BACKGROUND
eggplant  ranks third in commercial importance among the solanaceous crops after potato and tomato, and it is cultivated in many countries, particularly in southern asia, middle east and northern africa.
global production in  <dig> was about  <dig> mt  <cit> , and the largest european producer is italy.
despite its commercial importance, little research effort has been devoted to the genetic analysis of key breeding and quality traits with respect to the other solanaceae crops tomato, potato and capiscum
.
several quantitative trait loci  underlying fruit color and shape have been described by nunome et al.
<cit> , while doganlar et al.
<cit>  and frary et al.
<cit>  succeeded in identifying qtl for certain fruit- and plant-related traits, but only in a population derived from an inter-specific cross, which is of reduced utility in eggplant improvement.
more recently, the genetic basis of parthenocarpy  <cit> , ralstonia solanacearum resistance  <cit> , anthocyanin content  <cit>  and a group of agronomic traits  <cit>  has been elucidated.
the genome-wide association  approach represents an alternative to biparental linkage mapping for the determination of the genetic basis of traits  <cit> .
both approaches rely on recombination to re-arrange the genome  <cit> , and seek to establish correlations between phenotype and genotype, based on the non-random association of alleles at two or more loci, termed linkage disequilibrium .
in a bi-parental population, only the polymorphisms between the two parents can be queried, whereas in a gwa population the number of polymorphisms is determined by the genetic diversity of the entire germplasm panel.
in a gwa population, ld is determined not only by recombination frequency, but also by genetic drift, by the mating system of the plant and by the history of selection .
the major advantages of gwa over biparental linkage mapping lie in the much wider variability in phenotype and genotype made accessible, a history of multiple rounds of recombination in many different lineages and the inclusion of germplasm of direct relevance to crop improvement.
the real risk that the genetic architecture of the germplasm panel may cloud the analysis has to be allayed by a prior evaluation of the population’s structure  <cit> .
to date only one gwa-based study of variation in eggplant has been performed  <cit>  but the present study used a larger number of accessions  and a more densely populated genetic map  vs  <dig> microsatellites) and targeted traits related to anthocyanin accumulation and fruit color.
methods
plant material and dna isolation
a core set of  <dig> accessions , chosen from a large collection of breeding lines, old varieties and landrace selections by cericola et al.
<cit> , was established to represent germplasm grown in east asia  and in the mediterranean basin .
the entries were all highly homozygous and thus phenotypically stable.
genomic dna was extracted from fresh young leaves harvested from three individuals of each accession, using an e.z.n.a.™ plant dna mini kit , according to the manufacturer’s protocol.
the quality of each dna sample was monitored by electrophoresis through an  <dig> % agarose gel and its dna concentration estimated spectrophotometrically .
snp data acquisition
each accession was genotyped at  <dig> snp loci as reported by barchi et al.
<cit> ;  <dig> of these have been genetically mapped  <cit> .
snps were selected taking into account a quality score, based on the probability of good performance using the illumina golden gate assay ; the score > <dig>  indicates a high probability of success.
a blastx search was carried out against the tair <dig> dataset using the  <dig>  highest quality score snps as query; the  <dig> sequences having the highest e-value were then chosen.
the goldengate assay was carried out at the uc davis genome center .
automatic allele calling was handled by gencall software .
two of the entries were included in duplicate as an internal control.
snp loci in which the minimum allele frequency  fell below 5%, along with those where >10% of the data were missing, were discarded.
each snp locus was scored as a binary data point, and the pic  of each was estimated following anderson et al.
<cit> .
population structure
genetic similarities between pairs of entries were quantified by the dice  <cit>  similarity index, then used to describe genetic relationships using both the unweighted pair-group arithmetic mean  method, and principal coordinate analysis  by means of past  <dig>  software  <cit> .
structure v <dig>  software  <cit>  was used to estimate the number of sub-populations in the panel, applying the admixture model for the ancestry of individuals and correlated allele frequencies.
the population structure was modelled with a burn-in of  <dig>  cycles followed by  <dig>  markov chain monte carlo  repeats.
the evanno et al.
<cit>  transformation method was then used to infer k, the most likely number of populations.
pair-wise kinship coefficients between the accessions were estimated using spagedi software  <cit> .
the diagonal of the matrix was set to two, and negative values were set to  <dig>  following yu et al.
<cit> .
ld analysis
ld decay was quantified by plotting pair-wise r <dig> values against the distance  between adjacent snp loci, based on the genetic map developed by barchi et al.
<cit> .
the effect of population structure on ld was investigated with three approaches as suggested by mangin et al.
<cit> : r <dig> ; r2s  and r2sv .
to quantify the reach of ld, an r <dig> threshold of  <dig>  was set  <cit> .
the relationship between the baseline r <dig> values and genetic distance was determined using a locally weighted scatter plot smoothing line  <cit> .
to visualize ld throughout the genome, heat maps were produced based on pair-wise r <dig>  r2s and r2sv values  <cit> .
the estimation of all ld measures was carried out by programs implemented in the r package ldcorsv
<cit> .
acquisition and analysis of phenotypic and morphological data
the accessions were grown in field both at montanaso lombardo  and at monsampolo del tronto  in  <dig> and again in  <dig>  in each trial, the material was set out as two randomized complete blocks with six plants per entry per block, and standard horticultural practices were applied.
phenotyping methodology was based on the european cooperative programme for plant genetic resources solanaceae descriptors  and the international board for plant genetic resource descriptors for eggplant .
the traits assayed were adaxial leaf lamina anthocyanin , stem anthocyanin , abaxial leaf lamina anthocyanin , calyx anthocyanin , corolla color , adaxial leaf venation anthocyanin , abaxial leaf venation anthocyanin , peduncle anthocyanin , fruit color  and fruit glossiness .
the anthocyanin content of the vegetative part of the plant was scored on a 0– <dig> scale, with “0” representing no visible anthocyanin pigmentation , and “5” representing dark violet tissue.
corcol was scored as “0” for white, “1” for pink and “2” for violet.
fcol was measured using a cr- <dig> chroma-meter  to generate three hunter color coordinates , averaged across three regions of the surface on each fruit; the measurements were reduced to a single variable by calculating the euclidean distance from white , following prohens et al.
<cit> .
fglo was scored as “1” for opaque, “2” for intermediate and “3” for bright peel color.
the trait data were treated as adjusted entry means .
several multivariate linear mixed models were tested using a combination of the f-test  and the akaike test .
the model shown to best fit the data was: pijsb
= lj
+ ys
+ rbjs
+ gi
+ mij
+ nis
+ eijs, where pijsb represents the phenotype of the bth replicate of the ith genotype at the jth location in the sth year; lj represents the fixed effect of the jth location, ys the fixed effect of the sth year, rbjs the fixed effect of the bth replicate within the jth location in the sth year, gi the random effect of the ith genotype, mij the random effect of the genotype by location interaction, nis the random effect of the genotype by year interaction and e the residual.
broad-sense heritabilities were calculated from the expression
where σ2g represented the genotypic variance, σ2y the genotype x year interaction, σ2l the genotype x location interaction, σ2e the residual variance, ny the number of years  and nl the number of locations .
pearson correlation coefficients were calculated between each pair of phenotypic traits.
all analyses were carried out by algorithms implemented in r software  <cit> .
gwa mapping
the gwa analysis was performed using tassel v <dig> .
<dig> software  <cit> .
three models were tested: the simple general linear model , the structured association model , based on the structure output, and the mixed linear model , taking into account both the structure output and the kinship matrix  <cit> .
the mixed-model approach has been used elsewhere  to analyse variation in qualitative traits by treating them as quantitative ones, on the assumption that averaging across replicates would produce normality.
the cumulative density function was applied to assess the efficiency of the various models in correcting for population structure.
the false positive rate  was converted into a false discovery rate  <cit> , using the qvalue package implemented in r. the estimation of the overall proportion of true null hypothesis π <dig> was based on λ range set from  <dig> to  <dig>  by  <dig>  and the smoother method was applied  <cit> .
q-values < <dig>  were considered as significant.
for each significantly associated snp locus, a general linear model with all fixed effect terms was applied to estimate the proportion of the phenotypic variance explained .
in order to visualize the associations and to compare them with established qtl  <cit> , all snps associated with a particular trait mapping within less than double the mean ld stretch were considered as a single unit defining association groups.
the resulting genetic map, incorporating the associations and qtl detected here into an f2-based linkage map  <cit> , was drawn using mapchart v <dig>  software  <cit> .
synteny between tomato and eggplant chromosomal regions was investigated by aligning the rad tag sequences  <cit>  surrounding snps against the tomato sl <dig>  genome sequence  using the burrows-wheeler alignment tool  <cit> .
alignments with a mapping quality value > <dig> were considered as valid.
RESULTS
genotypic characterization and population structure
the  <dig> accessions were initially genotyped at  <dig> snp loci, of which  <dig> were retained after quality control.
the two replicated accessions gave uniformly consistent allele calls.
maf at most of the snp loci ranged from 10% to 50% , with only  <dig> displaying a maf value <5%.
these were discarded, leaving a genotypic matrix of  <dig> entries by  <dig> snp loci, of which  <dig> have been placed on the barchi et al.
<cit>  genetic map.
the global average pic value was  <dig> ; loci on chromosome e <dig> had a low mean pic value , while the mean pic for the remaining loci on a chromsome-by-chromosome basis lay between  <dig>  and  <dig>  .
the structure analysis resulted in a prediction for k of either  <dig> or  <dig> .
the upgma-based dendrogram  and the pcoa  show the genetic relationships between the  <dig> accessions.
their form, as well as the Δk analysis provided by the evanno et al.
<cit>  transformation, suggested a population structure comprising two subgroups.
according to the level of membership provided by structure, cluster a contained 91% of the ea accessions, while 96% of the cluster b membership comprised wa accessions.
the remaining  <dig> accessions  had ambiguous membership and were thus classified as admixed.
pcoas carried out separately on the ea accessions showed some clustering among the chinese entries, and among the indian and se asian ones .
the wa entries were grouped according to previously described morphology-based groups   <cit> , where group  <dig> accessions produced long, light and curved fruits, group  <dig> oblong shaped fruits of medium weight and group  <dig> round, heavy fruits.figure  <dig>
the genetic architecture of the full germplasm panel.
a) ln and dk plots derived from the snp data.
b) upgma dendrogram derived after taking account of the structure analysis.
c) pcoa visualization of the genetic relationships between members of the association panel.
cluster a is shown in orange, cluster b in blue and admixtures in grey.
ld evaluation
an r <dig> threshold of  <dig>  was applied to define which snp loci were significantly associated with one another.
on the basis of the r <dig> model , the mean genetic length of these associated groups was  <dig>  cm.
a mean r <dig> of  <dig>  was observed between all pairs of linked loci, with a mean maximum r <dig> value of  <dig> .
the mean ld between unlinked loci was  <dig> .
when the r2s model was applied , the ld stretch was reduced to  <dig>  cm, with a mean level of  <dig>  between adjacent loci; an average of the highest r2s value of each marker with any other of  <dig> , and the mean ld between unlinked loci was  <dig> .
on the basis of the r2sv model , ld extended over  <dig>  cm, with a whole genome mean of  <dig>  between adjacent  and a mean ld between unlinked loci of less than  <dig> .
heat maps produced for each of the three models showed that strong ld was limited to certain regions, mostly aligned to the diagonal .
apparent high levels of ld between loci mapping to a different chromosome were suggested in the r <dig> model, but this phenomenon was largely absent in the r2s and r2sv models.figure  <dig>
ld decay.
the curve was fitted using a locally weighted scatterplot smooth regression with the threshold set at  <dig> .
a) r <dig> model, b) r2
s model, c) r2
sv model.
heat maps indicating genome-wide variation in ld across the genome.
a) r <dig> model, b) r2
s model, c) r2
sv model.
phenotypic data analysis and association mapping
a summary of the accessions’ phenotypic performance is presented in table  <dig> together with the heritabilities for scored traits.
the pves are included in additional file 4: figure s <dig>  a wide range of variation was observed for most of the traits, and the genotypic variance component was substantial for all of them .
pedan, fcol and calan were the most genetically variable of the traits , and also the most highly heritable.
the gxe component of the variance  was small, with the exception of that for corcol and fglo, the two least heritable of the traits.
broad-sense heritabilities averaged  <dig>  , with the least heritable traits being fglo and corcol, and the most heritable pedan and fcol .
some significant inter-trait correlations were observed: traits associated with the anthocyanin content of the plant  were strongly and positively correlated with one another, but only moderately correlated with corcol.
the traits fglo and fcol were somewhat inter- correlated, and correlated with the anthocyanin content-related traits, but were uncorrelated with corcol.
both the correlation data and the distribution of each trait are reported in additional file 5: figure s <dig> table  <dig>
codes used to identify the various traits along with mean values, standard deviations , coefficients of variation  and broad-sense heritabilities
adlan
stean
ablan
calan
corcol
adlvean
ablvean
pedan
fcol
fglo
associations between snp alleles and morphology were acquired on the basis of three different models.
the glm naive-model, which involves no correction for population structure, identified several spurious associations.
this failing was improved by applying the glm q-model, but only the mlm k + q-model produced a distribution of p-values comparable to the theoretical one .
thus the latter model was pursued.
following q-value correction,  <dig> significant genotype/phenotype associations were detected.
regions carrying the presumed genes/qtl were identified on nine of the  <dig> chromosomes  , and involved eight of the ten traits .
the number of associations per trait ranged from four  to  <dig> , and the total number of snp loci involved was 20; these loci had a mean maf of  <dig> %.
the pve per association laid between 5% and 24% .
in order to match the associations with previously identified qtl, loci linked to one another by < <dig>  cm were considered as a unit, and their genomic location was obtained from the barchi et al.
<cit>  map.
overall,  <dig> association groups, comprising 1– <dig> snp loci, were defined in this way .
the most prominent clustering of traits occurred on chromosome e <dig>  which also proved to harbor the most genes/qtl underlying variation in anthocyanin content and fruit color.
the e <dig>  group  harbored genetic factors for adlan, stean, calan, adlevean, ablevean, pedan and fcol while e <dig>   included genetic factors for stean, ablevean, adlevean, pedan, fcol and fglo.
one of the two other large clusters was on chromosome e <dig> , which was influential for adlan, stean, calan and adlvean, and the other was on e <dig> , with genes/qtl determining stean, adlvean, pedan and fcol .figure  <dig>
cumulative density function using three alternative association models: the glm naive , glm q-model  and mlm .
traits showing significant associations are indicated.
the latter provided the most consistent p-values.
significant genotypic associations with anthocyanin content- and fruit color-related traits
adlan
stean
calan
adlvean
ablvean
pedan
fcol
fglo
the associated snps’ id, genomic location, relevant association group, the significance of the association , pve  and maf  are shown.
regions identified by gwa in comparison to qtl locations described by barchi et al.
<cit> .
the gwa outcome is given to the left of each chromosome  and the various association groups are indicated in panels and marked in red.
the qtl locations are shown to the right of each chromosome.
detailed information regarding the individual genotype/phenotype associations is given in table  <dig>  the snp locus most significantly associated with adlan was 24985_psti_l <dig> , with a maf of  <dig> % and a pve of 22%; for stean, calan, adlvean, ablvean, pedan and fcol 35442_psti_l <dig> , with a maf of  <dig> % and a pve of 14-24%.
for fglo, three highly significantly associated snps were detected, namely 3382_psti_l <dig>  19601_psti_l <dig> and 33571_psti_l387; these had a maf of 30% and each had a pve of 10%.
synteny with tomato and the identification of potential candidate genes
the regions of chromosomes e <dig>  e <dig> and e <dig> harboring genetic factors underlying anthocyanin content were aligned with the tomato genome sequence.
e <dig> and t <dig> are known to be syntenic, while part of e <dig> is syntenic with the lower section of t <dig> and the rest with the lower section of t12; e <dig> corresponds to the upper section of t <dig> and the lower one of t <dig>  <cit> .
genes in tomato encoding flavonone 3-hydroxylase and dihydroflavonol 4-reductase are present on t <dig> in a region homologous with group e <dig> , which harbors genes/qtl for adlan, stean, calan and adlvean .
the location of the e <dig>  group  corresponds to a segment of t <dig> in which a gene encoding the anthocyanin synthesis-associated enzyme udp glucose anthocyanidin 5– <dig> glucosyltransferase is located .
the tomato gene encoding udp glucose anthocyanidin 3– <dig> glucosyltransferase and the two myb transcription factors ant <dig> and an <dig> are present on t <dig>  in a region syntenic to groups e <dig>  and e <dig> ; genes/qtl in these groups had a strong influence over the pigmentation of both the vegetative parts of the plant and the fruit.figure  <dig>
synteny between tomato and eggplant chromosomes.
the latter are shown on the left with bars indicating the site of qtl for anthocyanin content and fruit color.
their tomato orthologs are shown on the right, along with the position of possible candidate genes.
discussion
genetic variation and population structure
the  <dig> members of the association panel were deliberately selected to represent the full range of phenotypic diversity in eggplant.
the panel was genotyped with a subset of  <dig> of the > <dig>  snps identified by sequencing rad tags generated from the genomic dna of 67/ <dig> and 305e <dig>  the parents of an established f <dig> mapping population  <cit> .
most of the snps included in the genotyping chip were unambiguously scorable and informative, producing average maf and pic values of  <dig>  and  <dig> , respectively.
a small number of snps had to be discarded for reasons of poor scoring quality or low informativeness.
particularly low pic values were obtained in the distal region of the chromosome e <dig> ; this chromosomal region is the site of the locus rfo-sa <dig>  which confers resistance against the soil-borne fungus fusarium oxysporum f. sp.
melongenae which was introgressed in the parental line 305e <dig> from solanum aethiopicum gr.
gilo
<cit> .
therefore, 305e <dig> carries some rather low frequency alleles which show a rather low frequency among the accession under study.
the information provided by these polymorphisms are of interest only in the artificial progeny in which they are well represented while may result of scarce usefulness in a germplasm panel.
the population structure of the panel  comprised two rather distinct sub-populations, which broadly matched the accessions’ provenance.
the species is thought to have been domesticated in asia  and introduced into the mediterranean basin by the arabs in the 7th to 8th century ce  <cit> .
the distinctness of the two gene-pools reflects a history of independent selection and adaptation to different environments and consumer preferences.
when the pco analysis was applied to just the “occidental” entries no evidence of correlation between provenance and genetic relatedness was found, while a clustering related to the three morphological groups we previously described , was detected   <cit> .
possibly, different uses of different types of fruits may have generated separate groups of varieties cultivated in different areas, with a different history of selection responsible of the observed genetic differentiation.
the picture was rather different for the “oriental” gene pool ; the asian material did form two recognizable clusters , with most of the indian, se asian and indonesian materials forming one group and the chinese ones the other.
this behavior replicated the outcome of a previous diversity study based on ssr markers  <cit> , thereby further supporting the hypothesis that eggplant was domesticated independently in the indian subcontinent and in china  <cit> .
ld in eggplant
in order to account for population structure, two different corrections to the r <dig> measure were attempted, as proposed by mangin et al.
<cit> .
the estimate of ld derived without these corrections was unreliable, as it included apparent associations over long intra-chromosomal distances , and even between loci mapping to two different chromosomes .
applying the r2s model reduced the extent of these clearly artefactual associations, but a more stringent method was still needed to correct for bias due to genetic relatedness.
this was provided by the r2sv model, which achieved a  <dig> fold reduction in associations between unlinked markers, leaving high ld values only between pairs of genetically linked snp loci.
the end estimate for the extension of ld was  <dig>  cm, which matches reasonably well with the level reported for eggplant by ge et al.
<cit> , and also with those documented in other self-pollinating species such as the near-relative tomato , arabidopsis thaliana , barley  and wheat .
ld was not uniformly distributed along the genome , a phenomenon which has also been noted elsewhere  <cit> .
its non-uniformity is thought to reflect the irregular distribution of recombination along the chromosome, but can also be influenced by positive selection  <cit> .
furthermore, the high level of ld and extended haplotype blocks in our material may be due to the high level of homozygosity which can hardly generate recombinations as well as the genetic bottleneck due to selection which drastically reduced the low frequent haplotypes and extended the association between markers.
such a level of ld is ideal for the gwa method, as it allows for an efficient coverage of the genome based on a relatively moderate number of markers, while still encouraging a high level of genetic diversity.
gwa mapping of genetic factors underlying anthocyanin pigmentation and fruit color
qtl discovery in eggplant has to date been achieved using linkage mapping in bi-parental inter and intra-specific populations  <cit> .
a first attempt to apply gwa has been published recently  <cit> , in which  <dig> marker associations related to eight traits were reported.
some potentially fatal weaknesses in the analysis can, however, be identified.
firstly, the issue of a maf threshold was not addressed, so it is not clear whether loci associated with a low maf were discarded or included; the effect of their inclusion would be to generate false associations caused by the co-incidence of variation for a trait and a statistically under-represented allele.
secondly, the glm model was used to estimate the significance of locus/trait associations, but this method has been shown to be incapable of adequately correcting for population structure  <cit> , unlike the mlm model used in the present study.
thirdly, no evidence of correcting for spurious associations  was provided.
fourthly, a threshold false positive rate  of  <dig>  was adopted as a threshold to validate the associations, instead of using a corrected threshold , or one for the false discovery rate.
the genetic basis of anthocyanin synthesis and accumulation has been widely explored in the solanaceae
.
in eggplant, this has long been thought to be rather complex  <cit> , involving at least three major and five minor loci, with the added complication of epistatic interactions and/or pleiotropic effects.
the gwa procedure generated  <dig> associations between snp loci and either anthocyanin content- or fruit color-related traits.
according to collard et al.
<cit> , a qtl associated with a pve of at least 10% should be considered as a “major” locus.
there was a lack of any significant association involving corcol and ablan, but at least one “major” qtl was putatively identified for each of the other eight traits.
the snp loci associated with one  traits were clustered into  <dig> groups, scattered over nine chromosomes.
the extent of some of the inter-trait correlations suggests that what appeared to be a cluster of qtl is more likely a single pleiotropic locus, although the presence of a set of linked qtl cannot be definitively ruled out.
broad-sense heritabilities were generally > <dig>   and corcol ) with a limited genotype by environment effect, which confirms the proposition that the influence of the growing environment on anthocyanin pigmentation is quite limited  <cit> .
the genomic location of eight of the  <dig> association groups overlapped that of a known qtl, showing how effective gwa can be in identifying the genetic basis of quantitative traits.
qtl identified via linkage analysis of bi-parental populations are generally considered to be experiment-specific, unless validated  <cit>  and have often proven to be genetic background specific as well.
of the  <dig> snp loci involved in the genotype/phenotype associations discovered here, six mapped to four genomic regions where no qtl related to anthocyanin content or fruit color has yet been reported .
some of these markers could only have been identified thanks to the extent of the genetic variability which the gwa approach makes accessible; following a validation exercise, they may well prove to provide viable indirect selection tools in a practical breeding programme.
the gwa study of ge et al.
<cit>  has located two marker/fcol associations, one on chromosome e <dig> and the other on e <dig>  in correspondence to the association groups e <dig>  and e <dig>  carrying qtls for the same trait.table  <dig>
genotype/phenotype associations and known qtl location
e <dig>
adlan
stean
fcol
adlan
stean
calan
adlvean
e <dig>
fglo
stean
adlvean
pedan
fcol
adlan
e <dig>
fcol
-
-
stean
calan
adlan
stean
calan
adlvean
ablvean
pedan
fcol
e <dig>
stean
adlvean
ablvean
pedan
fcol
fglo
adlan
adlvean
fcol
the four locations where no qtl related to anthocyanin content or fruit color have been located are shown in bold.
synteny and possible orthologs in other solanaceae species
to date most of the effort invested in the genetic analysis of anthocyanin pigmentation in the solanaceae has been focused on potato, sweet pepper and tomato.
in the latter crop,  <dig> genes  involved in anthocyanin synthesis have been described  <cit> .
the detailed understanding of this pathway, along with the well-established syntenic relationships between the tomato and eggplant chromosomes  <cit> , means that it is reasonable to search for candidate genes in eggplant by inspecting the gene content of the syntenic tomato sequence.
synteny was observed between e <dig>   and a portion of tomato chromosome t <dig>  where two genes encoding enzymes  involved in the anthocyanin production pathway reside.
one of these has been identified as the gene underlying the aw  qtl, in the presence of which there is a complete absence of anthocyanin throughout plant development  <cit> .
the same gene is responsible for the potato r qtl  <cit> , which produces red pelargonidin-based anthocyanin pigments.
e <dig>   lies in a region syntenic to a portion of t <dig> which harbors 5gt, a gene involved in the storage of betanidin  in the vacuole  <cit> .
e <dig>  and e <dig>   share synteny with a portion of t <dig> which carries several genes related to the anthocyanin production pathway, 3gt and the two myb transcription factors ant <dig> and an <dig>   <cit> .
ant <dig> regulates the genes encoding chalcone synthase and dihydroflavonol 4-reductase, key enzymes involved in the synthesis of anthocyanin compounds  <cit> .
ant <dig> is considered to be the prime candidate for the ag  qtl responsible for the delayed expression of anthocyanin  <cit> .
in potato, an2a is the likely candidate for qtl i, responsible for tissue-specific anthocyanin expression  <cit>  and an2b for f, a regulator of anthocyanin expression in the flower  <cit> .
in sweet pepper, a myb transcription factor encoded by a underlies the accumulation of anthocyanin pigment in the foliage, flower and immature fruit  <cit> .
CONCLUSIONS
the development of large-scale genotyping capacity has allowed the concept of gwa to become a viable approach for the genetic dissection of quantitative traits.
here, the technique has been applied to uncover the genomic regions harboring genes underlying anthocyanin pigmentation and fruit color traits in eggplant.
the gwa mapping approach was effective in validating a number of established qtl and, thanks to the wide diversity captured by the panel of genotypes in study, was able to detect a series of novel marker/trait associations.
synteny with tomato has allowed the ready identification of candidate orthologues for the chromosome e <dig>  e <dig> and e <dig> qtls related to anthocyanin accumulation.
availability of supporting data
the data sets supporting the results of this article are available in the labarchives repository at the following web address http://dx.doi.org/ <dig> /h4ng4nk <dig>
electronic supplementary material
additional file 1: table s1: list of the accessions used for the association mapping study.
additional file 2: figure s1:
a) snp performance.
loci with a maf < <dig>  were excluded from the gwa analysis.
b) snp pic values across chromosomes.
the solid line represents the average genome-wide pic, and the broken line the variation in pic value across chromosome e02; note the particularly low informativeness of loci at the distal end of this chromosome.
additional file 3: figure s2: the genetic architecture of the components of the germplasm panel.
a) pcoa of the ea accessions.
those of chinese origin cluster to the right of the plot, separated from those of s and se asian origin.
b) pcoa of the wa accessions cluster according to their fruit morphology: group  <dig> - long, light, curved fruits, group  <dig> – oblong fruits of intermediate weight, group  <dig> – round, heavy fruits as defined by cericola et al.
<cit> .
additional file 4: figure s3: pve values.
adlan = adaxial leaf lamina anthocyanin; stean = stem anthocyanin; ablan = abaxial leaf lamina anthocyanin; calan = calyx anthocyanin; corcol = corolla color; adlvean = adaxial leaf venation anthocyanin; ablvean = abaxial leaf venation anthocyanin; pedan = fruit peduncle anthocyanin; fcol = fruit color; fglo = fruit glossiness.
var = genotypic variance; var = genotype by location variance; var = genotype by year variance; var = residual variance.
additional file 5: figure s4: pearson’s inter-trait correlations  and regression coefficients .
the histograms included on the diagonal show the distribution of trait values .
competing interests
the authors declare that they have no competing interests.
authors’ contributions
glr, ep and sl planned and supervised the experimental work; fc, lt, lb, na, lp, ts and glr performed traits evaluation over the two years and over the two experimental fields; fc and lb performed the molecular experiment; fc and ep analyzed the data; fc, ep, sl, lt and glr drafted the manuscript; all authors read and approved the final version of the manuscript.
this research was partially supported by the italian ministry of agricultural alimentary and forest politics in the framework of the “esplora” project.
we thank ciriaci t., grazioli g., and tacconi m.g.
for the technical practices and data collection in the fields for eggplant production.

 eggplant ranks third in commercial importance among the solanaceous crops after potato and tomato, and it is cultivated in many countries, particularly in southern asia, middle east and northern africa. global production in was about mt, and the largest european producer is italy. despite its commercial importance, little research effort has been devoted to the genetic analysis of key breeding and quality traits with respect to the other solanaceae crops tomato, potato and capiscum. several quantitative trait loci underlying fruit color and shape have been described by nunome et al., while doganlar et al. and frary et al. succeeded in identifying qtl for certain fruit- and plant-related traits, but only in a population derived from an inter-specific cross, which is of reduced utility in eggplant improvement. more recently, the genetic basis of parthenocarpy, ralstonia solanacearum resistance, anthocyanin content and a group of agronomic traits has been elucidated. the genome-wide association approach represents an alternative to biparental linkage mapping for the determination of the genetic basis of traits. both approaches rely on recombination to re-arrange the genome, and seek to establish correlations between phenotype and genotype, based on the non-random association of alleles at two or more loci, termed linkage disequilibrium. in a bi-parental population, only the polymorphisms between the two parents can be queried, whereas in a gwa population the number of polymorphisms is determined by the genetic diversity of the entire germplasm panel. in a gwa population, ld is determined not only by recombination frequency, but also by genetic drift, by the mating system of the plant and by the history of selection. the major advantages of gwa over biparental linkage mapping lie in the much wider variability in phenotype and genotype made accessible, a history of multiple rounds of recombination in many different lineages and the inclusion of germplasm of direct relevance to crop improvement. the real risk that the genetic architecture of the germplasm panel may cloud the analysis has to be allayed by a prior evaluation of the population’s structure. to date only one gwa-based study of variation in eggplant has been performed but the present study used a larger number of accessions and a more densely populated genetic map vs microsatellites) and targeted traits related to anthocyanin accumulation and fruit color. methods plant material and dna isolation a core set of accessions, chosen from a large collection of breeding lines, old varieties and landrace selections by cericola et al., was established to represent germplasm grown in east asia and in the mediterranean basin. the entries were all highly homozygous and thus phenotypically stable. genomic dna was extracted from fresh young leaves harvested from three individuals of each accession, using an e.z.n.a.™ plant dna mini kit, according to the manufacturer’s protocol. the quality of each dna sample was monitored by electrophoresis through an % agarose gel and its dna concentration estimated spectrophotometrically. snp data acquisition each accession was genotyped at snp loci as reported by barchi et al.; of these have been genetically mapped. snps were selected taking into account a quality score, based on the probability of good performance using the illumina golden gate assay; the score > indicates a high probability of success. a blastx search was carried out against the tair dataset using the highest quality score snps as query; the sequences having the highest e-value were then chosen. the goldengate assay was carried out at the uc davis genome center. automatic allele calling was handled by gencall software. two of the entries were included in duplicate as an internal control. snp loci in which the minimum allele frequency fell below 5%, along with those where >10% of the data were missing, were discarded. each snp locus was scored as a binary data point, and the pic of each was estimated following anderson et al.. population structure genetic similarities between pairs of entries were quantified by the dice similarity index, then used to describe genetic relationships using both the unweighted pair-group arithmetic mean method, and principal coordinate analysis by means of past software. structure v software was used to estimate the number of sub-populations in the panel, applying the admixture model for the ancestry of individuals and correlated allele frequencies. the population structure was modelled with a burn-in of  cycles followed by markov chain monte carlo repeats. the evanno et al. transformation method was then used to infer k, the most likely number of populations. pair-wise kinship coefficients between the accessions were estimated using spagedi software. the diagonal of the matrix was set to two, and negative values were set to following yu et al.. ld analysis ld decay was quantified by plotting pair-wise r values against the distance between adjacent snp loci, based on the genetic map developed by barchi et al.. the effect of population structure on ld was investigated with three approaches as suggested by mangin et al.: r; r2s and r2sv. to quantify the reach of ld, an r threshold of was set. the relationship between the baseline r values and genetic distance was determined using a locally weighted scatter plot smoothing line. to visualize ld throughout the genome, heat maps were produced based on pair-wise r r2s and r2sv values. the estimation of all ld measures was carried out by programs implemented in the r package ldcorsv. acquisition and analysis of phenotypic and morphological data the accessions were grown in field both at montanaso lombardo and at monsampolo del tronto in and again in in each trial, the material was set out as two randomized complete blocks with six plants per entry per block, and standard horticultural practices were applied. phenotyping methodology was based on the european cooperative programme for plant genetic resources solanaceae descriptors and the international board for plant genetic resource descriptors for eggplant. the traits assayed were adaxial leaf lamina anthocyanin, stem anthocyanin, abaxial leaf lamina anthocyanin, calyx anthocyanin, corolla color, adaxial leaf venation anthocyanin, abaxial leaf venation anthocyanin, peduncle anthocyanin, fruit color and fruit glossiness. the anthocyanin content of the vegetative part of the plant was scored on a 0– scale, with “0” representing no visible anthocyanin pigmentation, and “5” representing dark violet tissue. corcol was scored as “0” for white, “1” for pink and “2” for violet. fcol was measured using a cr- chroma-meter to generate three hunter color coordinates, averaged across three regions of the surface on each fruit; the measurements were reduced to a single variable by calculating the euclidean distance from white, following prohens et al.. fglo was scored as “1” for opaque, “2” for intermediate and “3” for bright peel color. the trait data were treated as adjusted entry means. several multivariate linear mixed models were tested using a combination of the f-test and the akaike test. the model shown to best fit the data was: pijsb  = lj  + ys  + rbjs  + gi  + mij  + nis  + eijs, where pijsb represents the phenotype of the bth replicate of the ith genotype at the jth location in the sth year; lj represents the fixed effect of the jth location, ys the fixed effect of the sth year, rbjs the fixed effect of the bth replicate within the jth location in the sth year, gi the random effect of the ith genotype, mij the random effect of the genotype by location interaction, nis the random effect of the genotype by year interaction and e the residual. broad-sense heritabilities were calculated from the expression where σ2g represented the genotypic variance, σ2y the genotype x year interaction, σ2l the genotype x location interaction, σ2e the residual variance, ny the number of years and nl the number of locations. pearson correlation coefficients were calculated between each pair of phenotypic traits. all analyses were carried out by algorithms implemented in r software. gwa mapping the gwa analysis was performed using tassel v. software. three models were tested: the simple general linear model, the structured association model, based on the structure output, and the mixed linear model, taking into account both the structure output and the kinship matrix. the mixed-model approach has been used elsewhere to analyse variation in qualitative traits by treating them as quantitative ones, on the assumption that averaging across replicates would produce normality. the cumulative density function was applied to assess the efficiency of the various models in correcting for population structure. the false positive rate was converted into a false discovery rate, using the qvalue package implemented in r. the estimation of the overall proportion of true null hypothesis π was based on λ range set from to by and the smoother method was applied. q-values < were considered as significant. for each significantly associated snp locus, a general linear model with all fixed effect terms was applied to estimate the proportion of the phenotypic variance explained. in order to visualize the associations and to compare them with established qtl, all snps associated with a particular trait mapping within less than double the mean ld stretch were considered as a single unit defining association groups. the resulting genetic map, incorporating the associations and qtl detected here into an f2-based linkage map, was drawn using mapchart v software. synteny between tomato and eggplant chromosomal regions was investigated by aligning the rad tag sequences surrounding snps against the tomato sl genome sequence using the burrows-wheeler alignment tool. alignments with a mapping quality value > were considered as valid.  genotypic characterization and population structure the accessions were initially genotyped at snp loci, of which were retained after quality control. the two replicated accessions gave uniformly consistent allele calls. maf at most of the snp loci ranged from 10% to 50%, with only displaying a maf value <5%. these were discarded, leaving a genotypic matrix of entries by snp loci, of which have been placed on the barchi et al. genetic map. the global average pic value was; loci on chromosome e had a low mean pic value, while the mean pic for the remaining loci on a chromsome-by-chromosome basis lay between and. the structure analysis resulted in a prediction for k of either or. the upgma-based dendrogram and the pcoa show the genetic relationships between the accessions. their form, as well as the Δk analysis provided by the evanno et al. transformation, suggested a population structure comprising two subgroups. according to the level of membership provided by structure, cluster a contained 91% of the ea accessions, while 96% of the cluster b membership comprised wa accessions. the remaining accessions had ambiguous membership and were thus classified as admixed. pcoas carried out separately on the ea accessions showed some clustering among the chinese entries, and among the indian and se asian ones. the wa entries were grouped according to previously described morphology-based groups, where group accessions produced long, light and curved fruits, group oblong shaped fruits of medium weight and group round, heavy fruits.figure the genetic architecture of the full germplasm panel. a) ln and dk plots derived from the snp data. b) upgma dendrogram derived after taking account of the structure analysis. c) pcoa visualization of the genetic relationships between members of the association panel. cluster a is shown in orange, cluster b in blue and admixtures in grey. ld evaluation an r threshold of was applied to define which snp loci were significantly associated with one another. on the basis of the r model, the mean genetic length of these associated groups was  cm. a mean r of was observed between all pairs of linked loci, with a mean maximum r value of. the mean ld between unlinked loci was. when the r2s model was applied, the ld stretch was reduced to  cm, with a mean level of between adjacent loci; an average of the highest r2s value of each marker with any other of, and the mean ld between unlinked loci was. on the basis of the r2sv model, ld extended over  cm, with a whole genome mean of between adjacent and a mean ld between unlinked loci of less than. heat maps produced for each of the three models showed that strong ld was limited to certain regions, mostly aligned to the diagonal. apparent high levels of ld between loci mapping to a different chromosome were suggested in the r model, but this phenomenon was largely absent in the r2s and r2sv models.figure ld decay. the curve was fitted using a locally weighted scatterplot smooth regression with the threshold set at. a) r model, b) r2 s model, c) r2 sv model. heat maps indicating genome-wide variation in ld across the genome. a) r model, b) r2 s model, c) r2 sv model. phenotypic data analysis and association mapping a summary of the accessions’ phenotypic performance is presented in table  together with the heritabilities for scored traits. the pves are included in additional file 4: figure s a wide range of variation was observed for most of the traits, and the genotypic variance component was substantial for all of them. pedan, fcol and calan were the most genetically variable of the traits, and also the most highly heritable. the gxe component of the variance was small, with the exception of that for corcol and fglo, the two least heritable of the traits. broad-sense heritabilities averaged, with the least heritable traits being fglo and corcol, and the most heritable pedan and fcol. some significant inter-trait correlations were observed: traits associated with the anthocyanin content of the plant were strongly and positively correlated with one another, but only moderately correlated with corcol. the traits fglo and fcol were somewhat inter- correlated, and correlated with the anthocyanin content-related traits, but were uncorrelated with corcol. both the correlation data and the distribution of each trait are reported in additional file 5: figure s table codes used to identify the various traits along with mean values, standard deviations, coefficients of variation and broad-sense heritabilities adlan stean ablan calan corcol adlvean ablvean pedan fcol fglo associations between snp alleles and morphology were acquired on the basis of three different models. the glm naive-model, which involves no correction for population structure, identified several spurious associations. this failing was improved by applying the glm q-model, but only the mlm k + q-model produced a distribution of p-values comparable to the theoretical one. thus the latter model was pursued. following q-value correction, significant genotype/phenotype associations were detected. regions carrying the presumed genes/qtl were identified on nine of the chromosomes, and involved eight of the ten traits. the number of associations per trait ranged from four to, and the total number of snp loci involved was 20; these loci had a mean maf of %. the pve per association laid between 5% and 24%. in order to match the associations with previously identified qtl, loci linked to one another by <  cm were considered as a unit, and their genomic location was obtained from the barchi et al. map. overall, association groups, comprising 1– snp loci, were defined in this way. the most prominent clustering of traits occurred on chromosome e which also proved to harbor the most genes/qtl underlying variation in anthocyanin content and fruit color. the e group harbored genetic factors for adlan, stean, calan, adlevean, ablevean, pedan and fcol while e included genetic factors for stean, ablevean, adlevean, pedan, fcol and fglo. one of the two other large clusters was on chromosome e, which was influential for adlan, stean, calan and adlvean, and the other was on e, with genes/qtl determining stean, adlvean, pedan and fcol.figure cumulative density function using three alternative association models: the glm naive, glm q-model and mlm. traits showing significant associations are indicated. the latter provided the most consistent p-values. significant genotypic associations with anthocyanin content- and fruit color-related traits adlan stean calan adlvean ablvean pedan fcol fglo the associated snps’ id, genomic location, relevant association group, the significance of the association, pve and maf are shown. regions identified by gwa in comparison to qtl locations described by barchi et al. . the gwa outcome is given to the left of each chromosome and the various association groups are indicated in panels and marked in red. the qtl locations are shown to the right of each chromosome. detailed information regarding the individual genotype/phenotype associations is given in table  the snp locus most significantly associated with adlan was 24985_psti_l, with a maf of % and a pve of 22%; for stean, calan, adlvean, ablvean, pedan and fcol 35442_psti_l, with a maf of % and a pve of 14-24%. for fglo, three highly significantly associated snps were detected, namely 3382_psti_l 19601_psti_l and 33571_psti_l387; these had a maf of 30% and each had a pve of 10%. synteny with tomato and the identification of potential candidate genes the regions of chromosomes e e and e harboring genetic factors underlying anthocyanin content were aligned with the tomato genome sequence. e and t are known to be syntenic, while part of e is syntenic with the lower section of t and the rest with the lower section of t12; e corresponds to the upper section of t and the lower one of t. genes in tomato encoding flavonone 3-hydroxylase and dihydroflavonol 4-reductase are present on t in a region homologous with group e, which harbors genes/qtl for adlan, stean, calan and adlvean. the location of the e group corresponds to a segment of t in which a gene encoding the anthocyanin synthesis-associated enzyme udp glucose anthocyanidin 5– glucosyltransferase is located. the tomato gene encoding udp glucose anthocyanidin 3– glucosyltransferase and the two myb transcription factors ant and an are present on t in a region syntenic to groups e and e; genes/qtl in these groups had a strong influence over the pigmentation of both the vegetative parts of the plant and the fruit.figure synteny between tomato and eggplant chromosomes. the latter are shown on the left with bars indicating the site of qtl for anthocyanin content and fruit color. their tomato orthologs are shown on the right, along with the position of possible candidate genes. discussion genetic variation and population structure the members of the association panel were deliberately selected to represent the full range of phenotypic diversity in eggplant. the panel was genotyped with a subset of of the > snps identified by sequencing rad tags generated from the genomic dna of 67/ and 305e the parents of an established f mapping population. most of the snps included in the genotyping chip were unambiguously scorable and informative, producing average maf and pic values of and, respectively. a small number of snps had to be discarded for reasons of poor scoring quality or low informativeness. particularly low pic values were obtained in the distal region of the chromosome e; this chromosomal region is the site of the locus rfo-sa which confers resistance against the soil-borne fungus fusarium oxysporum f. sp. melongenae which was introgressed in the parental line 305e from solanum aethiopicum gr. gilo. therefore, 305e carries some rather low frequency alleles which show a rather low frequency among the accession under study. the information provided by these polymorphisms are of interest only in the artificial progeny in which they are well represented while may result of scarce usefulness in a germplasm panel. the population structure of the panel comprised two rather distinct sub-populations, which broadly matched the accessions’ provenance. the species is thought to have been domesticated in asia and introduced into the mediterranean basin by the arabs in the 7th to 8th century ce. the distinctness of the two gene-pools reflects a history of independent selection and adaptation to different environments and consumer preferences. when the pco analysis was applied to just the “occidental” entries no evidence of correlation between provenance and genetic relatedness was found, while a clustering related to the three morphological groups we previously described, was detected. possibly, different uses of different types of fruits may have generated separate groups of varieties cultivated in different areas, with a different history of selection responsible of the observed genetic differentiation. the picture was rather different for the “oriental” gene pool; the asian material did form two recognizable clusters, with most of the indian, se asian and indonesian materials forming one group and the chinese ones the other. this behavior replicated the outcome of a previous diversity study based on ssr markers, thereby further supporting the hypothesis that eggplant was domesticated independently in the indian subcontinent and in china. ld in eggplant in order to account for population structure, two different corrections to the r measure were attempted, as proposed by mangin et al.. the estimate of ld derived without these corrections was unreliable, as it included apparent associations over long intra-chromosomal distances, and even between loci mapping to two different chromosomes. applying the r2s model reduced the extent of these clearly artefactual associations, but a more stringent method was still needed to correct for bias due to genetic relatedness. this was provided by the r2sv model, which achieved a fold reduction in associations between unlinked markers, leaving high ld values only between pairs of genetically linked snp loci. the end estimate for the extension of ld was  cm, which matches reasonably well with the level reported for eggplant by ge et al., and also with those documented in other self-pollinating species such as the near-relative tomato, arabidopsis thaliana, barley and wheat. ld was not uniformly distributed along the genome, a phenomenon which has also been noted elsewhere. its non-uniformity is thought to reflect the irregular distribution of recombination along the chromosome, but can also be influenced by positive selection. furthermore, the high level of ld and extended haplotype blocks in our material may be due to the high level of homozygosity which can hardly generate recombinations as well as the genetic bottleneck due to selection which drastically reduced the low frequent haplotypes and extended the association between markers. such a level of ld is ideal for the gwa method, as it allows for an efficient coverage of the genome based on a relatively moderate number of markers, while still encouraging a high level of genetic diversity. gwa mapping of genetic factors underlying anthocyanin pigmentation and fruit color qtl discovery in eggplant has to date been achieved using linkage mapping in bi-parental inter and intra-specific populations. a first attempt to apply gwa has been published recently, in which marker associations related to eight traits were reported. some potentially fatal weaknesses in the analysis can, however, be identified. firstly, the issue of a maf threshold was not addressed, so it is not clear whether loci associated with a low maf were discarded or included; the effect of their inclusion would be to generate false associations caused by the co-incidence of variation for a trait and a statistically under-represented allele. secondly, the glm model was used to estimate the significance of locus/trait associations, but this method has been shown to be incapable of adequately correcting for population structure, unlike the mlm model used in the present study. thirdly, no evidence of correcting for spurious associations was provided. fourthly, a threshold false positive rate of was adopted as a threshold to validate the associations, instead of using a corrected threshold, or one for the false discovery rate. the genetic basis of anthocyanin synthesis and accumulation has been widely explored in the solanaceae. in eggplant, this has long been thought to be rather complex, involving at least three major and five minor loci, with the added complication of epistatic interactions and/or pleiotropic effects. the gwa procedure generated associations between snp loci and either anthocyanin content- or fruit color-related traits. according to collard et al., a qtl associated with a pve of at least 10% should be considered as a “major” locus. there was a lack of any significant association involving corcol and ablan, but at least one “major” qtl was putatively identified for each of the other eight traits. the snp loci associated with one traits were clustered into groups, scattered over nine chromosomes. the extent of some of the inter-trait correlations suggests that what appeared to be a cluster of qtl is more likely a single pleiotropic locus, although the presence of a set of linked qtl cannot be definitively ruled out. broad-sense heritabilities were generally > and corcol ) with a limited genotype by environment effect, which confirms the proposition that the influence of the growing environment on anthocyanin pigmentation is quite limited. the genomic location of eight of the association groups overlapped that of a known qtl, showing how effective gwa can be in identifying the genetic basis of quantitative traits. qtl identified via linkage analysis of bi-parental populations are generally considered to be experiment-specific, unless validated and have often proven to be genetic background specific as well. of the snp loci involved in the genotype/phenotype associations discovered here, six mapped to four genomic regions where no qtl related to anthocyanin content or fruit color has yet been reported. some of these markers could only have been identified thanks to the extent of the genetic variability which the gwa approach makes accessible; following a validation exercise, they may well prove to provide viable indirect selection tools in a practical breeding programme. the gwa study of ge et al. has located two marker/fcol associations, one on chromosome e and the other on e in correspondence to the association groups e and e carrying qtls for the same trait.table genotype/phenotype associations and known qtl location e adlan stean fcol adlan stean calan adlvean e fglo stean adlvean pedan fcol adlan e fcol - - stean calan adlan stean calan adlvean ablvean pedan fcol e stean adlvean ablvean pedan fcol fglo adlan adlvean fcol the four locations where no qtl related to anthocyanin content or fruit color have been located are shown in bold. synteny and possible orthologs in other solanaceae species to date most of the effort invested in the genetic analysis of anthocyanin pigmentation in the solanaceae has been focused on potato, sweet pepper and tomato. in the latter crop, genes involved in anthocyanin synthesis have been described. the detailed understanding of this pathway, along with the well-established syntenic relationships between the tomato and eggplant chromosomes, means that it is reasonable to search for candidate genes in eggplant by inspecting the gene content of the syntenic tomato sequence. synteny was observed between e and a portion of tomato chromosome t where two genes encoding enzymes involved in the anthocyanin production pathway reside. one of these has been identified as the gene underlying the aw qtl, in the presence of which there is a complete absence of anthocyanin throughout plant development. the same gene is responsible for the potato r qtl, which produces red pelargonidin-based anthocyanin pigments. e lies in a region syntenic to a portion of t which harbors 5gt, a gene involved in the storage of betanidin in the vacuole. e and e share synteny with a portion of t which carries several genes related to the anthocyanin production pathway, 3gt and the two myb transcription factors ant and an. ant regulates the genes encoding chalcone synthase and dihydroflavonol 4-reductase, key enzymes involved in the synthesis of anthocyanin compounds. ant is considered to be the prime candidate for the ag qtl responsible for the delayed expression of anthocyanin. in potato, an2a is the likely candidate for qtl i, responsible for tissue-specific anthocyanin expression and an2b for f, a regulator of anthocyanin expression in the flower. in sweet pepper, a myb transcription factor encoded by a underlies the accumulation of anthocyanin pigment in the foliage, flower and immature fruit.  the development of large-scale genotyping capacity has allowed the concept of gwa to become a viable approach for the genetic dissection of quantitative traits. here, the technique has been applied to uncover the genomic regions harboring genes underlying anthocyanin pigmentation and fruit color traits in eggplant. the gwa mapping approach was effective in validating a number of established qtl and, thanks to the wide diversity captured by the panel of genotypes in study, was able to detect a series of novel marker/trait associations. synteny with tomato has allowed the ready identification of candidate orthologues for the chromosome e e and e qtls related to anthocyanin accumulation. availability of supporting data the data sets supporting the results of this article are available in the labarchives repository at the following web address http://dx.doi.org/ /h4ng4nk electronic supplementary material additional file 1: table s1: list of the accessions used for the association mapping study. additional file 2: figure s1: a) snp performance. loci with a maf < were excluded from the gwa analysis. b) snp pic values across chromosomes. the solid line represents the average genome-wide pic, and the broken line the variation in pic value across chromosome e02; note the particularly low informativeness of loci at the distal end of this chromosome. additional file 3: figure s2: the genetic architecture of the components of the germplasm panel. a) pcoa of the ea accessions. those of chinese origin cluster to the right of the plot, separated from those of s and se asian origin. b) pcoa of the wa accessions cluster according to their fruit morphology: group - long, light, curved fruits, group – oblong fruits of intermediate weight, group – round, heavy fruits as defined by cericola et al.. additional file 4: figure s3: pve values. adlan = adaxial leaf lamina anthocyanin; stean = stem anthocyanin; ablan = abaxial leaf lamina anthocyanin; calan = calyx anthocyanin; corcol = corolla color; adlvean = adaxial leaf venation anthocyanin; ablvean = abaxial leaf venation anthocyanin; pedan = fruit peduncle anthocyanin; fcol = fruit color; fglo = fruit glossiness. var = genotypic variance; var = genotype by location variance; var = genotype by year variance; var = residual variance. additional file 5: figure s4: pearson’s inter-trait correlations and regression coefficients. the histograms included on the diagonal show the distribution of trait values. competing interests the authors declare that they have no competing interests. authors’ contributions glr, ep and sl planned and supervised the experimental work; fc, lt, lb, na, lp, ts and glr performed traits evaluation over the two years and over the two experimental fields; fc and lb performed the molecular experiment; fc and ep analyzed the data; fc, ep, sl, lt and glr drafted the manuscript; all authors read and approved the final version of the manuscript. this research was partially supported by the italian ministry of agricultural alimentary and forest politics in the framework of the “esplora” project. we thank ciriaci t., grazioli g., and tacconi m.g. for the technical practices and data collection in the fields for eggplant production.
the major advantages of gwa lie in being able to sample a much wider range of the phenotypic and genotypic variation present, in being able to exploit multiple rounds of historical recombination in many different lineages and to include multiple accessions of direct relevance to crop improvement. the genome-wide association approach represents an alternative to biparental linkage mapping for determining the genetic basis of trait variation. a accessions eggplant association panel, comprising a mixture of breeding lines, old varieties and landrace selections originating from asia and the mediterranean basin, was snp genotyped and scored for anthocyanin pigmentation and fruit color at two locations over two years. a mixed linear model appeared to be the most appropriate for gwa. a set of snp locus/phenotype associations was identified and the genomic regions harboring these loci were distributed over nine of the eggplant chromosomes. the gwa mapping approach was effective in validating a number of established qtl and, thanks to the wide diversity captured by the panel, was able to detect a series of novel marker/trait associations. the global level of linkage disequilibrium was  cm.
 the genome-wide association approach represents an alternative to biparental linkage mapping for determining the genetic basis of trait variation. both approaches rely on recombination to re-arrange the genome, and seek to establish correlations between phenotype and genotype. the major advantages of gwa lie in being able to sample a much wider range of the phenotypic and genotypic variation present, in being able to exploit multiple rounds of historical recombination in many different lineages and to include multiple accessions of direct relevance to crop improvement.  a accessions eggplant association panel, comprising a mixture of breeding lines, old varieties and landrace selections originating from asia and the mediterranean basin, was snp genotyped and scored for anthocyanin pigmentation and fruit color at two locations over two years. the panel formed two major clusters, reflecting geographical provenance and fruit type. the global level of linkage disequilibrium was  cm. a mixed linear model appeared to be the most appropriate for gwa. a set of snp locus/phenotype associations was identified and the genomic regions harboring these loci were distributed over nine of the eggplant chromosomes. the associations were compared with the location of known qtl for the same traits.  the gwa mapping approach was effective in validating a number of established qtl and, thanks to the wide diversity captured by the panel, was able to detect a series of novel marker/trait associations. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 