Transcriptional, hormonal, and metabolic changes in susceptible grape berries under powdery mildew infection.

Grapevine (Vitis vinifera) berries are extremely sensitive to infection by the biotrophic pathogen Erysiphe necator, causing powdery mildew disease with deleterious effects on grape and wine quality. The combined analysis of the transcriptome and metabolome associated with this common fungal infection has not been previously carried out in any fruit. In order to identify the molecular, hormonal, and metabolic mechanisms associated with infection, healthy and naturally infected V. vinifera cv. Carignan berries were collected at two developmental stages: late green (EL33) and early véraison (EL35). RNA sequencing combined with GC-electron impact ionization time-of-flight MS, GC-electron impact ionization/quadrupole MS, and LC-tandem MS analyses revealed that powdery mildew-susceptible grape berries were able to activate defensive mechanisms with the involvement of salicylic acid and jasmonates and to accumulate defense-associated metabolites (e.g. phenylpropanoids, fatty acids). The defensive strategies also indicated organ-specific responses, namely the activation of fatty acid biosynthesis. However, defense responses were not enough to restrict fungal growth. The fungal metabolic program during infection involves secretion of effectors related to effector-triggered susceptibility, carbohydrate-active enzymes and activation of sugar, fatty acid, and nitrogen uptake, and could be under epigenetic regulation. This study also identified potential metabolic biomarkers such as gallic, eicosanoic, and docosanoic acids and resveratrol, which can be used to monitor early stages of infection.

Expression of grapevine AINTEGUMENTA-like genes is associated with variation in ovary and berry size.

Fruit size is a highly important trait for most fruit and vegetable crops. This trait has been a main selection target and could be involved in divergent selection processes leading to the differentiation between modern table and wine cultivars. Even though its determination is highly influenced by cultural practices, several regions within the grapevine genome have been identified affecting berry size, either directly or indirectly through their effect on seed content. Using grapevine seeded cultivars, we have analyzed the relationship between ovary cell number and the final size of ovaries and berry fruits. We also performed the characterization of the grapevine AINTEGUMENTA-LIKE family, since it is well reported in Arabidopsis that AINTEGUMENTA (ANT) regulates cell proliferation and organ growth in flower organ primordia by maintaining the meristematic competence of cells during organogenesis. Here we show that orthologous grapevine gene expression associate with flower developmental stages suggesting a similar biological role for this gene family in this species. Moreover, we detected a correlation between those organs size and the level of expression of VviANT1 the grapevine homolog of AtANT. This grapevine gene also co-localizes in linkage group 18 with the confidence interval of a previously detected QTL for berry size. Thus our results suggest the involvement of ANT in the regulation of berry size in grapevine.

Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco.

Seedlessness is a relevant trait in grapevine cultivars intended for fresh consumption or raisin production. Previous DNA marker analysis indicated that Corinto bianco (CB) is a parthenocarpic somatic variant of the seeded cultivar Pedro Ximenes (PX). This study compared both variant lines to determine the basis of this parthenocarpic phenotype. At maturity, CB seedless berries were 6-fold smaller than PX berries. The macrogametophyte was absent from CB ovules, and CB was also pollen sterile. Occasionally, one seed developed in 1.6% of CB berries. Microsatellite genotyping and flow cytometry analyses of seedlings generated from these seeds showed that most CB viable seeds were formed by fertilization of unreduced gametes generated by meiotic diplospory, a process that has not been described previously in grapevine. Microarray and RNA-sequencing analyses identified 1958 genes that were differentially expressed between CB and PX developing flowers. Genes downregulated in CB were enriched in gametophyte-preferentially expressed transcripts, indicating the absence of regular post-meiotic germline development in CB. RNA-sequencing was also used for genetic variant calling and 14 single-nucleotide polymorphisms distinguishing the CB and PX variant lines were detected. Among these, CB-specific polymorphisms were considered as candidate parthenocarpy-responsible mutations, including a putative deleterious substitution in a HAL2-like protein. Collectively, these results revealed that the absence of a mature macrogametophyte, probably due to meiosis arrest, coupled with a process of fertilization-independent fruit growth, caused parthenocarpy in CB. This study provides a number of grapevine parthenocarpy-responsible candidate genes and shows how genomic approaches can shed light on the genetic origin of woody crop somatic variants.

The flowering repressor SVP underlies a novel Arabidopsis thaliana QTL interacting with the genetic background.

The timing of flowering initiation is a fundamental trait for the adaptation of annual plants to different environments. Large amounts of intraspecific quantitative variation have been described for it among natural accessions of many species, but the molecular and evolutionary mechanisms underlying this genetic variation are mainly being determined in the model plant Arabidopsis thaliana. To find novel A. thaliana flowering QTL, we developed introgression lines from the Japanese accession Fuk, which was selected based on the substantial transgression observed in an F(2) population with the reference strain Ler. Analysis of an early flowering line carrying a single Fuk introgression identified Flowering Arabidopsis QTL1 (FAQ1). We fine-mapped FAQ1 in an 11 kb genomic region containing the MADS transcription factor gene SHORT VEGETATIVE PHASE (SVP). Complementation of the early flowering phenotype of FAQ1-Fuk with a SVP-Ler transgen demonstrated that FAQ1 is SVP. We further proved by directed mutagenesis and transgenesis that a single amino acid substitution in SVP causes the loss-of-function and early flowering of Fuk allele. Analysis of a worldwide collection of accessions detected FAQ1/SVP-Fuk allele only in Asia, with the highest frequency appearing in Japan, where we could also detect a potential ancestral genotype of FAQ1/SVP-Fuk. In addition, we evaluated allelic and epistatic interactions of SVP natural alleles by analysing more than one hundred transgenic lines carrying Ler or Fuk SVP alleles in five genetic backgrounds. Quantitative analyses of these lines showed that FAQ1/SVP effects vary from large to small depending on the genetic background. These results support that the flowering repressor SVP has been recently selected in A. thaliana as a target for early flowering, and evidence the relevance of genetic interactions for the intraspecific evolution of FAQ1/SVP and flowering time.

Transcriptome and metabolome reprogramming in Vitis vinifera cv. Trincadeira berries upon infection with Botrytis cinerea.

Vitis vinifera berries are sensitive towards infection by the necrotrophic pathogen Botrytis cinerea, leading to important economic losses worldwide. The combined analysis of the transcriptome and metabolome associated with fungal infection has not been performed previously in grapes or in another fleshy fruit. In an attempt to identify the molecular and metabolic mechanisms associated with the infection, peppercorn-sized fruits were infected in the field. Green and veraison berries were collected following infection for microarray analysis complemented with metabolic profiling of primary and other soluble metabolites and of volatile emissions. The results provided evidence of a reprogramming of carbohydrate and lipid metabolisms towards increased synthesis of secondary metabolites involved in plant defence, such as trans-resveratrol and gallic acid. This response was already activated in infected green berries with the putative involvement of jasmonic acid, ethylene, polyamines, and auxins, whereas salicylic acid did not seem to be involved. Genes encoding WRKY transcription factors, pathogenesis-related proteins, glutathione S-transferase, stilbene synthase, and phenylalanine ammonia-lyase were upregulated in infected berries. However, salicylic acid signalling was activated in healthy ripening berries along with the expression of proteins of the NBS-LRR superfamily and protein kinases, suggesting that the pathogen is able to shut down defences existing in healthy ripening berries. Furthermore, this study provided metabolic biomarkers of infection such as azelaic acid, a substance known to prime plant defence responses, arabitol, ribitol, 4-amino butanoic acid, 1-O-methyl- glucopyranoside, and several fatty acids that alone or in combination can be used to monitor Botrytis infection early in the vineyard.

Solar ultraviolet radiation is necessary to enhance grapevine fruit ripening transcriptional and phenolic responses.

BACKGROUND: Ultraviolet (UV) radiation modulates secondary metabolism in the skin of Vitis vinifera L. berries, which affects the final composition of both grapes and wines. The expression of several phenylpropanoid biosynthesis-related genes is regulated by UV radiation in grape berries. However, the complete portion of transcriptome and ripening processes influenced by solar UV radiation in grapes remains unknown. RESULTS: Whole genome arrays were used to identify the berry skin transcriptome modulated by the UV radiation received naturally in a mid-altitude Tempranillo vineyard. UV radiation-blocking and transmitting filters were used to generate the experimental conditions. The expression of 121 genes was significantly altered by solar UV radiation. Functional enrichment analysis of altered transcripts mainly pointed out that secondary metabolism-related transcripts were induced by UV radiation including VvFLS1, VvGT5 and VvGT6 flavonol biosynthetic genes and monoterpenoid biosynthetic genes. Berry skin phenolic composition was also analysed to search for correlation with gene expression changes and UV-increased flavonols accumulation was the most evident impact. Among regulatory genes, novel UV radiation-responsive transcription factors including VvMYB24 and three bHLH, together with known grapevine UV-responsive genes such as VvMYBF1, were identified. A transcriptomic meta-analysis revealed that genes up-regulated by UV radiation in the berry skin were also enriched in homologs of Arabidopsis UVR8 UV-B photoreceptor-dependent UV-B -responsive genes. Indeed, a search of the grapevine reference genomic sequence identified UV-B signalling pathway homologs and among them, VvHY5-1, VvHY5-2 and VvRUP were up-regulated by UV radiation in the berry skin. CONCLUSIONS: Results suggest that the UV-B radiation-specific signalling pathway is activated in the skin of grapes grown at mid-altitudes. The biosynthesis and accumulation of secondary metabolites, which are appreciated in winemaking and potentially confer cross-tolerance, were almost specifically triggered. This draws attention to viticultural practices that increase solar UV radiation on vineyards as they may improve grape features.

The genetic structure of Arabidopsis thaliana in the south-western Mediterranean range reveals a shared history between North Africa and southern Europe.

BACKGROUND: Deciphering the genetic structure of Arabidopsis thaliana diversity across its geographic range provides the bases for elucidating the demographic history of this model plant. Despite the unique A. thaliana genomic resources currently available, its history in North Africa, the extreme southern limit in the biodiversity hotspot of the Mediterranean Basin, remains virtually unknown. RESULTS: To approach A. thaliana evolutionary history in North Africa, we have analysed the genetic diversity and structure of 151 individuals collected from 20 populations distributed across Morocco. Genotyping of 249 genome-wide SNPs indicated that Morocco contains substantially lower diversity than most analyzed world regions. However, IBD, STRUCTURE and PCA clustering analyses showed that genetic variation is strongly geographically structured. We also determined the genetic relationships between Morocco and the closest European region, the Iberian Peninsula, by analyses of 201 populations from both regions genotyped with the same SNPs. These analyses detected four genetic groups, but all Moroccan accessions belonged to a common Iberian/Moroccan cluster that appeared highly differentiated from the remaining groups. Thus, we identified a genetic lineage with an isolated demographic history in the south-western Mediterranean region. The existence of this lineage was further supported by the study of several flowering genes and traits, which also found Moroccan accessions similar to the same Iberian group. Nevertheless, genetic diversity for neutral SNPs and flowering genes was higher in Moroccan than in Iberian populations of this lineage. Furthermore, we analyzed the genetic relationships between Morocco and other world regions by joint analyses of a worldwide collection of 337 accessions, which detected an additional weak relationship between North Africa and Asia. CONCLUSIONS: The patterns of genetic diversity and structure of A. thaliana in Morocco show that North Africa is part of the species native range and support the occurrence of a glacial refugium in the Atlas Mountains. In addition, the identification of a genetic lineage specific of Morocco and the Iberian Peninsula indicates that the Strait of Gibraltar has been an A. thaliana migration route between Europe and Africa. Finally, the genetic relationship between Morocco and Asia suggests another migration route connecting north-western Africa and Asia.

Thermotolerance responses in ripening berries of Vitis vinifera L. cv Muscat Hamburg.

Berry organoleptic properties are highly influenced by ripening environmental conditions. In this study, we used grapevine fruiting cuttings to follow berry ripening under different controlled conditions of temperature and irradiation intensity. Berries ripened at higher temperatures showed reduced anthocyanin accumulation and hastened ripening, leading to a characteristic drop in malic acid and total acidity. The GrapeGen GeneChip® combined with a newly developed GrapeGen 12Xv1 MapMan version were utilized for the functional analysis of berry transcriptomic differences after 2 week treatments from veraison onset. These analyses revealed the establishment of a thermotolerance response in berries under high temperatures marked by the induction of heat shock protein (HSP) chaperones and the repression of transmembrane transporter-encoding transcripts. The thermotolerance response was coincident with up-regulation of ERF subfamily transcription factors and increased ABA levels, suggesting their participation in the maintenance of the acclimation response. Lower expression of amino acid transporter-encoding transcripts at high temperature correlated with balanced amino acid content, suggesting a transcriptional compensation of temperature effects on protein and membrane stability to allow for completion of berry ripening. In contrast, the lower accumulation of anthocyanins and higher malate metabolization measured under high temperature might partly result from imbalance in the expression and function of their specific transmembrane transporters and expression changes in genes involved in their metabolic pathways. These results open up new views to improve our understanding of berry ripening under high temperatures.

Genetic diversity and population structure assessed by SSR and SNP markers in a large germplasm collection of grape.

BACKGROUND: The economic importance of grapevine has driven significant efforts in genomics to accelerate the exploitation of Vitis resources for development of new cultivars. However, although a large number of clonally propagated accessions are maintained in grape germplasm collections worldwide, their use for crop improvement is limited by the scarcity of information on genetic diversity, population structure and proper phenotypic assessment. The identification of representative and manageable subset of accessions would facilitate access to the diversity available in large collections. A genome-wide germplasm characterization using molecular markers can offer reliable tools for adjusting the quality and representativeness of such core samples. RESULTS: We investigated patterns of molecular diversity at 22 common microsatellite loci and 384 single nucleotide polymorphisms (SNPs) in 2273 accessions of domesticated grapevine V. vinifera ssp. sativa, its wild relative V. vinifera ssp. sylvestris, interspecific hybrid cultivars and rootstocks. Despite the large number of putative duplicates and extensive clonal relationships among the accessions, we observed high level of genetic variation. In the total germplasm collection the average genetic diversity, as quantified by the expected heterozygosity, was higher for SSR loci (0.81) than for SNPs (0.34). The analysis of the genetic structure in the grape germplasm collection revealed several levels of stratification. The primary division was between accessions of V. vinifera and non-vinifera, followed by the distinction between wild and domesticated grapevine. Intra-specific subgroups were detected within cultivated grapevine representing different eco-geographic groups. The comparison of a phenological core collection and genetic core collections showed that the latter retained more genetic diversity, while maintaining a similar phenotypic variability. CONCLUSIONS: The comprehensive molecular characterization of our grape germplasm collection contributes to the knowledge about levels and distribution of genetic diversity in the existing resources of Vitis and provides insights into genetic subdivision within the European germplasm. Genotypic and phenotypic information compared in this study may efficiently guide further exploration of this diversity for facilitating its practical use.

Regulation of flowering time by FVE, a retinoblastoma-associated protein.

The initiation of flowering in plants is controlled by environmental and endogenous signals. Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC, a gene encoding a MADS-box protein that inhibits flowering. We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastoma-associated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering.

Transcriptome variation along bud development in grapevine (Vitis vinifera L.).

BACKGROUND: Vegetative buds provide plants in temperate environments the possibility for growth and reproduction when environmental conditions are favorable. In grapevine, crucial developmental events take place within buds during two growing seasons in consecutive years. The first season, the shoot apical meristem within the bud differentiates all the basic elements of the shoot including flowering transition in lateral primordia and development of inflorescence primordia. These events practically end with bud dormancy. The second season, buds resume shoot growth associated to flower formation and development. Gene expression has been previously monitored at specific stages of bud development but has never been followed along the two growing seasons. RESULTS: Gene expression changes were analyzed along the bud annual cycle at eight different time points. Principal Components Analysis (PCA) revealed that the main factors explaining the global gene expression differences were the processes of bud dormancy and active growth as well as stress responses. Accordingly, non dormant buds showed an enrichment in functional categories typical of actively proliferating and growing cells together with the over abundance of transcripts belonging to stress response pathways. Differential expression analyses performed between consecutive time points indicated that major transcriptional changes were associated to para/endodormancy, endo/ecodormancy and ecodormancy/bud break transitions. Transcripts encoding key regulators of reproductive development were grouped in three major expression clusters corresponding to: (i) transcripts associated to flowering induction, (ii) transcripts associated to flower meristem specification and initiation and (iii) transcripts putatively involved in dormancy. Within this cluster, a MADS-box gene (VvFLC2) and other transcripts with similar expression patterns could participate in dormancy regulation. CONCLUSIONS: This work provides a global view of major transcriptional changes taking place along bud development in grapevine, highlighting those molecular and biological functions involved in the main events of bud development. As reported in other woody species, the results suggest that genes regulating flowering could also be involved in dormancy regulatory pathways in grapevine.

Altitudinal and climatic adaptation is mediated by flowering traits and FRI, FLC, and PHYC genes in Arabidopsis.

Extensive natural variation has been described for the timing of flowering initiation in many annual plants, including the model wild species Arabidopsis (Arabidopsis thaliana), which is presumed to be involved in adaptation to different climates. However, the environmental factors that might shape this genetic variation, as well as the molecular bases of climatic adaptation by modifications of flowering time, remain mostly unknown. To approach both goals, we characterized the flowering behavior in relation to vernalization of 182 Arabidopsis wild genotypes collected in a native region spanning a broad climatic range. Phenotype-environment association analyses identified strong altitudinal clines (0-2600 m) in seven out of nine flowering-related traits. Altitudinal clines were dissected in terms of minimum winter temperature and precipitation, indicating that these are the main climatic factors that might act as selective pressures on flowering traits. In addition, we used an association analysis approach with four candidate genes, FRIGIDA (FRI), FLOWERING LOCUS C (FLC), PHYTOCHROME C (PHYC), and CRYPTOCHROME2, to decipher the genetic bases of this variation. Eleven different loss-of-function FRI alleles of low frequency accounted for up to 16% of the variation for most traits. Furthermore, an FLC allelic series of six novel putative loss- and change-of-function alleles, with low to moderate frequency, revealed that a broader FLC functional diversification might contribute to flowering variation. Finally, environment-genotype association analyses showed that the spatial patterns of FRI, FLC, and PHYC polymorphisms are significantly associated with winter temperatures and spring and winter precipitations, respectively. These results support that allelic variation in these genes is involved in climatic adaptation.

Novel natural alleles at FLC and LVR loci account for enhanced vernalization responses in Arabidopsis thaliana.

Vernalization, the induction of flowering by low winter temperatures, is likely to be involved in plant climatic adaptation. However, the genetic, molecular and ecological bases underlying the quantitative variation that tunes vernalization sensitivity to natural environments are largely unknown. To address these questions, we have studied the enhanced vernalization response shown by the Ll-0 accession of Arabidopsis thaliana. Quantitative trait locus (QTL) mapping for several flowering initiation traits in relation to vernalization, in a new Ler × Ll-0 recombinant inbred line (RIL) population, identified large effect alleles at FRI, FLC and HUA2, together with two small effect loci named as Llagostera vernalization response (LVR) 1 and 2. Phenotypic analyses of near isogenic lines validated LVR1 effect on flowering vernalization responses. To further characterize the FLC allele from Ll-0, we carried out genetic association analyses using a regional collection of wild genotypes. FLC-Ll-0 appeared as a low-frequency allele that is distinguished by polymorphism Del(-57), a 50-bp-deletion in the 5'-UTR. Del(-57) was significantly associated with enhanced vernalization responses and FLC RNA expression, as well as with altitude and minimum temperatures. These results are consistent with Del(-57) acting as a novel cis-regulatory FLC polymorphism that may confer climatic adaptation by increasing vernalization sensitivity.

Transposon-induced gene activation as a mechanism generating cluster shape somatic variation in grapevine.

We have characterized the genetic and molecular origin of the reiterated reproductive meristem (RRM) somatic variant phenotype of grapevine cultivar Carignan. Here, we show that the extreme cluster proliferation and delayed anthesis observed in this somatic variant is caused by a single dominant mutation. Transcriptional profiling of Carignan and RRM plants during early stages of inflorescence development demonstrated the overexpression of a few regulatory genes, including VvTFL1A, a close TFL1 Arabidopsis homolog, in RRM inflorescences. Genetic and molecular analyses correlated the insertion of a class-II transposable element, Hatvine1-rrm, in the VvTFL1A promoter, with upregulation of the corresponding VvTFL1A allele in reproductive and vegetative organs of the shoot apex. These results suggest a role for this TFL1 grapevine homolog in the determination of inflorescence structure, with a critical effect on the size and branching pattern of grapevine fruit clusters. Our results demonstrate the existence of spontaneous cis-activation processes caused by class-II transposable elements in grapevine plants, and point to their possible role as a mechanism to generate somatic cell variation in perennial plants. This mechanism is expected to generate dominant phenotypes in chimeric sectors that can be readily exposed to natural selection.

EARLY IN SHORT DAYS 7 (ESD7) encodes the catalytic subunit of DNA polymerase epsilon and is required for flowering repression through a mechanism involving epigenetic gene silencing.

We have characterized a mutation affecting the Arabidopsis EARLY IN SHORT DAYS 7 (ESD7) gene encoding the catalytic subunit of DNA polymerase epsilon (epsilon), AtPOL2a. The esd7-1 mutation causes early flowering independently of photoperiod, shortened inflorescence internodes and altered leaf and root development. esd7-1 is a hypomorphic allele whereas knockout alleles displayed an embryo-lethal phenotype. The esd7 early flowering phenotype requires functional FT and SOC1 proteins and might also be related to the misregulation of AG and AG-like gene expression found in esd7. Genes involved in the modulation of chromatin structural dynamics, such as LHP1/TFL2 and EBS, which negatively regulate FT expression, were found to interact genetically with ESD7. In fact a molecular interaction between the carboxy terminus of ESD7 and TFL2 was demonstrated in vitro. Besides, fas2 mutations suppressed the esd7 early flowering phenotype and ICU2 was found to interact with ESD7. Discrete regions of the chromatin of FT and AG loci were enriched in activating epigenetic marks in the esd7-1 mutant. We concluded that ESD7 might be participating in processes involved in chromatin-mediated cellular memory.

Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening.

BACKGROUND: Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses. RESULTS: Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening. CONCLUSIONS: Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.

A 48 SNP set for grapevine cultivar identification.

BACKGROUND: Rapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification. RESULTS: We analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification. CONCLUSIONS: We have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable.

Transcriptome changes in grapevine (Vitis vinifera L.) cv. Malbec leaves induced by ultraviolet-B radiation.

BACKGROUND: Ultraviolet-B radiation (UV-B, 280-315 nm) is a natural component of sunlight, which has numerous regulatory effects on plant physiology. The nature of the response to UV-B is dependent on fluence rate, dose, duration and wavelength of the UV-B treatment. Some reports have analyzed the changes in gene expression caused by UV-B light on several plant species using microarray technology. However, there is no information on the transcriptome response triggered by UV-B in grapevine. In this paper we investigate the gene expression responses of leaves from in vitro cultured Vitis vinifera cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m-2 d-1) administered at two different fluence rates (16 h at ≅ 8.25 µW cm-2, 4 h at ≅ 33 µW cm-2) using a new custom made GrapeGen Affymetrix GeneChip®. RESULTS: The number of genes modulated by high fluence rate UV-B doubled the number of genes modulated by low fluence UV-B. Their functional analyses revealed several functional categories commonly regulated by both UV-B treatments as well as categories more specifically modulated depending on UV-B fluence rate. General protective responses, namely the induction of pathways regulating synthesis of UV-B absorbing compounds such as the Phenylpropanoid pathway, the induction of different antioxidant defense systems and the activation of pathways commonly associated with pathogen defense and abiotic stress responses seem to play critical roles in grapevine responses against UV-B radiation. Furthermore, high fluence rate UV-B seemed to specifically modulate additional pathways and processes in order to protect grapevine plantlets against UV-B-induced oxidative stress, stop the cell cycle progression, and control protein degradation. On the other hand, low fluence rate UV-B regulated the expression of specific responses in the metabolism of auxin and abscisic acid as well as in the modification of cell walls that could be involved in UV-B acclimation-like processes. CONCLUSION: Our results show the UV-B radiation effects on the leaf transcriptome of grapevine (Vitis vinifera cv. Malbec) plantlets. Functional categories commonly modulated under both UV-B treatments as well as transcripts specifically regulated in an UV-B-intensity dependent way were identified. While high fluence rate UV-B had regulatory effects mainly on defense or general multiple-stress responses pathways, low fluence rate UV-B promoted the expression of genes that could be involved in UV-B protection or the amelioration of the UV-B-induced damage. This study also provides an extensive list of genes regulating multiple metabolic pathways involved in the response of grapevine to UV-B that can be used for future researches.

Temporal analysis of natural variation for the rate of leaf production and its relationship with flowering initiation in Arabidopsis thaliana.

Vegetative growth and flowering initiation are two crucial developmental processes in the life cycle of annual plants that are closely associated. The timing of both processes affects several presumed adaptive traits, such as flowering time (FT), total leaf number (TLN), or the rate of leaf production (RLP). However, the interactions among these complex processes and traits, and their mechanistic bases, remain largely unknown. To determine the genetic relationships between them, the natural genetic variation between A. thaliana accessions Fei-0 and Ler has been studied using a new population of 222 LerxFei-0 recombinant inbred lines. Temporal analysis of the parental development under a short day photoperiod distinguishes two vegetative phases differing in their RLP. QTL mapping of RLP in consecutive time intervals of vegetative development indicates that Ler/Fei-0 variation is caused by 10 loci whose small to moderate effects mainly display two different temporal patterns. Further comparative QTL analyses show that most of the genomic regions affecting FT or TLN also alter RLP. In addition, the partially independent genetic bases observed for FT and TLN appear determined by several genomic regions with two different patterns of phenotypic effects: regions with a larger effect on FT than TLN, and vice versa. The distinct temporal and pleiotropic patterns of QTL effects suggest that natural variation for flowering time is caused by different genetic mechanisms involved in vegetative and/or reproductive phase changes, most of them interacting with the control of leaf production rate. Thus, natural selection might contribute to maintain this genetic variation due to its phenotypic effects not only on the timing of flowering initiation but also on the rate of vegetative growth.

Genetic Relationships Among Portuguese Cultivated and Wild Vitis vinifera L. Germplasm.

The domesticated grapevine spread along the Mediterranean basin from the primary Near East domestication area, where the greatest genetic diversity is found in its ancestor, the wild vine populations. Portuguese wild populations are on the southwestern fringe of the distribution of the Vitis vinifera L. ssp. sylvestris (C.C. Gmel.) Hegi in Europe. During the last Glacial Period they became isolated from the previous continuum that had been the territory of wild vine populations. Archaeological remains of domesticated vinifera grapevines in Portugal date back from 795 Before Common Era (BCE) in the lower Tagus river basin. In this work, 258 Portuguese vinifera varieties and sylvestris plants were characterized using 261 single nucleotide polymorphism (SNP) markers. The study of the genetic diversity of this local germplasm, its population structure and kinship, all framed in their historical and geographical backgrounds, revealed a complex network of first-degree relationships, where only Iberian varieties are involved. Some Iberian genotypes, like Alfrocheiro (Bruñal, in Spain), Sarigo (Cayetana Blanca), Mourisco Branco (Hebén), Amaral (Caiño Bravo), and Marufo (Moravia Dulce) are ancestors of a considerable fraction of all the autochthonous analyzed varieties. A part of the diversity developed was mostly local in some cases as shown by the closeness of several varieties (Vinhos Verdes) to the wild cluster in different analyses. Besides, several evidences of introgression of domesticated germplasm into wild vines was found, substantiating the high risk of genetic contamination of the sylvestris subspecies. All these findings together to the known matching between the wild maternal lineage of the Iberian Peninsula and an important number of Portuguese grapevine varieties (chlorotype A), point out that some of these varieties derive, directly or indirectly, from originally local wild populations, supporting the possible occurrence of secondary events of local domestication, or, at least, of an introgression process of wild into cultivated grapevines.