BACKGROUND
grape  is the main fruit crop of temperate regions, reaching nearly  <dig> million tons of fruit produced throughout the world in  <dig>  <cit> .
it also exhibits a high level of genetic diversity; the genus vitis includes more than  <dig> species .
berry weight is considered as one of the main selection criteria in table grape breeding, due to the consumer preferences for large and seedless berries along with organoleptic quality traits such as flavor and aroma  <cit> .
however, berry weight is a quantitative and polygenic trait, probably determined by numerous processes such as cell multiplication, cell wall modification, water and sugar transport.
despite its relatively high heritability which is mostly additive, the genetic determination of berry weight was until recently scarcely documented  <cit> .
therefore, considering the economic importance of berry weight for table grapes, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in the expression of its driver genes.
this information is required for the development of new cultivars involving the combination of desirable traits, which include not just berry size and lack of seeds, but also cluster architecture compatible with a proper berry spatial distribution  <cit> , response to gibberellic acid   <cit> , yield  <cit>  and tolerance to fungal diseases  <cit> , among others production traits.
as in other plant species, growth and cell proliferation of grape berries correspond to different processes which together determine the final fruit dimensions  <cit> .
the development and maturation of grapevine berries has been studied as a model because of the uniqueness of this process in plant biology and its molecular regulation  <cit> .
berry development presents a characteristic double sigmoid curve with three main phases, encompassing a series of physical and biochemical changes such as cell division and elongation, primary and secondary metabolism and resistance/susceptibility to biotic/abiotic stress  <cit> .
phase i involves events associated with cell division and cell elongation  <cit> , the latter based on the accumulation of organic acids into the vacuole  <cit> .
in this stage the berry is hard, green and grows slowly  <cit> ; malic acid is the predominant metabolite.
in phase ii, slower growth is observed and berry softening begins; numerous changes occur associated with gene expression and berry physiology reprogramming.
phase iii is when berries reach their mature weight.
this stage is characterized by the onset of sugar accumulation, a decrease in organic acid content and concomitantly, accumulation of anthocyanins in colored cultivars and volatile secondary metabolites associated with flavor and aroma  <cit> .
a positive correlation has been described between the final berry weight and seed content  <cit>  in segregating populations , possibly being the result of growth regulators produced by seeds  <cit> .
interestingly, in stenospermocarpic varieties pollination occurs normally although the embryo development process aborts early, approximately  <dig> to  <dig> weeks after fertilization, while berry development continues normally  <cit> .
however, seedless varieties such as cv.
sultanina exhibit a reduced berry weight at harvest  <cit> , requiring two or three exogenous applications of gibberellic acid along with cluster thinning in order to maximize the potential berry growth; both practices demand high labor force, which increases production costs.
in relation to hormonal regulation, ethylene, auxins, aba, cytokinins and gibberellins can influence berry development and ripening  <cit> .
the concentration of auxins, cytokinins and gibberellins tends to increase during phase i, in pre-véraison stages, and later decreasing in véraison, where a peak of abscisic acid has been described  <cit> .
previous studies have described qtls associated with berry weight in chromosomes  <dig> and  <dig>  <cit> ,  <dig> and  <dig>  <cit> ,  <dig>   <dig> and  <dig>  <cit> ,  <dig>  <cit>  and  <dig>  <cit> .
in addition,  <cit>  recently reported the vvceb <dig> gene, a bhlh transcription factor, as possibly involved in the regulation of cell size in cv.
cabernet sauvignon.
also, the vvnac <dig> gene has been proposed as probably associated with berry weight variation in v. vinifera  <cit> .
however, the genetics and information on the molecular mechanisms behind berry development in table grapes are still scarce and limited.
diverse transcriptome studies based on microarrays  as well as high-throughput rna-seq sequencing  <cit>  have been developed in grapes, focused on understanding the developmental and maturation process of the berry.
however, these studies were directed to improve the understanding of organic acids, resveratrol, anthocyanin and tannin content and metabolism in relation to wine quality .
due to the economic importance of berry weight in table grapes, it is relevant to determine the underlying mechanisms controlling this trait, in order to reveal positive and negative genetic factors involved in the expression of this complex trait.
we carried out the first transcriptome analysis with the aim of elucidating the mechanisms involved in berry weight determination.
we contrasted seedless table grape segregants with opposite phenotypes for this trait in order to explore its genetic architecture.
this comparative transcriptome profiling revealed candidate genes associated with berry weight, which could be evaluated as selection tools in table grape breeding programs.
RESULTS
rna isolation from contrasting segregants for berry weight and library construction
the feasibility of this study was based on the availability of seedless segregants for berry weight , maintained under the same climatic and agronomic conditions, which offer a unique opportunity to analyze transcriptome changes associated with this complex trait.
in order to study the underlying differences between large and small berries, six seedless segregants derived from a ‘ruby seedless’ x ‘sultanina’ crossing  with contrasting phenotypes for berry weight were selected and phenotyped during three seasons, 2009– <dig> to 2011– <dig> .
according to anova, the genotype effect was the most significant , the season effect corresponding to  <dig>  % and the genotype x season interaction was  <dig>  %.
the linear model explained 97 % of the phenotypic variance .fig.
<dig> berry fresh weight at harvest  of six rxs segregants exhibiting contrasting phenotypes, including parents cv.
ruby seedless and sultanina.
each value corresponded to phenotypic mean values during the 2009– <dig>  2010– <dig> and 2011– <dig> seasons.
error bars represent one standard error of the mean
significance codes according to anova : ***0– <dig> ; ** <dig> – <dig> ; * <dig> – <dig> ; n.s.
not significant .
coefficient of determinations  based on mean squares of each factor, error and model according to anova
thus a transcriptome experiment based on illumina platform  was undertaken focused on early stages of berry development, i.e., fruit setting  and berry 6–8 mm stages   <cit> ; mrna samples isolated in both stages were sequenced independently .
these two stages are part of phase i of the double sigmoid curve during berry growth, when the final number of cells is being defined, followed by cell expansion associated with water and organic acid accumulation in the vacuole  <cit> , critical processes defining the final fruit size  <cit> .
during the fst stage the berry cell machinery is receptive to exogenous gibberellin  applications, increasing berry weight and reducing seed weight  <cit> .
ga <dig> and ga <dig>  the two endogenous bioactive gas synthetized in the berry, have their maximum peaks in the fst and b <dig> stages, respectively .fig.
<dig> experimental design, gene differential expression and hierarchical clustering of differentially expressed genes.
a phenological stages considered for the transcriptomic study.
rna samples were obtained from large  and small  berry genotypes, at phenological stages of fruit-setting  and berry 6–8 mm stages , modified from  <cit> .
b differentially expressed genes after comparison between rxs segregants with contrasting phenotypes for berry weight in both phenological stages.
c hierarchical clustering of a group of  <dig> differentially expressed genes among lb and sb segregants in the fst and b <dig> stages.
pearson correlation was used as distance and five clusters were identified
illumina gaii mrna sequencing
a total of  <dig> libraries were analyzed;  <dig> , <dig> reads of 50 bp were obtained .
after quality trimming  <dig> , <dig> reads were kept, representing a loss of about 2 % of the reads for each library .
of this total, 91 % of the reads were mapped as unique and multiple alignments .
the total of mapped reads corresponded to  <dig>  million reads, of which  <dig> to 69 % mapped in exons,  <dig> to 19 % in utr regions,  <dig> to 9 % within intron regions, and  <dig> to 9 % in intergenic regions; the percentage of usable reads  varied from  <dig> to 85 % .
a total of  <dig>  million reads obtained from the  <dig> libraries were not mapped to the reference genome pn <dig>  they were used to construct  <dig>  de novo contigs, with an average length of 673 bp.
of them,  <dig> contigs were mapped to the reference genome and reanalyzed .
global analysis of gene expression changes from fruit set  to berry 6–8 mm  stages
to determine which genes are changing their expression profiles and at what stage, comparisons between individuals with contrasting phenotypes for berry weight were performed .
a group of  <dig> differentially expressed genes  was identified comparing large  versus small berry  segregants in the two phenological stages  .
in particular,  <dig> genes were identified from fst   and  <dig> genes from b <dig>  .
interestingly,  <dig> of these were differentially expressed in both stages, with  <dig> coincidentally raising or decreasing their expression level, including transcripts coding for stilbene synthases   ; this is equivalent to what has been observed in previous transcriptome studies during berry development in cv.
corvina  <cit>  and cv.
cabernet sauvignon  <cit> .
a hierarchical clustering was performed using gene expression measured as fpkm observed in the group of  <dig> de genes , and using pearson correlation as distance in the transcriptional dendrogram.
according to the expression profiles, five groups of de genes were identified containing  <dig>   <dig>   <dig>   <dig> and  <dig> de genes .
in addition, a functional enrichment analysis  was developed to assess main processes over-represented in each cluster of transcripts using the agrigo platform  <cit>  .
no over-represented category was identified in the case of cluster  <dig>  concomitantly, go analysis of the groups of  <dig> de genes identified in the fst and  <dig> in the b <dig> stage were performed and the results agreed with the global analysis.
functional analysis of de genes comparing large and small berry segregants at fruit set  and berry 6–8 mm  development stages
selection of a subset of candidate genes able to explain the difference in berry size
in order to identify the genes involved in berry size determination, a principal components analysis was performed considering the  <dig> de genes.
the results showed that two components explained 87 % of the phenotypic variance .
the first component explained 55 % of the variation and clearly discriminates between contrasting phenotypes.
the second component explained  <dig>  % of the observed variation and discriminated between phenological stages .
subsequently, correlation analyses were performed and significant correlations  between de genes and the two components were performed in order to select candidate genes, defined as transcripts whose expression level discriminates between individual classes  <cit> .fig.
<dig> principal components analysis  using normalized expression data .
analysis included the group of  <dig> de genes derived from comparison between lb  and sb segregants  in the fst and b <dig> stages
a group of  <dig> de genes were significantly correlated with component  <dig> and  <dig> with component  <dig> .
interestingly, both subsets of de genes were identified in the b <dig> stage .table  <dig> differentially expressed genes  significantly correlated with pca components  <dig>  and  <dig>
one of the most relevant functional categories associated with this group of genes was stress/defense response , encompassing hsp and chaperonins up-regulated in lb segregants .
in addition, protein kinase modifications and transcription categories were also relevant, possibly associated with the reprogramming of genes controlling transcription and translation rate in order to remodel the set of cell proteins.
four genes coding for receptor kinase-like  were up-regulated in sb segregants .
rlks play a pivotal role in sensing external stimuli, activating downstream signaling pathways and regulating cell behavior involved in response to pathogens  <cit>  growth and development processes in plants as well as biotic and abiotic stresses, suggesting a possible participation in the defense response in plants  <cit> .
this evidence suggests that a transcriptome reprogramming process is taking place during berry maturation, involving changes in synthesis and activation of proteins, processes that have been previously described in cv.
corvina, as well as a possible compensatory adaptation  <cit> .
indeed, increments in hsps and chaperonin expression towards véraison have been reported, with a peak at véraison and subsequent reduction during berry maturation, associated with massive changes in metabolism at this phenological stage which demand the synthesis of new proteins  <cit> .
considering the observed evidence from other genetic backgrounds such as cv.
corvina, the higher expression level of hsp and chaperonins in lb segregants may be reflecting the adaptation of the berry to environmental stresses such as higher temperatures in the field.
furthermore, evidence of a strong transcriptional control was found, with seven genes associated with the transcription category, two of them up-regulated in lb segregants, the heat stress transcription factor a-2b and a gcn5-related n-acetyltransferase  family protein.
interestingly, the former corresponds to a transcriptional regulator whose orthologue in rice is the protein oshsfa2e, induced by heat stress and specifically bound to the promotor of heat shock elements and possibly responsible for tolerance to high temperatures.
considering this, its introgression could be considered useful, in order to improve crop tolerance to climate change-associated stresses  <cit> .
the latter gene, a histone acetyltransferase , is responsible for lysine residue acetylation in histones h2b, h <dig> and h <dig>  and also acts as a transcriptional activator, implicated in chromatin assembly and dna replication  <cit> .
in addition, a gene coding for a nac domain-containing protein  <dig> was found up-regulated in sb segregants, which are plant-specific transcription factors .
members of this gene family have been related to plant development  <cit> .
in particular in vitis vinifera, vvnac <dig> gene has been associated with the early development of grape flowers and berries  <cit> , possibly contributing to berry size variation  <cit> .
in the transport category six de genes were found, two up-regulated in lb segregants, the sugar carrier protein a and the putative mitochondrial 2-oxoglutarate/malate carrier protein, probably associated with the transport of malate to the vacuole and cell turgor; both could be key for cell expansion.
malate is the main organic acid stored in the vacuole of grape berry cells, from fst to véraison  <cit> .
associated with cell wall metabolism, we found de genes coding for a probable xyloglucan endotransglucosylase/hydrolase proteins, a lichenase and a probable galacturonosyltransferase  <dig>  up-regulated in sb segregants, and an expansin-a <dig>  up-regulated in lb segregants .
this result is concordant with the top over-represented category ‘xyloglucan:xyloglucosyl transferase’ associated with cluster  <dig> .
this evidence could be related to cell expansion events described in the b <dig> stage, which initially requires cell wall softening and later the incorporation of recently synthetized material  <cit> .
cell wall softening occurs as a result of disruption of chemical bonds between structural cell wall components, by acidification and hydrolase enzymes, modifications which require an accurate and coordinated transcriptional regulation of genes involved in biosynthesis and cell wall adaptations  <cit> .
these enzymes modify hemicelluloses during cell expansion and fruit softening, suggesting a direct influence on growth.
furthermore, cell expansion involves changes in composition as well as the accumulation of different compounds which maintain osmotic pressure and water flux in cells in expansion  <cit> .
evidence obtained in this study agreed with these events where a strong induction of genes associated with cell expansion was observed, which probably results in larger berry weights.
our results suggest a relevant role of expansins in the lb phenotype during the b <dig> stage.
in the case of sb segregants, genes with xyloglucan:xyloglucosyl transferase activity were found up-regulated in the same stage .
this evidence suggests a differentiation in cell wall modifications, considering that expansins have been proposed as cell wall activator agents without hydrolytic activity.
likewise, up-regulated endoglucanases were identified in lb segregants, which are also associated with cell wall dynamics.
concomitantly, in the b <dig> stage genes related to auxin metabolism were also identified, up-regulated in the lb phenotype, in line with the putative role of auxins in cell expansion, involved in acid growth mediated by expansins  <cit>  .
evidence obtained from the transcriptome analysis suggested that major differences among lb and sb seedless segregants are triggered at the b <dig> stage, which may be responsible for the final berry weight observed at harvest.
in this stage berry diameter increases by cell expansion  <cit> .
other functional categories were associated with secondary metabolism, transport of inorganic ions and metals, proteosome-protein degradation, hormone metabolism and signaling, development and chlorophyll biosynthesis .
regarding the group of  <dig> genes significantly correlated with component  <dig> , two genes were identified coding for aspartic proteinase nepenthesin- <dig>  possibly associated with aspartic-type endopeptidase activity  <cit> , and senescence process ; as well as a serine carboxypeptidase-like  <dig> and endoglucanase  <dig>  both related to cell wall metabolism .
furthermore, three genes were found associated with hormonal metabolism and signaling, coding for auxin-induced protein aux <dig> and arg <dig>  and pirin-like protein, related to calcium signaling.
co-expression network analysis
network analyses were performed to identify co-expression genes associated with the separation between lb and sb segregants.
subsequently, correlation analyses results lead to identify a total of  <dig>  partial correlations,  <dig> of them significant .
correlograms were plotted with the total observed correlations , and correlations of over 90 % were considered as significant .
furthermore, 15 % of the significant correlations were negative and more variable .
positive significant correlations represented 85 % and were less variable .
five interconnected clusters of nodes were identified  .fig.
<dig> nodes of co-expressed genes among lb and sb segregants identified using a network analysis.
main components of each node are n1: hsps, chaperonins; n2: stbs, thaumatins; n3: monooxygenases; n4: cell wall modifications; n5: vacuolar transporters.
lines in red and green represent negative and positive correlations, respectively
these results were concordant with those obtained from hierarchical clustering and pca; the seven de genes selected as candidate markers for berry weight from pca analysis were also present in the network analysis .
in addition, according to the cluster connectivity our results agreed with previous studies which described that highly connected genes were usually involved in the same biological pathways  <cit> .
cluster one was conformed mostly of genes coding for hsps and chaperonin proteins, including also a gene coding for gdsl esterase/lipase and expansin-a <dig> , all of them up-regulated in lb segregants.
this result is concordant with identification of the category ‘protein folding’ over-represented in cluster  <dig> , a process mediated by hsp  <cit> .
as these genes have been associated with heat stress during berry development  <cit>  and the response to microclimate changes in bunches  <cit> , this evidence suggests that lb segregants could respond more efficiently to heat stress.
negative correlations were among genes coding for major allergen pru av  <dig>  associated with defense responses  <cit> , and expansin-a8; genes coding for chaperonins or hsps were also found .
cluster two was composed mainly of genes coding for pals and sts, a gene co-expression previously reported in cv.
syrah  <cit> ; they were up-regulated in sb segregants in this study, in both phenological stages .
these results are concordant with the identification of the over-represented categories ‘l-phenylalanine catabolic process’ and ‘response to biotic stimulus’ found in cluster  <dig> .
sts expression has been considered as a response to stress factors such as fungal diseases, wounding and uv light  <cit> , and a shift in phenylpropanoid pathway metabolites is highly sensitive to temperature changes  <cit> .
the differential expression of those genes during berry development and maturation have been described in cv.
corvina  <cit> , norton  <cit>  and moscatel de hamburgo  <cit> .
hence these results suggest that sb segregants presented a higher stress level during berry development than lb segregants, possibly environmental due to high temperatures.
however, positive correlation was observed between genes coding for expansin-a <dig>  and f-box/lrr-repeat protein  <dig> , both negatively correlated with genes coding for stilbene synthases in the cluster.
f-box proteins act as regulators of the ubiquitin kinase dependent pathway associated with protein degradation, an important post-translational mechanism.
thus the removal of unfolded or non-functional proteins facilitates the adaptation of organisms to environmental changes, through rapid intracellular signaling  <cit> .
in particular, expansin-a <dig> also showed negative correlation with genes coding for thaumatins, proteosome subunits, inorganic transporters and proteins related to pathogenesis , identified up-regulated in sb segregants .
cluster three was composed mostly of genes with monooxygenase and oxide-reductase activities, including cytochrome p <dig>  pr <dig> protease inhibitor and eugenol synthase .
genes belonging to the cytochrome p <dig> family were found up-regulated in sb segregants , associated with phenylpropanoids, flavonoids, brassinosteroids and lignin synthesis.
interestingly, it has been reported that cytochrome p450-78a partially controls fruit size in tomato and possibly has a role in the domestication of this species  <cit> .
biosynthetic enzymes, redox regulators and hsp have been described as effector genes related to abiotic stress responses  <cit> .
however, genes coding for chloroplast beta-amylase  <dig>  gibberellin receptor gid <dig> and protein wax <dig>  up-regulated in sb segregants, were negatively correlated .
wax <dig> protein plays a role in the conversion or secretion of common precursors for cutins and wax metabolic pathways; it is also related to cuticle formation and stomata, both involved in transpiration control and drought tolerance as well  <cit> .
cluster  <dig> included a cohort of candidate enzymes related to cell wall modification, with xyloglucan endotransglucosylase/hydrolase protein  <dig>  and glucan endo-13-beta-glucosidase activities, positively correlated .
interestingly, cluster  <dig> presented no edges with the remaining clusters.
two branches were observed, the first composed of genes coding for 60s ribosomal protein l <dig> and abscisic acid 8'-hydroxylase  <dig>  all of them positively correlated.
the ribosomal protein modulation suggests that the transcriptome reprogramming that occurs during berry maturation involves changes in protein synthesis  <cit>  .
a second branch included genes coding for cysteine-rich receptor-like protein kinase 10; vacuolar amino acid transporter  <dig>  up-regulated in lb segregants , possibly associated with amino acid compartmentalization in the vacuole  <cit> ; cytokinin dehydrogenase  <dig>  as well as galactinol-sucrose galactosyltransferase; glutathione s-transferase, associated with the cellular response induced by heat shock stress and auxins, and metals such as cadmium, silver and copper  <cit> ; and isoflavone-7-o-methyltransferase  <dig>  related with flavonoid/isoflavonoid metabolism and biotic stress responses  <cit> , which were positively regulated .
expression analysis of a group of candidate genes associated with berry weight using qpcr
the expression profiles of seven de genes were experimentally validated by real-time qpcr experiments, in the phenological stages of anthesis , fruit-setting  and berry 6–8 mm  , in order to select candidate genes as putative factors associated with berry weight determination.fig.
<dig> validation of differentially expressed  genes among lb and sb segregants by real-time pcr.
lb, large berries, in black; sb, small berries, in grey.
phenological stages were anthesis , fruit setting  and berry 6–8 mm .
genes are a gdsl esterase/lipase; b cytokinin dehydrogenase 3; c stilbene synthase 6; d gene coding for  <dig>  kda class ii hsp; e tf-bhlh60; f tf-bhlh93; g tf-bhlh96; different letters on top of bars indicate significant differences  according to one-way anova and tukey’s multiple comparison test among phenotypic category/phenological stage; values are the results of  <dig> observations categorized by phenotype.
the tcpb gene  was used as reference gene and gene expression was expressed as relative expression
the results of the network and pca were considered in the selection of candidate genes.
genes coding for gdsl esterase/lipase, cytokinin dehydrogenase  <dig> and stilbene synthase  <dig> were selected from the network analysis.
in addition, the gene coding for hsp  <dig>  kda class ii was significantly correlated with pca component  <dig>
in the case of the gene coding for gdsl esterase/lipase, experimental results confirmed its up-regulated expression in lb segregants in the b <dig> stage , suggesting an increase in its expression in this stage in both lb and sb segregants .
in addition, the gene coding for cytokinin dehydrogenase  <dig> was significantly up-regulated in lb segregants in the fst stage , with lower expression in both groups of segregants at the b <dig> stage .
interestingly, the gene coding for stilbene synthase  <dig> showed a tendency to be up-regulated in sb segregants in the three evaluated stages.
significant differences were confirmed during fl and b <dig> , being higher in the latter stage .
in addition, the gene coding for hsp  <dig>  kda class ii  showed similar expression in fl in lb and sb segregants.
however, in the fst and b <dig> stages it was significantly up-regulated in lb segregants .
considering that hsp <dig> -d was also highly correlated with component  <dig> of the pca, it could be considered as a potential candidate gene for berry weight.
participation of bhlh proteins in plant organ size determination has been described.
in particular in v. vinifera, the cell elongation protein bhlh  has been recently associated with berry weight in cabernet sauvignon, possibly involved in cell expansion during berry development  <cit> .
therefore, in order to evaluate the possible role of members of this transcription factor family in the differences between lb and sb segregants, three de genes coding for bhlh <dig>  bhlh <dig> and bhlh <dig> were selected to be experimentally confirmed by real-time qpcr experiments.
the results showed that in the case of genes coding for transcription factors , tf-bhlh <dig> was significantly up-regulated in sb segregants during the fl stage.
however, in the fst stage it was up-regulated in lb segregants  .
there was an inflexion in the fst stage, with maximum expression for lb segregants and minimum for sb segregants.
the same tendency was detected for tf-bhlh <dig>  but with a significant differential expression during the fst and b <dig> stages, up-regulated in lb segregants .
a similar expression profile was observed in the gene coding for tf-bhlh <dig>  which was up-regulated in lb segregants at the fst stage .
interestingly, our results differ from previous reports that proposed the gene vvceb <dig> as a candidate marker for berry size, whose transcripts are predominantly accumulated in berries, especially with minimum auxin content  <cit> .
indeed, the three tfs evaluated showed higher expression in the fst stage comparing lb vs. sb segregants, suggesting a possible role in early stages of development.
experimental validation in advanced phenological stages would confirm their expression profile in berries in order to determine if, as with vvceb <dig>  these tfs plays a role in cell expansion in a wider genetic background.
CONCLUSIONS
we have carried out the first transcriptome analysis focused on seedless table grape segregants with contrasting phenotypes for berry weight.
a group of  <dig> differentially expressed genes potentially associated with berry size was identified,  <dig> genes in the fst stage and  <dig> genes in the b <dig> stage.
the integration of differential expression, pca, correlation and network analysis provided a wide characterization of overall regulation and dynamic remodeling of the gene expression in berry development in pre-véraison stages.
a survey of candidate genes was also performed, and expression profiles of seven candidate genes were validated.

 grape is the main fruit crop of temperate regions, reaching nearly million tons of fruit produced throughout the world in. it also exhibits a high level of genetic diversity; the genus vitis includes more than species. berry weight is considered as one of the main selection criteria in table grape breeding, due to the consumer preferences for large and seedless berries along with organoleptic quality traits such as flavor and aroma. however, berry weight is a quantitative and polygenic trait, probably determined by numerous processes such as cell multiplication, cell wall modification, water and sugar transport. despite its relatively high heritability which is mostly additive, the genetic determination of berry weight was until recently scarcely documented. therefore, considering the economic importance of berry weight for table grapes, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in the expression of its driver genes. this information is required for the development of new cultivars involving the combination of desirable traits, which include not just berry size and lack of seeds, but also cluster architecture compatible with a proper berry spatial distribution, response to gibberellic acid, yield and tolerance to fungal diseases, among others production traits. as in other plant species, growth and cell proliferation of grape berries correspond to different processes which together determine the final fruit dimensions. the development and maturation of grapevine berries has been studied as a model because of the uniqueness of this process in plant biology and its molecular regulation. berry development presents a characteristic double sigmoid curve with three main phases, encompassing a series of physical and biochemical changes such as cell division and elongation, primary and secondary metabolism and resistance/susceptibility to biotic/abiotic stress. phase i involves events associated with cell division and cell elongation, the latter based on the accumulation of organic acids into the vacuole. in this stage the berry is hard, green and grows slowly; malic acid is the predominant metabolite. in phase ii, slower growth is observed and berry softening begins; numerous changes occur associated with gene expression and berry physiology reprogramming. phase iii is when berries reach their mature weight. this stage is characterized by the onset of sugar accumulation, a decrease in organic acid content and concomitantly, accumulation of anthocyanins in colored cultivars and volatile secondary metabolites associated with flavor and aroma. a positive correlation has been described between the final berry weight and seed content in segregating populations, possibly being the result of growth regulators produced by seeds. interestingly, in stenospermocarpic varieties pollination occurs normally although the embryo development process aborts early, approximately to weeks after fertilization, while berry development continues normally. however, seedless varieties such as cv. sultanina exhibit a reduced berry weight at harvest, requiring two or three exogenous applications of gibberellic acid along with cluster thinning in order to maximize the potential berry growth; both practices demand high labor force, which increases production costs. in relation to hormonal regulation, ethylene, auxins, aba, cytokinins and gibberellins can influence berry development and ripening. the concentration of auxins, cytokinins and gibberellins tends to increase during phase i, in pre-véraison stages, and later decreasing in véraison, where a peak of abscisic acid has been described. previous studies have described qtls associated with berry weight in chromosomes and, and, and, and. in addition, recently reported the vvceb gene, a bhlh transcription factor, as possibly involved in the regulation of cell size in cv. cabernet sauvignon. also, the vvnac gene has been proposed as probably associated with berry weight variation in v. vinifera. however, the genetics and information on the molecular mechanisms behind berry development in table grapes are still scarce and limited. diverse transcriptome studies based on microarrays as well as high-throughput rna-seq sequencing have been developed in grapes, focused on understanding the developmental and maturation process of the berry. however, these studies were directed to improve the understanding of organic acids, resveratrol, anthocyanin and tannin content and metabolism in relation to wine quality. due to the economic importance of berry weight in table grapes, it is relevant to determine the underlying mechanisms controlling this trait, in order to reveal positive and negative genetic factors involved in the expression of this complex trait. we carried out the first transcriptome analysis with the aim of elucidating the mechanisms involved in berry weight determination. we contrasted seedless table grape segregants with opposite phenotypes for this trait in order to explore its genetic architecture. this comparative transcriptome profiling revealed candidate genes associated with berry weight, which could be evaluated as selection tools in table grape breeding programs.  rna isolation from contrasting segregants for berry weight and library construction the feasibility of this study was based on the availability of seedless segregants for berry weight, maintained under the same climatic and agronomic conditions, which offer a unique opportunity to analyze transcriptome changes associated with this complex trait. in order to study the underlying differences between large and small berries, six seedless segregants derived from a ‘ruby seedless’ x ‘sultanina’ crossing with contrasting phenotypes for berry weight were selected and phenotyped during three seasons, 2009– to 2011–. according to anova, the genotype effect was the most significant, the season effect corresponding to  % and the genotype x season interaction was  %. the linear model explained 97 % of the phenotypic variance.fig. berry fresh weight at harvest of six rxs segregants exhibiting contrasting phenotypes, including parents cv. ruby seedless and sultanina. each value corresponded to phenotypic mean values during the 2009– 2010– and 2011– seasons. error bars represent one standard error of the mean significance codes according to anova: ***0–; ** –; * –; n.s. not significant. coefficient of determinations based on mean squares of each factor, error and model according to anova thus a transcriptome experiment based on illumina platform was undertaken focused on early stages of berry development, i.e., fruit setting and berry 6–8 mm stages; mrna samples isolated in both stages were sequenced independently. these two stages are part of phase i of the double sigmoid curve during berry growth, when the final number of cells is being defined, followed by cell expansion associated with water and organic acid accumulation in the vacuole, critical processes defining the final fruit size. during the fst stage the berry cell machinery is receptive to exogenous gibberellin applications, increasing berry weight and reducing seed weight. ga and ga the two endogenous bioactive gas synthetized in the berry, have their maximum peaks in the fst and b stages, respectively.fig. experimental design, gene differential expression and hierarchical clustering of differentially expressed genes. a phenological stages considered for the transcriptomic study. rna samples were obtained from large and small berry genotypes, at phenological stages of fruit-setting and berry 6–8 mm stages, modified from. b differentially expressed genes after comparison between rxs segregants with contrasting phenotypes for berry weight in both phenological stages. c hierarchical clustering of a group of differentially expressed genes among lb and sb segregants in the fst and b stages. pearson correlation was used as distance and five clusters were identified illumina gaii mrna sequencing a total of libraries were analyzed;, reads of 50 bp were obtained. after quality trimming, reads were kept, representing a loss of about 2 % of the reads for each library. of this total, 91 % of the reads were mapped as unique and multiple alignments. the total of mapped reads corresponded to million reads, of which to 69 % mapped in exons, to 19 % in utr regions, to 9 % within intron regions, and to 9 % in intergenic regions; the percentage of usable reads varied from to 85 %. a total of million reads obtained from the libraries were not mapped to the reference genome pn they were used to construct de novo contigs, with an average length of 673 bp. of them, contigs were mapped to the reference genome and reanalyzed. global analysis of gene expression changes from fruit set to berry 6–8 mm stages to determine which genes are changing their expression profiles and at what stage, comparisons between individuals with contrasting phenotypes for berry weight were performed. a group of differentially expressed genes was identified comparing large versus small berry segregants in the two phenological stages. in particular, genes were identified from fst and genes from b. interestingly, of these were differentially expressed in both stages, with coincidentally raising or decreasing their expression level, including transcripts coding for stilbene synthases; this is equivalent to what has been observed in previous transcriptome studies during berry development in cv. corvina and cv. cabernet sauvignon. a hierarchical clustering was performed using gene expression measured as fpkm observed in the group of de genes, and using pearson correlation as distance in the transcriptional dendrogram. according to the expression profiles, five groups of de genes were identified containing and de genes. in addition, a functional enrichment analysis was developed to assess main processes over-represented in each cluster of transcripts using the agrigo platform. no over-represented category was identified in the case of cluster concomitantly, go analysis of the groups of de genes identified in the fst and in the b stage were performed and the results agreed with the global analysis. functional analysis of de genes comparing large and small berry segregants at fruit set and berry 6–8 mm development stages selection of a subset of candidate genes able to explain the difference in berry size in order to identify the genes involved in berry size determination, a principal components analysis was performed considering the de genes. the results showed that two components explained 87 % of the phenotypic variance. the first component explained 55 % of the variation and clearly discriminates between contrasting phenotypes. the second component explained  % of the observed variation and discriminated between phenological stages. subsequently, correlation analyses were performed and significant correlations between de genes and the two components were performed in order to select candidate genes, defined as transcripts whose expression level discriminates between individual classes.fig. principal components analysis using normalized expression data. analysis included the group of de genes derived from comparison between lb and sb segregants in the fst and b stages a group of de genes were significantly correlated with component and with component. interestingly, both subsets of de genes were identified in the b stage.table differentially expressed genes significantly correlated with pca components and one of the most relevant functional categories associated with this group of genes was stress/defense response, encompassing hsp and chaperonins up-regulated in lb segregants. in addition, protein kinase modifications and transcription categories were also relevant, possibly associated with the reprogramming of genes controlling transcription and translation rate in order to remodel the set of cell proteins. four genes coding for receptor kinase-like were up-regulated in sb segregants. rlks play a pivotal role in sensing external stimuli, activating downstream signaling pathways and regulating cell behavior involved in response to pathogens growth and development processes in plants as well as biotic and abiotic stresses, suggesting a possible participation in the defense response in plants. this evidence suggests that a transcriptome reprogramming process is taking place during berry maturation, involving changes in synthesis and activation of proteins, processes that have been previously described in cv. corvina, as well as a possible compensatory adaptation. indeed, increments in hsps and chaperonin expression towards véraison have been reported, with a peak at véraison and subsequent reduction during berry maturation, associated with massive changes in metabolism at this phenological stage which demand the synthesis of new proteins. considering the observed evidence from other genetic backgrounds such as cv. corvina, the higher expression level of hsp and chaperonins in lb segregants may be reflecting the adaptation of the berry to environmental stresses such as higher temperatures in the field. furthermore, evidence of a strong transcriptional control was found, with seven genes associated with the transcription category, two of them up-regulated in lb segregants, the heat stress transcription factor a-2b and a gcn5-related n-acetyltransferase family protein. interestingly, the former corresponds to a transcriptional regulator whose orthologue in rice is the protein oshsfa2e, induced by heat stress and specifically bound to the promotor of heat shock elements and possibly responsible for tolerance to high temperatures. considering this, its introgression could be considered useful, in order to improve crop tolerance to climate change-associated stresses. the latter gene, a histone acetyltransferase, is responsible for lysine residue acetylation in histones h2b, h and h and also acts as a transcriptional activator, implicated in chromatin assembly and dna replication. in addition, a gene coding for a nac domain-containing protein was found up-regulated in sb segregants, which are plant-specific transcription factors. members of this gene family have been related to plant development. in particular in vitis vinifera, vvnac gene has been associated with the early development of grape flowers and berries, possibly contributing to berry size variation. in the transport category six de genes were found, two up-regulated in lb segregants, the sugar carrier protein a and the putative mitochondrial 2-oxoglutarate/malate carrier protein, probably associated with the transport of malate to the vacuole and cell turgor; both could be key for cell expansion. malate is the main organic acid stored in the vacuole of grape berry cells, from fst to véraison. associated with cell wall metabolism, we found de genes coding for a probable xyloglucan endotransglucosylase/hydrolase proteins, a lichenase and a probable galacturonosyltransferase up-regulated in sb segregants, and an expansin-a up-regulated in lb segregants. this result is concordant with the top over-represented category ‘xyloglucan:xyloglucosyl transferase’ associated with cluster. this evidence could be related to cell expansion events described in the b stage, which initially requires cell wall softening and later the incorporation of recently synthetized material. cell wall softening occurs as a result of disruption of chemical bonds between structural cell wall components, by acidification and hydrolase enzymes, modifications which require an accurate and coordinated transcriptional regulation of genes involved in biosynthesis and cell wall adaptations. these enzymes modify hemicelluloses during cell expansion and fruit softening, suggesting a direct influence on growth. furthermore, cell expansion involves changes in composition as well as the accumulation of different compounds which maintain osmotic pressure and water flux in cells in expansion. evidence obtained in this study agreed with these events where a strong induction of genes associated with cell expansion was observed, which probably results in larger berry weights. our results suggest a relevant role of expansins in the lb phenotype during the b stage. in the case of sb segregants, genes with xyloglucan:xyloglucosyl transferase activity were found up-regulated in the same stage. this evidence suggests a differentiation in cell wall modifications, considering that expansins have been proposed as cell wall activator agents without hydrolytic activity. likewise, up-regulated endoglucanases were identified in lb segregants, which are also associated with cell wall dynamics. concomitantly, in the b stage genes related to auxin metabolism were also identified, up-regulated in the lb phenotype, in line with the putative role of auxins in cell expansion, involved in acid growth mediated by expansins. evidence obtained from the transcriptome analysis suggested that major differences among lb and sb seedless segregants are triggered at the b stage, which may be responsible for the final berry weight observed at harvest. in this stage berry diameter increases by cell expansion. other functional categories were associated with secondary metabolism, transport of inorganic ions and metals, proteosome-protein degradation, hormone metabolism and signaling, development and chlorophyll biosynthesis. regarding the group of genes significantly correlated with component, two genes were identified coding for aspartic proteinase nepenthesin- possibly associated with aspartic-type endopeptidase activity, and senescence process; as well as a serine carboxypeptidase-like and endoglucanase both related to cell wall metabolism. furthermore, three genes were found associated with hormonal metabolism and signaling, coding for auxin-induced protein aux and arg and pirin-like protein, related to calcium signaling. co-expression network analysis network analyses were performed to identify co-expression genes associated with the separation between lb and sb segregants. subsequently, correlation analyses results lead to identify a total of partial correlations, of them significant. correlograms were plotted with the total observed correlations, and correlations of over 90 % were considered as significant. furthermore, 15 % of the significant correlations were negative and more variable. positive significant correlations represented 85 % and were less variable. five interconnected clusters of nodes were identified.fig. nodes of co-expressed genes among lb and sb segregants identified using a network analysis. main components of each node are n1: hsps, chaperonins; n2: stbs, thaumatins; n3: monooxygenases; n4: cell wall modifications; n5: vacuolar transporters. lines in red and green represent negative and positive correlations, respectively these results were concordant with those obtained from hierarchical clustering and pca; the seven de genes selected as candidate markers for berry weight from pca analysis were also present in the network analysis. in addition, according to the cluster connectivity our results agreed with previous studies which described that highly connected genes were usually involved in the same biological pathways. cluster one was conformed mostly of genes coding for hsps and chaperonin proteins, including also a gene coding for gdsl esterase/lipase and expansin-a, all of them up-regulated in lb segregants. this result is concordant with identification of the category ‘protein folding’ over-represented in cluster, a process mediated by hsp. as these genes have been associated with heat stress during berry development and the response to microclimate changes in bunches, this evidence suggests that lb segregants could respond more efficiently to heat stress. negative correlations were among genes coding for major allergen pru av associated with defense responses, and expansin-a8; genes coding for chaperonins or hsps were also found. cluster two was composed mainly of genes coding for pals and sts, a gene co-expression previously reported in cv. syrah; they were up-regulated in sb segregants in this study, in both phenological stages. these results are concordant with the identification of the over-represented categories ‘l-phenylalanine catabolic process’ and ‘response to biotic stimulus’ found in cluster. sts expression has been considered as a response to stress factors such as fungal diseases, wounding and uv light, and a shift in phenylpropanoid pathway metabolites is highly sensitive to temperature changes. the differential expression of those genes during berry development and maturation have been described in cv. corvina, norton and moscatel de hamburgo. hence these results suggest that sb segregants presented a higher stress level during berry development than lb segregants, possibly environmental due to high temperatures. however, positive correlation was observed between genes coding for expansin-a and f-box/lrr-repeat protein, both negatively correlated with genes coding for stilbene synthases in the cluster. f-box proteins act as regulators of the ubiquitin kinase dependent pathway associated with protein degradation, an important post-translational mechanism. thus the removal of unfolded or non-functional proteins facilitates the adaptation of organisms to environmental changes, through rapid intracellular signaling. in particular, expansin-a also showed negative correlation with genes coding for thaumatins, proteosome subunits, inorganic transporters and proteins related to pathogenesis, identified up-regulated in sb segregants. cluster three was composed mostly of genes with monooxygenase and oxide-reductase activities, including cytochrome p pr protease inhibitor and eugenol synthase. genes belonging to the cytochrome p family were found up-regulated in sb segregants, associated with phenylpropanoids, flavonoids, brassinosteroids and lignin synthesis. interestingly, it has been reported that cytochrome p450-78a partially controls fruit size in tomato and possibly has a role in the domestication of this species. biosynthetic enzymes, redox regulators and hsp have been described as effector genes related to abiotic stress responses. however, genes coding for chloroplast beta-amylase gibberellin receptor gid and protein wax up-regulated in sb segregants, were negatively correlated. wax protein plays a role in the conversion or secretion of common precursors for cutins and wax metabolic pathways; it is also related to cuticle formation and stomata, both involved in transpiration control and drought tolerance as well. cluster included a cohort of candidate enzymes related to cell wall modification, with xyloglucan endotransglucosylase/hydrolase protein and glucan endo-13-beta-glucosidase activities, positively correlated. interestingly, cluster presented no edges with the remaining clusters. two branches were observed, the first composed of genes coding for 60s ribosomal protein l and abscisic acid 8'-hydroxylase all of them positively correlated. the ribosomal protein modulation suggests that the transcriptome reprogramming that occurs during berry maturation involves changes in protein synthesis. a second branch included genes coding for cysteine-rich receptor-like protein kinase 10; vacuolar amino acid transporter up-regulated in lb segregants, possibly associated with amino acid compartmentalization in the vacuole; cytokinin dehydrogenase as well as galactinol-sucrose galactosyltransferase; glutathione s-transferase, associated with the cellular response induced by heat shock stress and auxins, and metals such as cadmium, silver and copper; and isoflavone-7-o-methyltransferase related with flavonoid/isoflavonoid metabolism and biotic stress responses, which were positively regulated. expression analysis of a group of candidate genes associated with berry weight using qpcr the expression profiles of seven de genes were experimentally validated by real-time qpcr experiments, in the phenological stages of anthesis, fruit-setting and berry 6–8 mm, in order to select candidate genes as putative factors associated with berry weight determination.fig. validation of differentially expressed genes among lb and sb segregants by real-time pcr. lb, large berries, in black; sb, small berries, in grey. phenological stages were anthesis, fruit setting and berry 6–8 mm. genes are a gdsl esterase/lipase; b cytokinin dehydrogenase 3; c stilbene synthase 6; d gene coding for  kda class ii hsp; e tf-bhlh60; f tf-bhlh93; g tf-bhlh96; different letters on top of bars indicate significant differences according to one-way anova and tukey’s multiple comparison test among phenotypic category/phenological stage; values are the results of observations categorized by phenotype. the tcpb gene was used as reference gene and gene expression was expressed as relative expression the results of the network and pca were considered in the selection of candidate genes. genes coding for gdsl esterase/lipase, cytokinin dehydrogenase and stilbene synthase were selected from the network analysis. in addition, the gene coding for hsp  kda class ii was significantly correlated with pca component in the case of the gene coding for gdsl esterase/lipase, experimental results confirmed its up-regulated expression in lb segregants in the b stage, suggesting an increase in its expression in this stage in both lb and sb segregants. in addition, the gene coding for cytokinin dehydrogenase was significantly up-regulated in lb segregants in the fst stage, with lower expression in both groups of segregants at the b stage. interestingly, the gene coding for stilbene synthase showed a tendency to be up-regulated in sb segregants in the three evaluated stages. significant differences were confirmed during fl and b, being higher in the latter stage. in addition, the gene coding for hsp  kda class ii showed similar expression in fl in lb and sb segregants. however, in the fst and b stages it was significantly up-regulated in lb segregants. considering that hsp -d was also highly correlated with component of the pca, it could be considered as a potential candidate gene for berry weight. participation of bhlh proteins in plant organ size determination has been described. in particular in v. vinifera, the cell elongation protein bhlh has been recently associated with berry weight in cabernet sauvignon, possibly involved in cell expansion during berry development. therefore, in order to evaluate the possible role of members of this transcription factor family in the differences between lb and sb segregants, three de genes coding for bhlh bhlh and bhlh were selected to be experimentally confirmed by real-time qpcr experiments. the results showed that in the case of genes coding for transcription factors, tf-bhlh was significantly up-regulated in sb segregants during the fl stage. however, in the fst stage it was up-regulated in lb segregants. there was an inflexion in the fst stage, with maximum expression for lb segregants and minimum for sb segregants. the same tendency was detected for tf-bhlh but with a significant differential expression during the fst and b stages, up-regulated in lb segregants. a similar expression profile was observed in the gene coding for tf-bhlh which was up-regulated in lb segregants at the fst stage. interestingly, our results differ from previous reports that proposed the gene vvceb as a candidate marker for berry size, whose transcripts are predominantly accumulated in berries, especially with minimum auxin content. indeed, the three tfs evaluated showed higher expression in the fst stage comparing lb vs. sb segregants, suggesting a possible role in early stages of development. experimental validation in advanced phenological stages would confirm their expression profile in berries in order to determine if, as with vvceb these tfs plays a role in cell expansion in a wider genetic background.  we have carried out the first transcriptome analysis focused on seedless table grape segregants with contrasting phenotypes for berry weight. a group of differentially expressed genes potentially associated with berry size was identified, genes in the fst stage and genes in the b stage. the integration of differential expression, pca, correlation and network analysis provided a wide characterization of overall regulation and dynamic remodeling of the gene expression in berry development in pre-véraison stages. a survey of candidate genes was also performed, and expression profiles of seven candidate genes were validated. 
however, this is a quantitative and polygenic trait, and its genetic determination is still poorly understood. a group of differentially expressed genes were identified, by comparing seedless segregants with contrasting phenotypes for berry weight: genes from the fst stage and from the b stage. also, we integrated differential expression, principal components analysis, correlations and network co-expression analyses to characterize the transcriptome profiling observed in segregants with contrasting phenotypes for berry weight. to approach this issue, an rna-seq experiment based on illumina platform was performed, including seedless segregants with contrasting phenotypes for berry weight at fruit setting and 6–8 mm berries phenological stages. we have carried out the first transcriptome analysis focused on table grape seedless segregants with contrasting phenotypes for berry weight. our findings contributed to the understanding of the mechanisms involved in berry weight determination. also, this comparative transcriptome profiling revealed candidate genes for berry weight which could be evaluated as selection tools in table grape breeding programs. rna-seqtable grapesberry weightfunctional genomicscandidate genesconicytg07i1002hinrichsen patricio fondecyt11208883150519muñoz-espinoza claudia hinrichsen patricio fondap15090007orellana ariel basal programpb-16orellana ariel issue-copyright-statement© the author 2016 berry size is considered as one of the main selection criteria in table grape breeding programs. considering its economic importance, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in its expression. after this, de genes were selected as candidate genes, and seven candidate genes were validated by real time-pcr, confirming their expression profiles.
 berry size is considered as one of the main selection criteria in table grape breeding programs. however, this is a quantitative and polygenic trait, and its genetic determination is still poorly understood. considering its economic importance, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in its expression. to approach this issue, an rna-seq experiment based on illumina platform was performed, including seedless segregants with contrasting phenotypes for berry weight at fruit setting and 6–8 mm berries phenological stages.  a group of differentially expressed genes were identified, by comparing seedless segregants with contrasting phenotypes for berry weight: genes from the fst stage and from the b stage. also, we integrated differential expression, principal components analysis, correlations and network co-expression analyses to characterize the transcriptome profiling observed in segregants with contrasting phenotypes for berry weight. after this, de genes were selected as candidate genes, and seven candidate genes were validated by real time-pcr, confirming their expression profiles.  we have carried out the first transcriptome analysis focused on table grape seedless segregants with contrasting phenotypes for berry weight. our findings contributed to the understanding of the mechanisms involved in berry weight determination. also, this comparative transcriptome profiling revealed candidate genes for berry weight which could be evaluated as selection tools in table grape breeding programs. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rna-seqtable grapesberry weightfunctional genomicscandidate genesconicytg07i1002hinrichsen patricio fondecyt11208883150519muñoz-espinoza claudia hinrichsen patricio fondap15090007orellana ariel basal programpb-16orellana ariel issue-copyright-statement© the author 2016 