomic dna was quantified using qubit™ quantitation assay. dna integrity was checked on a 1% agarose gel. genotyping by sequencing genomic dna from the individuals of the ibl population and parental accessions were subjected to gbs analysis by the university of wisconsin biotechnology center. sequencing library preparation protocol, including restriction enzyme digestion, use of barcode adapters, sample pooling and amplification, was performed as described by elshire et al.. the 95-plex library was sequenced with the illumina hiseq next-generation sequencing platform. gbs data analysis and snp identification gbs data analysis was performed using the gbs discovery pipeline of tassel version software. the fastq and sample key files generated from raw sequence reads by the casava. software package were used as input for processing in the pipeline. before the analysis, 64-base reads were generated by trimming reads having the barcodes for each genotype followed by an apeki cut site using the fastqtotagcountplugin of the pipeline. reads with unidentified bases were excluded from analysis. the barcoded sequence reads were collapsed into unique sequence tags with counts using the fastqtotagcountplugin with default parameters with the exception that minimum number of times a tag must be present was set to tag count files that contained the sequence tags that passed the minimum count threshold of were merged into a master file using the mergemultipletagcountplugin. the master tags in fastq format generated by tagcounttofastqplugin were aligned to the tomato s. lycopersicum reference genome using the bowtie plugin with default parameters. samconverterplugin generated the “tags on physical map” file which contained information about the physical positions of the master tags which had the best unique alignments with the reference genome. in addition to the topm file, the “tags by taxa” file that contained tag counts of each barcode generated by fastqtotbtplugin was used for snp calling according to the parameters of the tagstosnpbyalignmentplugin. snps were recorded in a hapmap file for each chromosome. mergeduplicatesnpsplugin was used to merge the duplicate snps. snps were filtered based on minimum taxon coverage, minimum site coverage, linkage disequilibrium with neighboring snps, minimum r value for the ld filter:, and minimum bonferroni-corrected p-value for the ld filter:. a physical map of the identified snps was drawn using mapchart software. phenotypic evaluation tomato fruits at the normal market stage were evaluated for qualitative fruit quality traits: fruit weight, dry matter weight, external color, internal color, locule number, wall thickness, firmness, fruit shape, stem scar, total soluble solids content and ph. phenotyping was performed as described in the first such work using s. pimpinellifolium and most of the studies thereafter. fruit weight was determined by bulking the fruit from plants and calculating the mean weight of representative tomato fruits. fruits from plants per genotype were bulked and characterized for external and internal color, fruit firmness, shape, stem scar, locule number, wall thickness and total soluble solids content. external and internal fruit colour were visually determined for each individual using a scale from to. a total of 100 g tomato fruits were dried and weighed to calculate fruit dry matter. fruit firmness was determined by hand squeezing using a scale of to. ratio of fruit length to fruit width represented fruit shape with a scale from to. stem scar diameter represented stem scar size. locule number was counted in transversely-cut fruits. fruit wall thickness was visually determined using a scale from to. total soluble solids content of the tomato fruits was measured using a refractometer. the ph of juice from the fruits was measured with a ph meter. qtl mapping qgene version was used for qtl analysis. the cim qtl analysis method uses both interval mapping and multiple regression analysis and was performed with automatic forward cofactor selection and a scan interval of  mb. a total of random permutations were performed with parameters to calculate the genome-wide lod threshold. correlation analysis between traits was performed using pasw software.  gbs a total of, reads were generated by sequencing of the 95-plex library. from these reads, sequence tags were generated. a majority of the sequence tags were uniquely aligned to the tomato genome. the remaining tags were either aligned to multiple positions or could not be located to the tomato genome assembly. the sequence tags uniquely aligned to the genome were used for genome-wide high-throughput snp discovery. sequences of the merged gbs tags in sam file format can be accessed at sra database under accession number srp snp identification tag alignment to the reference genome revealed unique snp loci between the s. lycopersicum and s. pimpinellifolium genomes. the snp loci were found on all chromosomes of tomato. the physical map constructed with the identified snp loci had sufficient coverage for genome analysis such as qtl mapping and contained snps that were evenly distributed along the chromosomes with gaps in size of more than 2 mb. after filtration based on the proportion of missing data and parameters such as minimum taxon coverage, minimum site coverage, linkage disequilibrium with neighboring snps, minimum r value for the ld filter:, minimum bonferroni-corrected p-value for the ld filter:, a total of snp loci were retained. while the average distance between adjacent loci was  kb for the pre-filtration snps, frequency was reduced to one snp per  kb after filtering. chromosome t had the highest frequency of filtered snps with an average distance of  kb between adjacent markers.fig. physical map of the snps retained after the filtering process. detailed information about the physical map and snp locations is available in the additional file 2 anumber and frequency of snps physically mapped in tomato genome bnumber and frequency of snps retained after the filtration process the majority of the snps identified in this study were transition mutations as expected. the most frequently observed substitution types, a/g and c/t transitions, had similar frequencies: and %, respectively. c/g transversion was the least common substitution type. the observed transition/transversion ratio was. phenotypic variation a total of ibl individuals and parental accessions were characterized for fruit quality traits including fruit weight, dry matter weight, external and internal color, locule number, wall thickness, firmness, fruit shape, stem scar, soluble solids content and ph. the parents of the ibl population had extreme phenotypes for fruit weight, wall thickness, stem scar and soluble solids content traits. all of the traits segregated in the ibl population. fruit weight, wall thickness, firmness and stem scar traits displayed the highest variation in the population with coefficients of variation ranging from to %,. with the exception of soluble solids content and ph, the remaining traits had considerable variation in the population. the ph and soluble solids contents displayed the lowest variation with and % cv, respectively. all traits except external color, locule number and fruit shape displayed normal and continuous distributions.table statistics for fruit quality traits measured in ibl population and parents; s. lycopersicum cv. tueza and s. pimpinellifolium cv. la1589 correlation analysis demonstrated significant associations between some traits. fruit weight was correlated to all traits except dry matter weight, internal color, fruit shape and ph. fruit weight was positively correlated to locule number and wall thickness and negatively weakly correlated to external color and soluble solids content. dry matter weight was moderately correlated to soluble solids content. external color was weakly correlated to internal color and locule number. locule number was moderately correlated to stem scar. qtl mapping the genome-wide snp loci that were retained after filtering were used in qtl mapping of the fruit quality traits. cim analysis was performed and a logarithm of odds threshold generated by permutations was used to identify qtls for each trait. for fruit weight, the lod threshold was and three qtls were identified on chromosomes t t and t. the percentage of phenotypic variation explained by the qtls varied from to 26%. the qtl on chromosome t had the highest pve, 26%. three qtls were identified for dry matter weight on chromosome t based on a lod threshold of. the pves of these loci were and 14% for dw, dw and dw, respectively.table qtls identified for fruit quality traits effectc s. pimpinellifolium s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. lycopersicum s. lycopersicum s. pimpinellifolium s. pimpinellifolium s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum s. pimpinellifolium s. pimpinellifolium s. pimpinellifolium s. lycopersicum s. lycopersicum s. lycopersicum s. lycopersicum apeak position of qtl bpercentage of phenotypic variation explained by identified qtl cparental allele associated with increased trait value a total of qtls were identified for external and internal color in tomato based on lod thresholds of and respectively. for the external color, two qtls were identified on chromosomes t and t with a total pve of 21%. a total of nine qtls were identified for internal color on chromosomes t t t t t t and t the percentage of phenotypic variation explained by the loci varied from to 24%. two qtls were identified for locule number on chromosomes t and t with a lod threshold of. pves of the qtls were 30% for ln and 13% for ln. lod threshold for wall thickness was and two qtls were identified on chromosomes t and t pves of the qtls were and 13%, for wall and wall, respectively. a total of four qtls with lod scores higher than the threshold were identified for firmness on chromosomes t t and t pves of the qtls varied from to 24%. for fruit shape, four qtls were detected on chromosomes t t and t. pves of the qtls for the trait ranged from to 26%. the qtl on chromosome t had the highest pve. two qtls were determined for stem scar on chromosomes t and t. pves of the qtls were and 3% for sc and sc, respectively. lod thresholds for soluble solids content and ph traits were and, respectively. for soluble solids content, four qtls were identified on chromosomes t t t and t pves of the qtls varied from to 34%. the qtl on chromosome t had the highest pve. for ph, two qtls were identified on chromosomes t and t the qtl on chromosome t had a major allelic effect with a pve of 47%. the pve of the qtl on chromosome t was 14%. colocalization of the qtls indicates that a given qtl has an effect on more than one trait. in the present study, a few colocalized qtls were detected. qtls on chromosomes t and t for locule number colocalized with qtls for fruit weight and fruit shape, respectively. qtls on chromosomes t and t for wall thickness colocalized with qtls for soluble solids content and fruit shape, respectively. qtls for ph and external color colocalized on chromosome t. qtl mapping revealed several s. pimpinellifolium alleles potentially useful for improving fruit traits. a total of qtls for traits such as fruit weight, external and internal color, firmness, soluble solids content and stem scar had favourable alleles provided by the s. pimpinellifolium parent. s. pimpinellifolium had the highest breeding potential for internal color with six qtls on chromosomes t t t and t having s. pimpinellifolium alleles that improved the trait. all the qtls for external color and firmness on chromosomes t t t and t had favourable s. pimpinellifolium alleles with total pves of and 75%, respectively. for soluble solids content, s. pimpinellifolium alleles improved the trait for two qtls, with pves of 23% and 34%. also single qtls on chromosomes t and t for fruit weight and stem scar, respectively, had wild alleles which improved the traits = with pves of 15% and 3%,. discussion snp identification by gbs analysis the analysis of gbs data in conjunction with a well-established reference genome is a relatively straightforward route for snp calling and marker ordering along chromosomes. in this work, most of the sequence tags could be uniquely aligned to the tomato reference genome. this was expected because complete genome assemblies of tomato are available. in the present study, although snps were discovered by comparison of the s. lycopersicum and s. pimpinellifolium genome sequences, most of the snps were excluded due to a high proportion of missing data. nevertheless, a sufficient number of validated snp markers remained and were found to be useful for qtl mapping. in comparison with previous work, this study discovered fewer validated snps than are available in the solanaceae genomics network database and those reported by chen et al.. the current number of snps is also much lower than discovered by causse et al. and kim et al.; however, these snps were not validated. the average frequency of snps identified in this study was snp per  kb, much higher than reported for the s. lycopersicum × s. pimpinellifolium linkage maps of salinan et al., capel et al. and chen et al.. thus, the present research demonstrated that the gbs approach was efficient in constructing a snp-based physical map of sufficient resolution for qtl mapping in tomato. phenotypic variation the ibl population and parental genotypes were evaluated for fruit quality traits in order to identify associated qtls. sizable variation for all traits except soluble solids content and ph, and normal continuous distribution of all but three traits were observed in the ibl population. external color and locule number tended to skew toward more intense red color and higher locule numbers due to the unbalanced nature of the ibl population which favors the recurrent parent genotype. the parents of the ibl population had extreme alleles for fruit weight, wall thickness, stem scar and soluble solids content traits. although the parental alleles for soluble solids content were extreme, low variation was observed in the ibl population for the trait. this finding implies an unbalanced introgression of s. pimpinellifolium alleles for soluble solids content into the s. lycopersicum genome. the present study demonstrated correlations between fruit quality traits, however, most of the significant correlations were weak. correlations between fruit weight and all traits except fruit shape, internal color, dry matter weight and ph demonstrated that fruit weight was associated with fruit quality traits such as locule number, wall thickness, firmness and stem scar. fruit weight had a high positive correlation with locule number. this is expected because increased locule number has a direct effect on fruit size and weight. negative correlations of fruit weight with external color and soluble solids content indicate that intensity of external color decreases with increased fruit size due to decreased lycopene content and that sucrose content is negatively correlated with fruit volume. this negative correlation was also reported by chen et al., doganlar et al., sun et al. and fulton et al.. correlation results between fruit weight and quality traits were consistent with the results of lippman and tanksley, okmen et al. and fulton et al.. a direct effect of soluble solids content on dry matter weight was observed in the ibl population. the positive correlation between internal color and external color was expected and consistent with previous reports. these correlations can also be attributed to the pleiotropic effects of genes on different fruit quality traits. qtl mapping fruit quality parameters are important agronomic traits that increase the market value of both fresh market and processing tomatoes. thus, there are many reports on qtl identification for fruit quality traits. all previous qtl mapping studies were performed using low density linkage maps constructed with pcr-based markers and rflp probes. various parental lines and mapping populations such as bc2f ibl and ril were used in these previous studies. this is the first study in which qtls for fruit quality traits were identified by constructing a high density snp-based physical map using a recently developed ibl population that carries introgressions from the s. pimpinellifolium genome. the physical map of snp markers was useful for qtl mapping as ibls are unbalanced populations which are not suitable for linkage map construction. fruit weight is the focus of many studies because increased fruit weight has direct effects on tomato yield. fruit size is also an important trait that directs consumer preferences. medium and large tomatoes are usually preferred by consumers. in this work, three qtls were identified on chromosomes t t and t for fruit weight. previous studies identified three major and two minor qtls on chromosomes t t t t and t although qtl locations varied among these studies, all studies identified a qtl with major effect on chromosome t corresponding to a cloned gene that controls fruit weight. in the present study, the fruit weight qtl on chromosome t explained a variance of 15% for the trait, a value which is relatively low when compared with the same qtl in other studies. differences in qtl magnitudes of effect and locations are most likely due to differences in population type used in the studies. the present work is most similar to the work of doganlar et al. which also studied an ibl population, which used a processing tomato as the recurrent parent. pve of the qtl on chromosome t was the same as that reported by doganlar et al. due to the similarity of the genetic structures of the populations used in the two studies. identification of previously undetected qtls on chromosomes t and t in the present work can be attributed to variation in the genetic backgrounds of the two mapping populations which is due to the use of different recurrent parents. because dried tomatoes have a high economic value, fruit dry matter weight can be as important as fruit weight. a previous qtl mapping study performed by saliba-colombani et al. identified qtls on chromosomes t t and t in a ril population developed from the cross between a cherry tomato cultivar and s. lycopersicum. in other work, qtls were identified on chromosomes t t t and t using introgression lines carrying s. chmielewski introgressions in a s. lycopersicum genetic background. in the present study, none of the above mentioned qtls were detected. this result can be due to insufficient variation for dry matter weight between the parents and the moderate coefficient of variation detected for the trait in the mapping population. pves of identified qtls ranged from to 19%, suggesting that in contrast to fruit weight, dry matter weight is not controlled by major effect qtls. in the present study, while a total of nine qtls were identified for internal color, only two loci were identified for external color. the low number of qtls identified for external color might be due to the unbalanced segregation of the trait in the ibl population. previous work detected qtls for external color on chromosomes t t t t t t t and t. although a qtl was also identified on chromosome t in this work, the physical position of the closest marker to the locus on the same chromosome by okmen et al. reveals that the two qtls are not identical. for internal color, previous studies identified qtls on chromosomes t t t t t t and t with pves that ranged between to 30%. in the present work, qtls for internal color were identified on chromosomes t t and t the physical positions of the markers linked to the three qtls indicated that they do not overlap with the qtls identified in previous work. previous studies showed that locule number is controlled by six qtls on chromosomes t t t t t and t. in addition, a major gene for locule number was mapped at the  mb position on chromosome t. the major qtl containing this single gene was also identified in the present study. in addition to this major qtl, a new qtl with minor effect was identified on chromosome t wall thickness and firmness are important fruit quality traits that define the shelf life of tomatoes. qtls with minor effects on wall thickness were reported on chromosomes t t t and t, however, these loci do not overlap with those reported in the present work. previously, qtls for firmness were identified on chromosomes t t t t t t and t. in addition to these previously identified qtls, four new qtls were identified for firmness trait in this work. fruit shape and stem scar are appearance traits analysed in this study. globular fruits with small stem scar are favoured in the market. more than qtls for fruit shape were identified in previous studies. in addition to these qtls, a total of four new qtls were identified in this work with minor effects on fruit shape. for stem scar, seven qtls were previously identified in tomato. one of the two qtls identified in this study for the stem scar was previously reported at  mb position on chromosome t with a low pve of 8%. soluble solids content and ph are important traits for fresh market tomatoes as they help define flavor. a total of five qtls were detected on chromosomes t t t and t in previous studies for soluble solids content the present report demonstrated that different qtls control soluble solids content in fresh market tomatoes. for ph, a total of six qtls were identified in tomato on chromosomes t t t t t and t in previous studies. while the position of the previously identified qtl on chromosome t was at 86 mb, the major effect qtl identified on the same chromosome in this work was positioned at  mb. thus, the qtl identified in this study is close to the qtl previously identified by chen et al.. these two qtls might actually overlap because the snp-based map of the present study has much higher resolution than the linkage map of chen et al.. some qtls colocalized as expected. for example, qtls for locule number coincided with those for fruit weight and fruit shape because increased locule number results in larger tomatoes. however, colocalization of a qtl for ph with one for external color and colocalization of loci for wall thickness and soluble solids content were unexpected. these unexpected colocalizations might be due to linkage of the genes that control the traits. this present study confirmed the high breeding potential of s. pimpinellifolium by detecting useful alleles for breeding of fruit quality traits such as fruit weight, external and internal color, firmness, soluble solids content and stem scar. the findings were expected for external color and soluble solids content because s. pimpinellifolium had higher values than cultivated tomato. in contrast, although s. pimpinellifolium had lower values than s. lycopersicum for fruit weight, internal color and stem scar, favorable s. pimpinellifolium alleles were detected for these traits. this result was consistent with the work of top et al.. in that study, although s. pimpinellifolium had lower values than cultivated tomato for fruit weight and firmness, some individuals from an ibl population derived from the cross s. lycopersicum and s. pimpinellifolium showed higher values than s. lycopersicum.  the present research demonstrated that gbs was an efficient technique for construction of a snp-based physical map with sufficient resolution for mapping fruit quality qtls in tomato. the identified snps were well distributed in the tomato genome. this study also revealed valuable s. pimpinellifolium alleles for most of the traits. thus, in this work, valuable findings were obtained for unlocking the genetic potential of this wild species for tomato breeding. additional files additional file 1: table s tassel software parameters used for snp calling and filtering. table s types of substitutions represented by the identified snp loci. table s significant correlations between tomato fruit traits. correlations with p value >  were considered to be non-significant. fw = fruit weight, dw = dry matter weight, exc = external color, inc = internal color, ln = locule number, wall = wall thickness, firm = firmness, fs = fruit shape, scar = stem scar, ssc = soluble solids content. table s lod thresholds of fruit quality traits calculated by random permutations with parameters. table s fruit quality qtls that colocalized. figure s frequency distributions for the fruit traits in the ibl population. fw = fruit weight, dw = dry matter weight, exc = external color, inc = internal color, ln = locule number, wall = wall thickness, firm = firmness, fs = fruit shape, scar = stem scar, ssc = soluble solids content. all the traits except external color, locule number and fruit shape showed normal and continuous distribution additional file 2: additional excel file containing physical maps of identified and filtered snps and the linkage map. abbreviations abadvanced backcross cimcomposite interval mapping cosiiconserved ortholog set ii cvcoefficients of variation dwdry matter weight excexternal color firmfirmness fsfruit shape fwfruit weigh gbsgenotyping by sequencing hrmhigh-resolution melting iblinbred backcross lines incinternal color lnlocule number ngsnext-generation sequencing pvepercentage of phenotypic variation explained by the qtl qtlquantitative trait loci rflprestriction fragment length polymorphism scarstem scar snpsingle nucleotide polymorphism sscsoluble solids content ssrsimple sequence sepeats tbttags by taxa topmtags on physical map wallwall thickness 
the present work introduced a set of snps at sufficiently high density for qtl mapping in populations derived from s. pimpinellifolium. a total of unique snp loci were identified as a result of tag alignment to the genome assembly and were used in qtl analysis of fruit quality traits. solanum pimpinellifolium has high breeding potential for fruit quality traits and has been used as a donor in tomato breeding programs. a total of, reads were generated by the illumina hiseq next-generation sequencing platform. unlocking the genetic potential of s. pimpinellifolium requires high-throughput polymorphism identification protocols for qtl mapping and introgression of favourable alleles into cultivated tomato by both positive and background selection. s. pimpinellifolium contributed favourable alleles for qtls, thus confirming the high breeding potential of this wild species. in this study we identified snp loci using a genotyping by sequencing approach in an ibl mapping population derived from the cross between a high yielding fresh market tomato and s. pimpinellifolium as the recurrent and donor parents, respectively. moreover, this study demonstrated the high efficiency of the gbs approach for snp identification, genotyping and qtl mapping in an interspecific tomato population. a majority of the sequence tags were uniquely aligned to the tomato genome. fruit weightphsoluble solids contenttomato genomehttp://dx.doi.org/ /501100004410türkiye bilimsel ve teknolojik araştirma kurumu114z116doganlar sami issue-copyright-statement© the author 2017 from these reads sequence tags were generated.
 solanum pimpinellifolium has high breeding potential for fruit quality traits and has been used as a donor in tomato breeding programs. unlocking the genetic potential of s. pimpinellifolium requires high-throughput polymorphism identification protocols for qtl mapping and introgression of favourable alleles into cultivated tomato by both positive and background selection.  in this study we identified snp loci using a genotyping by sequencing approach in an ibl mapping population derived from the cross between a high yielding fresh market tomato and s. pimpinellifolium as the recurrent and donor parents, respectively. a total of, reads were generated by the illumina hiseq next-generation sequencing platform. from these reads sequence tags were generated. a majority of the sequence tags were uniquely aligned to the tomato genome. a total of unique snp loci were identified as a result of tag alignment to the genome assembly and were used in qtl analysis of fruit quality traits. as a result, qtls were identified. s. pimpinellifolium contributed favourable alleles for qtls, thus confirming the high breeding potential of this wild species.  the present work introduced a set of snps at sufficiently high density for qtl mapping in populations derived from s. pimpinellifolium. moreover, this study demonstrated the high efficiency of the gbs approach for snp identification, genotyping and qtl mapping in an interspecific tomato population. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords fruit weightphsoluble solids contenttomato genomehttp://dx.doi.org/ /501100004410türkiye bilimsel ve teknolojik araştirma kurumu114z116doganlar sami issue-copyright-statement© the author 2017 