BACKGROUND
vitis vinifera l genomic resources, including both released genomic sequences  <cit> , allow the characterization at molecular level of the biological function of genes involved in agronomically interesting traits  <cit> .
stenospermocarpic seedlessness  <cit> , found in popular table grape varieties for fresh or dried consumption such as sultanina , is one of these traits.
in stenospermocarpic berries, pollination and fertilization occur but both the seed coat and endosperm cease their normal development at early stages, leaving undeveloped seeds or seed traces  <cit> .
seed and berry size depend on genetic background, and they both segregate in experimental populations with a continuous distribution indicative of polygenic determinism  <cit> .
to increase the chances of obtaining new seedless varieties, breeding programs commonly cross two seedless parental genotypes and progeny are obtained through embryo rescue assisted by in vitro tissue culture  <cit> .
the progeny thus obtained  are used to investigate the genetic basis of grape seedlessness and berry size  <cit> .
the most accepted model proposed that genetic inheritance of seedlessness in grapevine is based on the expression of three independent recessive genes under the control of a dominant regulator gene named sdi   <cit> .
this model was partly confirmed by several studies that all reported a major qtl for seedlessness co-localizing with sdi on linkage group   <dig>  this major qtl explains 50% to 70% of the phenotypic variation of the trait  <cit> .
numerous other minor qtls were found on different lgs, but they were not reproducible across different seasons and were not present in all crosses.
thus, the molecular characterization of the sdi locus is a key step toward understanding the molecular mechanisms underlying seedlessness.
in arabidopsis and other model species, genes involved in flower, ovule, seed and fruit development have been isolated and characterized from loss of function mutants.
among them, the mads-box family plays an important role  <cit> .
most of the mads-box genes identified in arabidopsis seem to have counterparts in grapevine  <cit> .
in spite of grapevine particular features, characterized mads-box genes expressed during the reproductive development might have the same role than their functionally characterized orthologues in model species  <cit> .
among these mads-box genes, vvagl <dig>  shows homology to the stk/agl <dig> gene in arabidopsis and is expressed in mature carpels, developing seeds and pre- and post véraison fruits; this expression suggests a possible role for this gene in ovule, seed and berry development in grapevine  <cit> .
vvagl <dig> was also mapped in silico to the same contig that contains the sdi locus and the closest marker to a seedlessness qtl , suggesting that it might play an important role in seed development.
in parallel, a transcriptional analysis of genes differentially expressed in the flowers of seeded and seedless sultanina lines allowed the identification of a chloroplast chaperonin  whose silencing in tobacco and tomato resulted in seed abortion  <cit> , and of a ubiquitin extension protein  having a probable general role in the control of organ development in grapevine  <cit> .
none of these genes co-segregated with the sdi locus.
besides these works, no further evidence has been generated to unveil the genetic control of seedlessness in grapevine.
genetic analyses have also revealed a major qtl for berry size  <cit>  and ripening date  <cit>  that overlap with the major seedlessness qtl on lg  <dig>  the complex developmental process modified by genetic, physiological and environmental factors that underlies berry development was first reviewed by coombe  <cit>  and was very recently updated by carmona et al.
<cit> .
the relationship between seed number and berry size was reviewed by ollat et al.
<cit> .
these overlapping qtls detected by genetic experiments could be reflective of pleiotropic effects caused by hormones in developing seeds  <cit> .
however, most of the phenotypic variation for berry size is not explained by the sdi locus  <cit> , and there is still room for the identification of other loci involved in seed and berry development.
the molecular biology of fleshy fruit ripening has received considerable attention  <cit> , but little is known about the determinants of early fleshy fruit morphogenesis.
differential screening of ests and berry transcriptomic analysis identified several genes that show differential expression during young fruit development, the onset of véraison and ripening  <cit> .
in this work, we designed a strategy to test the hypothesis for a possible role of vvagl <dig> in seeddlessness.
we integrated multiple genomic resources as soon as they became available to contribute to the molecular characterization of the sdi locus: qtl mapping in seedless × seedless derived progeny  <cit> , physical mapping on a cabernet-sauvignon physical map  <cit>  and the released sequence of grapevine  <cit> , which gave further positional evidence for vvagl <dig> as being the major gene responsible for seedlessness  <cit> .
here, we provide genetic and transcriptional support for this hypothesis and discuss its potential for molecular-assisted breeding programs.
RESULTS
phenotypic evaluation
phenotypic evaluations of plants grown in their own roots  and over sultanina rootstocks  confirmed the distribution of seed and berry weight previously reported by mejía et al.
<cit>  for the same progeny .
neither of the two traits fit a normal distribution  according to the anderson-darling normality test.
non-parametric spearman analysis showed a correlation between mean seed fresh weight per berry  and mean berry weight  of  <dig> %,  <dig> % and  <dig> % for the  <dig>   <dig> and  <dig> seasons, respectively .
however, variations in bw values were explained by a weak linear relationship with sfw .
most of the heterozygous genotypes of the population, defined as such by the ssr vmc7f <dig> marker tightly linked to the sdi locus, were seedless and showed an average sfw below the population average, like  both heterozygous parental genotypes.
the calculated dominance effect d was negative, showing that the seedless allele presents incomplete dominance  over the seeded allele.
this partial dominance effect was also detected for berry weight, but the effect was lower.
finally, several offspring exhibited extreme phenotypes relative to the parents for both traits .
this phenotypic distribution was consistent with the heterozygosity in both parental genotypes of the sdi locus and the partial dominance of the seedless allele.
construction of linkage group 18
taking into account a former qtl detection experiment  <cit>  and other results  <cit>  that all showed the presence of a major qtl for seedlessness on lg  <dig>  we replaced dominant markers and increased marker density with available and newly developed co-dominant markers.
for this purpose,  <dig> new ssrs linked to the targeted regions were designed taking advantage of the available genomic resources , or the pinot noir pn <dig> 6x genome assembly), and they were genotyped in the same experimental population.
as an example, the microsatellite vmc7f <dig>  previously reported as the nearest marker to the sdi locus  <cit>  and the closest marker to the peak of the major qtl for seedlessness and berry size  <cit> , was localized on the cabernet-sauvignon physical map on bac contig_ <dig>  bes from this bac contig were searched against the 6x genomic assembly of the grapevine genome.
five ssrs  identified in these genomic sequences could be mapped  to the vicinity of vmc7f <dig> .
with this strategy, only  <dig> new markers were consistently positioned on both parental linkage maps .
the mapping data set for lg  <dig> in ruby seedless  and sultanina  included a total of  <dig> co-dominant markers , among which were six bes-derived ssrs and five genomic assembly-derived ssrs.
the consensus linkage map built with these data covers  <dig>  cm with a mean inter-locus distance of  <dig> cm .
no significant differences in distances or positions were observed between the two parental maps .
seedlessness and berry weight qtl analysis
improvements that were made based upon a former study  <cit>   resulted in more accurate qtl detection.
a narrower  and more reliable confidence interval  was established for the major qtl identified on lg  <dig> for seed and berry size .
for both traits, the qtl analysis was performed over three different seasons with and without intragenic vvagl <dig> markers.
the table shows the closest marker to the peak in the lod profile, the lod value for the same marker , the 1-lod support confidence interval , the proportion of phenotypic variance explained by qtls with parametric and non-parametric analysis , the p-value for the kruskal-wallis test , and the mean seed fresh weight or berry weight values for genotypic classes  detected in the rs × s progeny.
parametric qtl analyses  did not reveal significant differences between the parental genotypes in any of the evaluated seasons  for either of the two analyzed traits .
co-localizing qtls were detected for sfw and bw, both centered on the vmc7f <dig> marker that was used as a cofactor for mqm analysis .
these qtls explained most of the phenotypic variation in sfw , and a minor proportion of the phenotypic variation in bw .
non-parametric analysis performed with the same marker used as a cofactor in the mqm analysis  gave the highest kruskal-wallis values for sfw  and bw .
other minor qtls were found on other linkage groups.
however, none of them were consistent across seasons or in previous analyses performed in the same or other progeny  <cit> .
therefore, these other minor qtls were not further assessed in the present work.
positional candidate gene identification for sfw and bw
of the two co-localizing qtls for bw and sfw, bw defined the largest confidence interval , which was flanked by ssr markers vvp18b <dig> and vvp18b <dig>  defining a region equivalent to ~ <dig> kb  in the 12x genome assembly of pinot noir pn <dig>  this region contains four gene models  confirmed by alignments with vitis vinifera cdnas from public databases.
as expected, among these gene models, gsvivt <dig>  is an ortholog of the agamous-like  <dig> gene of arabidopsis , with 75% amino acid identity  and 86% positive matches .
agl <dig> belongs to the d-lineage mads box family responsible for ovule identity in monocotyledons and dicotyledons  <cit> .
the protein alignment of the c and d lineages of the agamous family from different plant families and the construction of a phylogram showed that these lineages evolved from a common ancestor during angiosperm evolution  <cit>  .
the alignment also indicated that vvmads <dig>  isolated and characterized in cv.
syrah  <cit> , is likely to be allelic  to the vvagl <dig> sequences obtained from sultanina .
lacking evidence that any of the remaining three annotated genes from this region could be involved in seed or berry development , we decided to concentrate our further analysis on vvagl <dig>  indeed, in grapevine, vvagl <dig> has been shown to have carpel-specific rna expression and to be highly expressed in flowers after the cap has been shed and in seeds  <cit> .
all these results and current knowledge of the possible functions of the genes in the region confirmed the former hypothesis of costantini et al.
<cit>  that vvagl <dig> is the best positional candidate gene for the control of seed development.
to obtain more evidence for a possible role of vvagl <dig> in seedless table grapes, this positional candidate gene was characterized at the molecular, genetic and transcriptional levels.
molecular characterization of vvagl <dig> alleles
based on their genotype at the vmc7f <dig> marker, both ruby seedless and sultanina are heterozygous in the vvagl <dig> region .
vvagl <dig> sequences  were thus isolated from homozygous genotypes showing a stable seeded or seedless phenotype among the rs × s progeny.
as ruby seedless inherited the seedless allele from sultanina, the isolated seedless allele was called an indifferently seedless allele whatever its origin .
the seeded allele from sultanina, ruby seedless or pinot noir  was called indifferently seeded allele.
the pedigree of each analysed genotype is indicated.
mean seed fresh weight/berry , sfw relative to the minimum value, the normalised expression of vvagl <dig> in berries at pea stage and the expression of vvagl <dig> relative to the minimum value.
sequence polymorphisms in the promoter region and in putative regulatory elements
in the reference genome pn <dig>  <cit> , vvagl11's putative regulatory region is defined as ~ <dig>  bp upstream of the tata box and by a 5'utr disrupted by an intron of ~  <dig>  bp .
flanked by the same 5' and 3' ends, the seeded and seedless regulatory regions are  <dig>  and  <dig>  bp long, respectively.
pn <dig> and the seeded allele share  <dig> % identity.
by contrast, the seeded and seedless regulatory sequences have  <dig> % identity with  <dig> indels and  <dig> snps differentiating the two alleles.
<dig> out of  <dig> cis-regulatory elements identified by place  <cit>  vary in number and position .
among them several n cis-regulatory elements were identified as polymorphic in the putative regulatory region of vvagl <dig> upstream and downstream from the transcription start site.
in the cauliflower mosaic virus 35s gene, ga-rich motifs positively affect promoter activity even when translocated upstream of the transcription start site  <cit> , and in arabidopsis, the first intron of agl <dig> contains ga-rich motifs required for ovule- and septum-specific expression  <cit> .
thus, the putative cis-regulatory elements identified in the 5'utr intron of vvagl <dig> might be functional.
the ssr markers vmc7f <dig>  and vvp18b <dig>  are located  <dig> and  <dig> bp, respectively, upstream of the tata-box of the vvagl <dig> gene, and the polymorphisms revealed by these ssr are n repeats .
sequence polymorphisms in the coding sequence
the cds region of vvagl <dig> was 100% identical between the seeded alleles isolated from the homozygous seeded individual and the predicted cdna sequence from pinot noir , whereas eight snps were identified between the seeded and seedless alleles .
six of them were located in exon  <dig>  two causing non-silent mutations .
the characterization of the progeny by sscp marker e7_vvagl <dig>  later revealed the existence of a second seeded allele segregating in the rs × s progeny.
e7_vvagl <dig> alleles were thus amplified and sequenced from the different genotypic classes identified in the rs × s progeny: ee, ef, eg, fg; where e denotes the seedless allele.
seeded f and g alleles differed by one snp in exon  <dig> that produced a silent mutation .
the c-domain, encoded in part by exon  <dig>  is the less conserved domain within this gene family  <cit>  .
the r > l mutation, detected only in the seedless sultanina-derived allele, affects one of the conserved motifs, and in arabidopsis it has been shown that this c-terminal region might be a transactivation domain or contribute to the formation of multimeric mads-box protein complexes  <cit> .
to check for a possible association between the r > l mutation and the seedless trait, exon  <dig> was sequenced in a collection of  <dig> individuals: one wild vitis vinifera genotype, five representatives of other species of the vitis genus and fifteen cultivated vitis vinifera, among which were one additional seedless variety , eight seeded table varieties and seven wine varieties.
no additional snps or indels other than those identified in the rs × s background were found in this exon in the whole set of genotypes, although they were arranged into six haplotypes instead of the three segregating in the rs × s population .
the most frequent haplotype was the seeded allele found in sultanina .
it seems to be conserved across the genus with nearly no variation observed at the interspecific level .
a t > a non-silent mutation was found in five table grapes  that are seedless and one wine variety .
the r > l mutation was observed in the seedless varieties  but also in the seeded variety assyl kara  .
these results suggest that this mutation does not by itself explain the seedless phenotype.
genetic characterization of vvagl <dig> alleles
to acquire more precise information about a possible role of the coding and/or putative regulatory region of vvagl <dig> in the seeded versus seedless phenotype, intragenic markers derived from allele sequencing were designed to perform a qtl analysis.
markers p <dig>  p <dig> and p3_vvagl <dig> were designed to genetically analyze indels in the regulatory region .
an indel revealed by p1_vvagl <dig> affects a putative o2-like box, p2_vvagl <dig> marks a putative tata-box near far the transcription start site and p3_vvagl <dig> marks a n motif.
finally, marker e7_vvagl <dig> was designed to test snps identified in exon  <dig> .
genetic mapping with intragenic markers reduced the sfw and bw qtl confidence intervals down to  <dig>  and  <dig>  cm, respectively .
the kruskal-wallis non-parametric method for qtl analysis was used to test the efficiency of these markers in the rs × s population.
for all three analyzed seasons, the markers showing the highest correlation with seedlessness were vmc7f <dig> and p3_vvagl <dig> .
a similar pattern was observed for berry weight, but with k values explaining 38% to 44% of the phenotypic variation .
a strong correlation was also found for both traits with p1_vvagl <dig>  p2_vvagl <dig> and e7_vvagl11; however, p3_vvagl <dig> ) was found to be the best marker in terms genotypic and phenotypic association across the three evaluated seasons, as no false positives or negatives were identified in the homozygous genotypes  or  .
this genetic evidence shows that the region delimited by marker vmc7f <dig> and the tata-box  makes the largest contribution to the seedless phenotype in the sultanina genetic background, suggesting that this region  might contain the causative genetic variation of the seedless phenotype.
the stratification of the progeny by genotype  defined by the p3_vvagl <dig> marker  revealed a partial dominant effect of the seedless allele  over the seeded allele , which is consistent with the dominance effect observed at the phenotypic level only.
this incomplete dominance effect is also observed for berry weight but with a minor effect .
transcriptional characterization of vvagl <dig> alleles
expression of vvagl <dig> was analyzed by real-time pcr analysis at three key developmental stages for ovule and seed development: pre-bloom, bloom and pea-size berries.
the samples came from seven genotypes: two seedless and two seeded homozygous seedlings of the rs × s progeny, both seedless heterozygous parental genotypes  and a common seeded table grape genotype that contains two different seeded alleles: red globe .
in the seeded genotypes, vvagl <dig> gene was expressed after anthesis, while in pre-bloom and bloom stages expression remained minimal.
during the pea-size stage, its expression was  <dig> times higher than in pre-bloom or bloom stages , which is consistent with previous results  <cit> .
within the pea stage of development, the level of vvagl <dig> expression was associated with the vvagl <dig> genotype : genotypes homozygous for the seeded allele showed transcription  <dig> times higher than genotypes homozygous for the seedless allele, and the basal level was detected at earlier developmental stages.
as expected, heterozygous genotypes showed an intermediate level of expression .
all these differences were statistically significant, whereas no statistically significant difference in vvagl <dig> expression in pea-stage berries was observed between the bb and bc seeded genotypes.
validation of intragenic vvagl <dig> markers in different genetic backgrounds
to extend the genetic analyses performed in the experimental progeny  to different genetic backgrounds, an association analysis was performed with a population of  <dig> genotypes characterized quantitatively for seed fresh weight.
the population, derived mainly from crosses of ten seedless varieties, revealed p3_vvagl <dig> as the marker that explains the largest proportion of phenotypic variation.
for markers vvp18b <dig>  vmc7f <dig>  p <dig>  p <dig>  p3_vvagl <dig> and vvp18b <dig>  the statistic kruskal-wallis values were  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig>  and  <dig>  , respectively.
the p3_vvagl <dig> marker revealed six different alleles  and seven main genotypes .
most of the genotypes harboring one or two copies of the 198-bp allele have a seedless phenotype .
as described for the experimental progeny , the seedless allele  has partial dominance over the  <dig> and  <dig> bp seeded alleles; however, the same effect was not detected with respect to the  <dig> bp seeded allele.
interestingly, all of the genotyped seedless varieties within this analysis were heterozygous for this locus .
discussion
genetic dissection of seedlessness
major qtls for seed and berry weight were previously detected on lg <dig> in a subset of this progeny  <cit> , in progeny derived from two other partially seedless genotypes  <cit>  and in progeny derived from a cross of seeded and seedless genotypes  <cit> .
for sfw, confidence intervals varied between  <dig> and  <dig> cm in doligez et al.
<cit> ,  <dig> and  <dig> cm in cabezas et al.
<cit>  and  <dig> cm in mejía et al.
<cit> .
in the present work, integration of all the available genomic resources allowed us to quickly develop new co-dominant markers in the targeted area and to further reduce the confidence interval for this trait down to  <dig>  cm with a segregating population of only ~  <dig> phenotyped individuals.
as the development of a well-balanced population in terms of phenotypic classes for seedlessness requires a step of in vitro embryo rescue  <cit> , any strategy aiming to increase the accuracy of qtl detection without increasing the population size is of great interest.
moreover, genetic mapping of intragenic vvagl <dig> markers, in addition to revealing a putative functional role of the regulatory of the coding region of vvagl <dig>  resulted in a narrower confidence interval  for the sfw qtl, so far the narrowest qtl identified for this trait.
according to the genetic size of the most comprehensive ssr-based map for vitis vinifera l.  <cit>  and to the genome size reported for the grapevine genome  <cit> , a confidence interval of  <dig>  cm should be equivalent to ~  <dig> kb.
in our study, the confidence interval is equivalent to ~ <dig> kb, indicating that this region may be hot spot for recombination, which allowed the mapping of intragenic vvagl <dig> markers in a small progeny set .
however, genotyping errors in data sets are the most common source of variation and inflated genetic distances  <cit> .
for instance, intragenic variation could be due to replication slippage  <cit> , the mutation mechanism that cause the hypervariability of microsatellites .
the putative regulatory region of vvagl <dig> contains at least nine intragenic microsatellites annotated as n boxes  with repeat units that vary from  <dig> to  <dig>  two genotypes of the rs × s experimental progeny presented a mutation, identified by ssr genotyping and sequence-verified, in the region amplified by marker p3_vvagl <dig> .
this mutation consists of one additional unit of the ga repeat, which could have arisen either by taq polymerase slippage during pcr or by a real mutation occurring in these genotypes.
the use of a proofreading polymerase for the amplification and sequencing supports the latter hypothesis .
the limited size of our experimental population is also a potential source of distortions in genetic distance and qtl effect estimations.
it is now well known that in such small populations, major effect qtls are detected properly, but mapping experiments should be refined with larger populations and/or experimental designs adapted for the detection of environmental effects and minor qtls  <cit> .
indeed, the minor qtls for bw and sfw detected in the present study, detected earlier in the same experimental population  <cit>  and detected in other studies are neither coincident with each other nor stable among years  <cit> .
the positive correlation of seed and berry weights and the co-localization of major qtls for both traits observed in this study was also detected and described in other progeny sets  <cit> .
as already discussed in these former papers, this correlation could be due to  one underlying gene having a direct effect over ovule and seed development and indirectly affecting berry development through growth regulators produced by the developing seed,  one or several genes having different and independent impacts on seed and berry development, or  to a combination of both alternatives.
the argument in favor of a pleiotropic effect of one gene is based on the fact that the growth of fleshy fruits mainly relies on cell division at early stages of berry development but on cell expansion after véraison  <cit> .
cell division and expansion are both controlled by gibberellins, cytokinins and auxins, which are imported from seeds or ovules  <cit> .
however, because the partial dominance effect observed for seedlessness was less pronounced with respect to berry weight, it is probable that the same or other underlying genes have an independent influence on berry development.
molecular dissection of the major qtl for seedlessness
the reduced confidence interval for the major seedlessness qtl corresponds to a  <dig> kb region of the grapevine genome sequence that contains four gene models.
one of these corresponds to an ortholog of the mads-box gene agl <dig> in arabidopsis thaliana  <cit>  and fbp <dig> in petunia hybrida  <cit> , which were shown to be involved in the control of ovule identity.
based on current knowledge, none of the other genes are candidates for seed or berry development.
moreover, earlier expression studies in grapevine  <cit>  suggested that vvagl <dig> might influence ovule and seed development and that an alteration in this gene could yield seedless grapes.
the expression profile described in this work for the seeded allele of vvagl <dig> is consistent with what had been already reported in syrah  <cit>  and in tempranillo  <cit> , but also with the expression of orthologous genes like agl <dig>  <cit> , fbp <dig>  <cit> , tagl <dig>  <cit>  and osmads <dig>  <cit> .
in arabidopsis, pynyopich et al.
<cit>  showed that agl <dig> is strongly expressed in the funiculus starting from the initial stages of ovule development, in mature ovules and after fertilization.
they also showed that in agl <dig> mutants, seeds are rounder and smaller than in the wild type, and that funicular cells are greater in number and size, indicating that agl <dig> is also required to prevent abnormal growth of the funiculus.
among the mads-box genes known to control ovule identity  <cit> , agl <dig> is the only one that seems to be both necessary and sufficient to promote ovule development  <cit> .
the others have proven to be redundant, suggesting that some of them evolved from a common ancestral gene  <cit> .
in arabidopsis, the ectopic expression of stk  promotes carpel development  <cit> , and in grapes vvagl <dig> is highly expressed in carpels  <cit>  which ultimately develop into fruit, supporting the hypothesis that vvagl <dig> might have a direct influence on berry development instead of merely a pleiotropic effect through seed development.
alignment of the vvagl <dig> and agl <dig> nucleic and protein sequences showed that although the two proteins share 75% amino acid identity, no significant similarity exists between their promoter sequences.
however, both predicted promoters are similar in length  and share 68%  of their cis-regulatory elements according to a signal scan performed with the place database  <cit>  over the agl <dig> and vvagl <dig>  regulatory regions.
also, the mm algorithm  <cit>   identified the following shared motifs: ttttcttt, with  <dig> and  <dig> motifs in vitis  and arabidopsis , respectively; gcaa, with  <dig>  and  <dig> ; and ccattc, with  <dig>  and  <dig> .
the first  motif corresponds to n putative regulatory elements, which are the binding site for basic pentacysteine <dig> , a regulator of the homeotic arabidopsis thaliana gene agl <dig>  which controls ovule identity  <cit> .
bpc <dig> induces conformational changes by cooperative binding to purine-rich elements  present in the agl <dig> regulatory sequence  <cit> .
interestingly, these purine-rich repeats are abundant in the putative regulatory region of vvagl11: at least six n were identified upstream of the tata-box and three in the 5'utr intron.
the closest n repeats to the tata-box correspond to three ssr markers segregating in the rs × s progeny, vmc7f <dig>  vvb18b <dig> and p3_vvagl <dig> .
in this experimental population, p3_vvagl <dig> and vmc7f <dig> explain up to 78% of the phenotypic variation in seedless, which make them very good candidates for being the main regulatory elements involved in the expression of the final seedless phenotype.
in a selection of  <dig> genotypes derived from crosses of seedless × seedless varieties within our breeding program, p3_vvagl <dig> yielded the highest kruskall-wallis value .
the proportion of phenotypic variation in seedlessness explained by vvagl <dig> is huge, much greater than the estimated effect of other genes identified as qtls from cultivated plants, like  ovate, which controls fruit shape in tomato , and se <dig>  which controls flowering time in rice   <cit> .
as discussed above, even though this result is quite consistent with previous results using similar experimental designs  <cit> , it must be taken carefully as some degree of distortion and/or overestimation of effects could exist due to the small size of our  progeny sets, genotyping and phenotyping errors and recombination slippage events in the regulatory region of vvagl <dig>  further analyses should be performed with larger experimental designs or by transgenic assays manipulating gene expression.
the mutations identified in regulatory elements of the seedless allele of vvagl <dig> explained a slightly higher degree of phenotypic variation than those identified in the coding region , suggesting that the seedless phenotype might be genetically controlled by this regulatory region.
transcriptional analyses performed in contrasting phenotypes as well as in homozygous seeded and seedless genotypes revealed that in seedless genotypes, the expression of vvagl <dig> was abolished during the period of rapid seed and berry growth after berry set.
as expected, in heterozygous genotypes like sultanina or ruby seedless, its expression was half that observed in homozygous seeded genotypes.
together, the genetic and transcriptional evidence suggest that seedlessness in table grapes might be due to misexpression of vvagl <dig> caused by indels in its regulatory elements.
defined by intragenic marker p3_vvagl <dig>  the seedless allele  exerts a partially dominant effect over the seeded alleles : most of the heterozygous genotypes are seedless.
the c-domain in the coding sequence has been described as the less conserved domain between the mads-box family members  <cit> .
however, each of the major mikc sub-families possesses short, highly conserved motifs  <cit>  whose specific function remains unknown  <cit> .
the c-domain has also been reported to be involved in the mediation of higher-order interactions among mads protein dimers  <cit> , in transcriptional activation  <cit> , and in post-translational modifications  <cit> .
a non-silent mutation identified in one of these conserved motifs of vvagl <dig> that did not by itself explain the seedless phenotype might be responsible for a structural change in the c-domain making the mutant transcription factor barely expressed during the initial stages of seed development and therefore dominant over its wild-type alleles.
altogether, these results are partially agree with the model proposed by bouquet and danglot  <cit>  and lahogue et al.
<cit>  for the control of seed development, where a single dominant locus, sdi, codes for a major regulatory gene.
the three remaining loci that interact with sdi, according to the proposed model, were not identified with the current experimental design.
gene-assisted selection
in a perennial species such as grapevine, markers that allow individuals not carrying the favorable allele for the most desirable given trait to be discarded before planting in the field for further evaluation are invaluable.
this is especially true for phenotypes that can only be screened in adult plants such as those affecting berries.
by identifying several interesting intragenic polymorphisms between seeded and seedless genotypes in the vvagl <dig> regulatory region , our study provides four new intragenic markers in a candidate gene for seedlessness for breeding purposes.
these intragenic markers displayed different relative efficiencies measured as the phenotypic variation explained by the marker and based on their efficiency to select positively seedless genotypes or negatively seeded genotypes.
the ssr marker vmc7f <dig>  already described as the closest marker to the sdi locus  <cit> , was confirmed as one of the best markers for progeny screening.
association analysis performed in the rs × s experimental progeny and over a population derived from several other seedless × seedless crosses revealed p3_vvagl <dig> as the most reliable marker for breeding purposes over three different seasons and across different genetic backgrounds.
the two most interesting markers identified in our work or former studies  need to be tested for their robustness in larger genetic backgrounds segregating for seedlessness.
lahogue et al.
<cit>  developed the scar marker scc <dig>  which is tightly linked to the sdi locus; however, scc <dig> was not useful in all the evaluated progeny  <cit>  or in the rs × s experimental population , as it often amplifies a null allele  <cit> .
in a controlled population derived from dominga × autumn seedless, cabezas et al.
<cit>  identified ssr markers closely linked to the sdi locus , and these markers results in 4% to 6% false positive identifications  and in 11% to 13% false negatives.
in the experimental population analysed in the present work, the use of marker p3_vvagl <dig> for the selection of homozygous genotypes resulted in 0% false positives , while vmc2f <dig> yielded 5% false positives .
haplotype analysis, defined either by combinations marker pairs or by all the intragenic markers for vvagl <dig>  failed to improve the efficiency achieved by p3_vvagl <dig> alone in our experimental population; any combination not only gave the same number of selected true seedless phenotypes but also increased the number of seeded phenotypes identified by mistake as true seedless .
CONCLUSIONS
vvagl <dig> belongs to the d-lineage of mads-box genes that control ovule identity.
a better understanding of its function would benefit other crops, as its function seems to be conserved across the plant species already studied .
however, its function in grapevine remains to be proven by genetic transformation of seeded cultivars.
whether its role in seedlessness is confirmed or not, vvagl <dig> has proven to be a very useful marker for assisted selection of seedless grapevine.

 vitis vinifera l genomic resources, including both released genomic sequences, allow the characterization at molecular level of the biological function of genes involved in agronomically interesting traits. stenospermocarpic seedlessness, found in popular table grape varieties for fresh or dried consumption such as sultanina, is one of these traits. in stenospermocarpic berries, pollination and fertilization occur but both the seed coat and endosperm cease their normal development at early stages, leaving undeveloped seeds or seed traces. seed and berry size depend on genetic background, and they both segregate in experimental populations with a continuous distribution indicative of polygenic determinism. to increase the chances of obtaining new seedless varieties, breeding programs commonly cross two seedless parental genotypes and progeny are obtained through embryo rescue assisted by in vitro tissue culture. the progeny thus obtained are used to investigate the genetic basis of grape seedlessness and berry size. the most accepted model proposed that genetic inheritance of seedlessness in grapevine is based on the expression of three independent recessive genes under the control of a dominant regulator gene named sdi. this model was partly confirmed by several studies that all reported a major qtl for seedlessness co-localizing with sdi on linkage group this major qtl explains 50% to 70% of the phenotypic variation of the trait. numerous other minor qtls were found on different lgs, but they were not reproducible across different seasons and were not present in all crosses. thus, the molecular characterization of the sdi locus is a key step toward understanding the molecular mechanisms underlying seedlessness. in arabidopsis and other model species, genes involved in flower, ovule, seed and fruit development have been isolated and characterized from loss of function mutants. among them, the mads-box family plays an important role. most of the mads-box genes identified in arabidopsis seem to have counterparts in grapevine. in spite of grapevine particular features, characterized mads-box genes expressed during the reproductive development might have the same role than their functionally characterized orthologues in model species. among these mads-box genes, vvagl shows homology to the stk/agl gene in arabidopsis and is expressed in mature carpels, developing seeds and pre- and post véraison fruits; this expression suggests a possible role for this gene in ovule, seed and berry development in grapevine. vvagl was also mapped in silico to the same contig that contains the sdi locus and the closest marker to a seedlessness qtl, suggesting that it might play an important role in seed development. in parallel, a transcriptional analysis of genes differentially expressed in the flowers of seeded and seedless sultanina lines allowed the identification of a chloroplast chaperonin whose silencing in tobacco and tomato resulted in seed abortion, and of a ubiquitin extension protein having a probable general role in the control of organ development in grapevine. none of these genes co-segregated with the sdi locus. besides these works, no further evidence has been generated to unveil the genetic control of seedlessness in grapevine. genetic analyses have also revealed a major qtl for berry size and ripening date that overlap with the major seedlessness qtl on lg the complex developmental process modified by genetic, physiological and environmental factors that underlies berry development was first reviewed by coombe and was very recently updated by carmona et al.. the relationship between seed number and berry size was reviewed by ollat et al.. these overlapping qtls detected by genetic experiments could be reflective of pleiotropic effects caused by hormones in developing seeds. however, most of the phenotypic variation for berry size is not explained by the sdi locus, and there is still room for the identification of other loci involved in seed and berry development. the molecular biology of fleshy fruit ripening has received considerable attention, but little is known about the determinants of early fleshy fruit morphogenesis. differential screening of ests and berry transcriptomic analysis identified several genes that show differential expression during young fruit development, the onset of véraison and ripening. in this work, we designed a strategy to test the hypothesis for a possible role of vvagl in seeddlessness. we integrated multiple genomic resources as soon as they became available to contribute to the molecular characterization of the sdi locus: qtl mapping in seedless × seedless derived progeny, physical mapping on a cabernet-sauvignon physical map and the released sequence of grapevine, which gave further positional evidence for vvagl as being the major gene responsible for seedlessness. here, we provide genetic and transcriptional support for this hypothesis and discuss its potential for molecular-assisted breeding programs.  phenotypic evaluation phenotypic evaluations of plants grown in their own roots and over sultanina rootstocks confirmed the distribution of seed and berry weight previously reported by mejía et al. for the same progeny. neither of the two traits fit a normal distribution according to the anderson-darling normality test. non-parametric spearman analysis showed a correlation between mean seed fresh weight per berry and mean berry weight of %, % and % for the and seasons, respectively. however, variations in bw values were explained by a weak linear relationship with sfw. most of the heterozygous genotypes of the population, defined as such by the ssr vmc7f marker tightly linked to the sdi locus, were seedless and showed an average sfw below the population average, like both heterozygous parental genotypes. the calculated dominance effect d was negative, showing that the seedless allele presents incomplete dominance over the seeded allele. this partial dominance effect was also detected for berry weight, but the effect was lower. finally, several offspring exhibited extreme phenotypes relative to the parents for both traits. this phenotypic distribution was consistent with the heterozygosity in both parental genotypes of the sdi locus and the partial dominance of the seedless allele. construction of linkage group 18 taking into account a former qtl detection experiment and other results that all showed the presence of a major qtl for seedlessness on lg we replaced dominant markers and increased marker density with available and newly developed co-dominant markers. for this purpose, new ssrs linked to the targeted regions were designed taking advantage of the available genomic resources, or the pinot noir pn 6x genome assembly), and they were genotyped in the same experimental population. as an example, the microsatellite vmc7f previously reported as the nearest marker to the sdi locus and the closest marker to the peak of the major qtl for seedlessness and berry size, was localized on the cabernet-sauvignon physical map on bac contig_ bes from this bac contig were searched against the 6x genomic assembly of the grapevine genome. five ssrs identified in these genomic sequences could be mapped to the vicinity of vmc7f. with this strategy, only new markers were consistently positioned on both parental linkage maps. the mapping data set for lg in ruby seedless and sultanina included a total of co-dominant markers, among which were six bes-derived ssrs and five genomic assembly-derived ssrs. the consensus linkage map built with these data covers cm with a mean inter-locus distance of cm. no significant differences in distances or positions were observed between the two parental maps. seedlessness and berry weight qtl analysis improvements that were made based upon a former study resulted in more accurate qtl detection. a narrower and more reliable confidence interval was established for the major qtl identified on lg for seed and berry size. for both traits, the qtl analysis was performed over three different seasons with and without intragenic vvagl markers. the table shows the closest marker to the peak in the lod profile, the lod value for the same marker, the 1-lod support confidence interval, the proportion of phenotypic variance explained by qtls with parametric and non-parametric analysis, the p-value for the kruskal-wallis test, and the mean seed fresh weight or berry weight values for genotypic classes detected in the rs × s progeny. parametric qtl analyses did not reveal significant differences between the parental genotypes in any of the evaluated seasons for either of the two analyzed traits. co-localizing qtls were detected for sfw and bw, both centered on the vmc7f marker that was used as a cofactor for mqm analysis. these qtls explained most of the phenotypic variation in sfw, and a minor proportion of the phenotypic variation in bw. non-parametric analysis performed with the same marker used as a cofactor in the mqm analysis gave the highest kruskal-wallis values for sfw and bw. other minor qtls were found on other linkage groups. however, none of them were consistent across seasons or in previous analyses performed in the same or other progeny. therefore, these other minor qtls were not further assessed in the present work. positional candidate gene identification for sfw and bw of the two co-localizing qtls for bw and sfw, bw defined the largest confidence interval, which was flanked by ssr markers vvp18b and vvp18b defining a region equivalent to ~ kb in the 12x genome assembly of pinot noir pn this region contains four gene models confirmed by alignments with vitis vinifera cdnas from public databases. as expected, among these gene models, gsvivt is an ortholog of the agamous-like gene of arabidopsis, with 75% amino acid identity and 86% positive matches. agl belongs to the d-lineage mads box family responsible for ovule identity in monocotyledons and dicotyledons. the protein alignment of the c and d lineages of the agamous family from different plant families and the construction of a phylogram showed that these lineages evolved from a common ancestor during angiosperm evolution. the alignment also indicated that vvmads isolated and characterized in cv. syrah, is likely to be allelic to the vvagl sequences obtained from sultanina. lacking evidence that any of the remaining three annotated genes from this region could be involved in seed or berry development, we decided to concentrate our further analysis on vvagl indeed, in grapevine, vvagl has been shown to have carpel-specific rna expression and to be highly expressed in flowers after the cap has been shed and in seeds. all these results and current knowledge of the possible functions of the genes in the region confirmed the former hypothesis of costantini et al. that vvagl is the best positional candidate gene for the control of seed development. to obtain more evidence for a possible role of vvagl in seedless table grapes, this positional candidate gene was characterized at the molecular, genetic and transcriptional levels. molecular characterization of vvagl alleles based on their genotype at the vmc7f marker, both ruby seedless and sultanina are heterozygous in the vvagl region. vvagl sequences were thus isolated from homozygous genotypes showing a stable seeded or seedless phenotype among the rs × s progeny. as ruby seedless inherited the seedless allele from sultanina, the isolated seedless allele was called an indifferently seedless allele whatever its origin. the seeded allele from sultanina, ruby seedless or pinot noir was called indifferently seeded allele. the pedigree of each analysed genotype is indicated. mean seed fresh weight/berry, sfw relative to the minimum value, the normalised expression of vvagl in berries at pea stage and the expression of vvagl relative to the minimum value. sequence polymorphisms in the promoter region and in putative regulatory elements in the reference genome pn, vvagl11's putative regulatory region is defined as ~ bp upstream of the tata box and by a 5'utr disrupted by an intron of ~ bp. flanked by the same 5' and 3' ends, the seeded and seedless regulatory regions are and bp long, respectively. pn and the seeded allele share % identity. by contrast, the seeded and seedless regulatory sequences have % identity with indels and snps differentiating the two alleles. out of cis-regulatory elements identified by place vary in number and position. among them several n cis-regulatory elements were identified as polymorphic in the putative regulatory region of vvagl upstream and downstream from the transcription start site. in the cauliflower mosaic virus 35s gene, ga-rich motifs positively affect promoter activity even when translocated upstream of the transcription start site, and in arabidopsis, the first intron of agl contains ga-rich motifs required for ovule- and septum-specific expression. thus, the putative cis-regulatory elements identified in the 5'utr intron of vvagl might be functional. the ssr markers vmc7f and vvp18b are located and bp, respectively, upstream of the tata-box of the vvagl gene, and the polymorphisms revealed by these ssr are n repeats. sequence polymorphisms in the coding sequence the cds region of vvagl was 100% identical between the seeded alleles isolated from the homozygous seeded individual and the predicted cdna sequence from pinot noir, whereas eight snps were identified between the seeded and seedless alleles. six of them were located in exon two causing non-silent mutations. the characterization of the progeny by sscp marker e7_vvagl later revealed the existence of a second seeded allele segregating in the rs × s progeny. e7_vvagl alleles were thus amplified and sequenced from the different genotypic classes identified in the rs × s progeny: ee, ef, eg, fg; where e denotes the seedless allele. seeded f and g alleles differed by one snp in exon that produced a silent mutation. the c-domain, encoded in part by exon is the less conserved domain within this gene family. the r > l mutation, detected only in the seedless sultanina-derived allele, affects one of the conserved motifs, and in arabidopsis it has been shown that this c-terminal region might be a transactivation domain or contribute to the formation of multimeric mads-box protein complexes. to check for a possible association between the r > l mutation and the seedless trait, exon was sequenced in a collection of individuals: one wild vitis vinifera genotype, five representatives of other species of the vitis genus and fifteen cultivated vitis vinifera, among which were one additional seedless variety, eight seeded table varieties and seven wine varieties. no additional snps or indels other than those identified in the rs × s background were found in this exon in the whole set of genotypes, although they were arranged into six haplotypes instead of the three segregating in the rs × s population. the most frequent haplotype was the seeded allele found in sultanina. it seems to be conserved across the genus with nearly no variation observed at the interspecific level. a t > a non-silent mutation was found in five table grapes that are seedless and one wine variety. the r > l mutation was observed in the seedless varieties but also in the seeded variety assyl kara. these results suggest that this mutation does not by itself explain the seedless phenotype. genetic characterization of vvagl alleles to acquire more precise information about a possible role of the coding and/or putative regulatory region of vvagl in the seeded versus seedless phenotype, intragenic markers derived from allele sequencing were designed to perform a qtl analysis. markers p p and p3_vvagl were designed to genetically analyze indels in the regulatory region. an indel revealed by p1_vvagl affects a putative o2-like box, p2_vvagl marks a putative tata-box near far the transcription start site and p3_vvagl marks a n motif. finally, marker e7_vvagl was designed to test snps identified in exon. genetic mapping with intragenic markers reduced the sfw and bw qtl confidence intervals down to and cm, respectively. the kruskal-wallis non-parametric method for qtl analysis was used to test the efficiency of these markers in the rs × s population. for all three analyzed seasons, the markers showing the highest correlation with seedlessness were vmc7f and p3_vvagl. a similar pattern was observed for berry weight, but with k values explaining 38% to 44% of the phenotypic variation. a strong correlation was also found for both traits with p1_vvagl p2_vvagl and e7_vvagl11; however, p3_vvagl ) was found to be the best marker in terms genotypic and phenotypic association across the three evaluated seasons, as no false positives or negatives were identified in the homozygous genotypes or. this genetic evidence shows that the region delimited by marker vmc7f and the tata-box makes the largest contribution to the seedless phenotype in the sultanina genetic background, suggesting that this region might contain the causative genetic variation of the seedless phenotype. the stratification of the progeny by genotype defined by the p3_vvagl marker revealed a partial dominant effect of the seedless allele over the seeded allele, which is consistent with the dominance effect observed at the phenotypic level only. this incomplete dominance effect is also observed for berry weight but with a minor effect. transcriptional characterization of vvagl alleles expression of vvagl was analyzed by real-time pcr analysis at three key developmental stages for ovule and seed development: pre-bloom, bloom and pea-size berries. the samples came from seven genotypes: two seedless and two seeded homozygous seedlings of the rs × s progeny, both seedless heterozygous parental genotypes and a common seeded table grape genotype that contains two different seeded alleles: red globe. in the seeded genotypes, vvagl gene was expressed after anthesis, while in pre-bloom and bloom stages expression remained minimal. during the pea-size stage, its expression was times higher than in pre-bloom or bloom stages, which is consistent with previous results. within the pea stage of development, the level of vvagl expression was associated with the vvagl genotype: genotypes homozygous for the seeded allele showed transcription times higher than genotypes homozygous for the seedless allele, and the basal level was detected at earlier developmental stages. as expected, heterozygous genotypes showed an intermediate level of expression. all these differences were statistically significant, whereas no statistically significant difference in vvagl expression in pea-stage berries was observed between the bb and bc seeded genotypes. validation of intragenic vvagl markers in different genetic backgrounds to extend the genetic analyses performed in the experimental progeny to different genetic backgrounds, an association analysis was performed with a population of genotypes characterized quantitatively for seed fresh weight. the population, derived mainly from crosses of ten seedless varieties, revealed p3_vvagl as the marker that explains the largest proportion of phenotypic variation. for markers vvp18b vmc7f p p p3_vvagl and vvp18b the statistic kruskal-wallis values were,,,, and, respectively. the p3_vvagl marker revealed six different alleles and seven main genotypes. most of the genotypes harboring one or two copies of the 198-bp allele have a seedless phenotype. as described for the experimental progeny, the seedless allele has partial dominance over the and bp seeded alleles; however, the same effect was not detected with respect to the bp seeded allele. interestingly, all of the genotyped seedless varieties within this analysis were heterozygous for this locus. discussion genetic dissection of seedlessness major qtls for seed and berry weight were previously detected on lg in a subset of this progeny, in progeny derived from two other partially seedless genotypes and in progeny derived from a cross of seeded and seedless genotypes. for sfw, confidence intervals varied between and cm in doligez et al., and cm in cabezas et al. and cm in mejía et al.. in the present work, integration of all the available genomic resources allowed us to quickly develop new co-dominant markers in the targeted area and to further reduce the confidence interval for this trait down to cm with a segregating population of only ~ phenotyped individuals. as the development of a well-balanced population in terms of phenotypic classes for seedlessness requires a step of in vitro embryo rescue, any strategy aiming to increase the accuracy of qtl detection without increasing the population size is of great interest. moreover, genetic mapping of intragenic vvagl markers, in addition to revealing a putative functional role of the regulatory of the coding region of vvagl resulted in a narrower confidence interval for the sfw qtl, so far the narrowest qtl identified for this trait. according to the genetic size of the most comprehensive ssr-based map for vitis vinifera l. and to the genome size reported for the grapevine genome, a confidence interval of cm should be equivalent to ~ kb. in our study, the confidence interval is equivalent to ~ kb, indicating that this region may be hot spot for recombination, which allowed the mapping of intragenic vvagl markers in a small progeny set. however, genotyping errors in data sets are the most common source of variation and inflated genetic distances. for instance, intragenic variation could be due to replication slippage, the mutation mechanism that cause the hypervariability of microsatellites. the putative regulatory region of vvagl contains at least nine intragenic microsatellites annotated as n boxes with repeat units that vary from to two genotypes of the rs × s experimental progeny presented a mutation, identified by ssr genotyping and sequence-verified, in the region amplified by marker p3_vvagl. this mutation consists of one additional unit of the ga repeat, which could have arisen either by taq polymerase slippage during pcr or by a real mutation occurring in these genotypes. the use of a proofreading polymerase for the amplification and sequencing supports the latter hypothesis. the limited size of our experimental population is also a potential source of distortions in genetic distance and qtl effect estimations. it is now well known that in such small populations, major effect qtls are detected properly, but mapping experiments should be refined with larger populations and/or experimental designs adapted for the detection of environmental effects and minor qtls. indeed, the minor qtls for bw and sfw detected in the present study, detected earlier in the same experimental population and detected in other studies are neither coincident with each other nor stable among years. the positive correlation of seed and berry weights and the co-localization of major qtls for both traits observed in this study was also detected and described in other progeny sets. as already discussed in these former papers, this correlation could be due to one underlying gene having a direct effect over ovule and seed development and indirectly affecting berry development through growth regulators produced by the developing seed, one or several genes having different and independent impacts on seed and berry development, or to a combination of both alternatives. the argument in favor of a pleiotropic effect of one gene is based on the fact that the growth of fleshy fruits mainly relies on cell division at early stages of berry development but on cell expansion after véraison. cell division and expansion are both controlled by gibberellins, cytokinins and auxins, which are imported from seeds or ovules. however, because the partial dominance effect observed for seedlessness was less pronounced with respect to berry weight, it is probable that the same or other underlying genes have an independent influence on berry development. molecular dissection of the major qtl for seedlessness the reduced confidence interval for the major seedlessness qtl corresponds to a kb region of the grapevine genome sequence that contains four gene models. one of these corresponds to an ortholog of the mads-box gene agl in arabidopsis thaliana and fbp in petunia hybrida, which were shown to be involved in the control of ovule identity. based on current knowledge, none of the other genes are candidates for seed or berry development. moreover, earlier expression studies in grapevine suggested that vvagl might influence ovule and seed development and that an alteration in this gene could yield seedless grapes. the expression profile described in this work for the seeded allele of vvagl is consistent with what had been already reported in syrah and in tempranillo, but also with the expression of orthologous genes like agl, fbp, tagl and osmads. in arabidopsis, pynyopich et al. showed that agl is strongly expressed in the funiculus starting from the initial stages of ovule development, in mature ovules and after fertilization. they also showed that in agl mutants, seeds are rounder and smaller than in the wild type, and that funicular cells are greater in number and size, indicating that agl is also required to prevent abnormal growth of the funiculus. among the mads-box genes known to control ovule identity, agl is the only one that seems to be both necessary and sufficient to promote ovule development. the others have proven to be redundant, suggesting that some of them evolved from a common ancestral gene. in arabidopsis, the ectopic expression of stk promotes carpel development, and in grapes vvagl is highly expressed in carpels which ultimately develop into fruit, supporting the hypothesis that vvagl might have a direct influence on berry development instead of merely a pleiotropic effect through seed development. alignment of the vvagl and agl nucleic and protein sequences showed that although the two proteins share 75% amino acid identity, no significant similarity exists between their promoter sequences. however, both predicted promoters are similar in length and share 68% of their cis-regulatory elements according to a signal scan performed with the place database over the agl and vvagl regulatory regions. also, the mm algorithm identified the following shared motifs: ttttcttt, with and motifs in vitis and arabidopsis, respectively; gcaa, with and; and ccattc, with and. the first motif corresponds to n putative regulatory elements, which are the binding site for basic pentacysteine, a regulator of the homeotic arabidopsis thaliana gene agl which controls ovule identity. bpc induces conformational changes by cooperative binding to purine-rich elements present in the agl regulatory sequence. interestingly, these purine-rich repeats are abundant in the putative regulatory region of vvagl11: at least six n were identified upstream of the tata-box and three in the 5'utr intron. the closest n repeats to the tata-box correspond to three ssr markers segregating in the rs × s progeny, vmc7f vvb18b and p3_vvagl. in this experimental population, p3_vvagl and vmc7f explain up to 78% of the phenotypic variation in seedless, which make them very good candidates for being the main regulatory elements involved in the expression of the final seedless phenotype. in a selection of genotypes derived from crosses of seedless × seedless varieties within our breeding program, p3_vvagl yielded the highest kruskall-wallis value. the proportion of phenotypic variation in seedlessness explained by vvagl is huge, much greater than the estimated effect of other genes identified as qtls from cultivated plants, like ovate, which controls fruit shape in tomato, and se which controls flowering time in rice. as discussed above, even though this result is quite consistent with previous results using similar experimental designs, it must be taken carefully as some degree of distortion and/or overestimation of effects could exist due to the small size of our progeny sets, genotyping and phenotyping errors and recombination slippage events in the regulatory region of vvagl further analyses should be performed with larger experimental designs or by transgenic assays manipulating gene expression. the mutations identified in regulatory elements of the seedless allele of vvagl explained a slightly higher degree of phenotypic variation than those identified in the coding region, suggesting that the seedless phenotype might be genetically controlled by this regulatory region. transcriptional analyses performed in contrasting phenotypes as well as in homozygous seeded and seedless genotypes revealed that in seedless genotypes, the expression of vvagl was abolished during the period of rapid seed and berry growth after berry set. as expected, in heterozygous genotypes like sultanina or ruby seedless, its expression was half that observed in homozygous seeded genotypes. together, the genetic and transcriptional evidence suggest that seedlessness in table grapes might be due to misexpression of vvagl caused by indels in its regulatory elements. defined by intragenic marker p3_vvagl the seedless allele exerts a partially dominant effect over the seeded alleles: most of the heterozygous genotypes are seedless. the c-domain in the coding sequence has been described as the less conserved domain between the mads-box family members. however, each of the major mikc sub-families possesses short, highly conserved motifs whose specific function remains unknown. the c-domain has also been reported to be involved in the mediation of higher-order interactions among mads protein dimers, in transcriptional activation, and in post-translational modifications. a non-silent mutation identified in one of these conserved motifs of vvagl that did not by itself explain the seedless phenotype might be responsible for a structural change in the c-domain making the mutant transcription factor barely expressed during the initial stages of seed development and therefore dominant over its wild-type alleles. altogether, these results are partially agree with the model proposed by bouquet and danglot and lahogue et al. for the control of seed development, where a single dominant locus, sdi, codes for a major regulatory gene. the three remaining loci that interact with sdi, according to the proposed model, were not identified with the current experimental design. gene-assisted selection in a perennial species such as grapevine, markers that allow individuals not carrying the favorable allele for the most desirable given trait to be discarded before planting in the field for further evaluation are invaluable. this is especially true for phenotypes that can only be screened in adult plants such as those affecting berries. by identifying several interesting intragenic polymorphisms between seeded and seedless genotypes in the vvagl regulatory region, our study provides four new intragenic markers in a candidate gene for seedlessness for breeding purposes. these intragenic markers displayed different relative efficiencies measured as the phenotypic variation explained by the marker and based on their efficiency to select positively seedless genotypes or negatively seeded genotypes. the ssr marker vmc7f already described as the closest marker to the sdi locus, was confirmed as one of the best markers for progeny screening. association analysis performed in the rs × s experimental progeny and over a population derived from several other seedless × seedless crosses revealed p3_vvagl as the most reliable marker for breeding purposes over three different seasons and across different genetic backgrounds. the two most interesting markers identified in our work or former studies need to be tested for their robustness in larger genetic backgrounds segregating for seedlessness. lahogue et al. developed the scar marker scc which is tightly linked to the sdi locus; however, scc was not useful in all the evaluated progeny or in the rs × s experimental population, as it often amplifies a null allele. in a controlled population derived from dominga × autumn seedless, cabezas et al. identified ssr markers closely linked to the sdi locus, and these markers results in 4% to 6% false positive identifications and in 11% to 13% false negatives. in the experimental population analysed in the present work, the use of marker p3_vvagl for the selection of homozygous genotypes resulted in 0% false positives, while vmc2f yielded 5% false positives. haplotype analysis, defined either by combinations marker pairs or by all the intragenic markers for vvagl failed to improve the efficiency achieved by p3_vvagl alone in our experimental population; any combination not only gave the same number of selected true seedless phenotypes but also increased the number of seeded phenotypes identified by mistake as true seedless.  vvagl belongs to the d-lineage of mads-box genes that control ovule identity. a better understanding of its function would benefit other crops, as its function seems to be conserved across the plant species already studied. however, its function in grapevine remains to be proven by genetic transformation of seeded cultivars. whether its role in seedlessness is confirmed or not, vvagl has proven to be a very useful marker for assisted selection of seedless grapevine. 
transcriptional analysis lent additional support to the putative role of vvagl11's regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. current knowledge of the function of its orthologous genes, its expression profile in vitis varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the d-lineage mads-box gene vvagl corresponds to the seed development inhibitor locus described earlier as a major locus for seedlessness. these results transform vvagl into a functional candidate gene for further analyses based on genetic transformation. these results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development. genetic and physical maps were integrated with the public genomic sequence of vitis vinifera l. to improve qtl analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. characterization of vvagl at the sequence level in the experimental progeny identified several snps and indels in both regulatory and coding regions. moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. stenospermocarpy has not yet been characterized at the molecular level. for breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site. functional information from model species including vitis suggested that vvagl included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development. in association analyses performed over three seasons, these snps and indels explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. we propose that vvagl is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. major qtls co-positioning for both traits on chromosome defined a 92-kb confidence interval.
 stenospermocarpy is a mechanism through which certain genotypes of vitis vinifera l. such as sultanina produce berries with seeds reduced in size. stenospermocarpy has not yet been characterized at the molecular level.  genetic and physical maps were integrated with the public genomic sequence of vitis vinifera l. to improve qtl analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. major qtls co-positioning for both traits on chromosome defined a 92-kb confidence interval. functional information from model species including vitis suggested that vvagl included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development. characterization of vvagl at the sequence level in the experimental progeny identified several snps and indels in both regulatory and coding regions. in association analyses performed over three seasons, these snps and indels explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. transcriptional analysis lent additional support to the putative role of vvagl11's regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. these results transform vvagl into a functional candidate gene for further analyses based on genetic transformation. for breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site.  we propose that vvagl is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. current knowledge of the function of its orthologous genes, its expression profile in vitis varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the d-lineage mads-box gene vvagl corresponds to the seed development inhibitor locus described earlier as a major locus for seedlessness. these results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development. 