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Grapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern., This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT): PICT2015-0822, MINCyT/ANPCyT (FONTAR)-CDTI ‘IBEROGEN’, CONICET (Bilateral PCB-II, CONICET-CSIC; MINECO BIO2017-86375-R). COST Action CA17111. Authors wish to thank M. Victoria Bertoldi for the assistance with laboratory work at IBAM-CONICET.

Grapevine phenology is being modified by climate change, particularly by the increase of temperatures that affect grape attributes for wine production. Besides the existing oenological and viticultural approaches, the thorough exploration of the current intra-cultivar genetic variability to select late-ripening genotypes emerges as an interesting alternative. In the present work, we have analyzed the natural genetic variation for phenology and agronomic traits among 21 'Malbec' clones and we demonstrated that fruiting cuttings are a useful tool for the analysis of such variation in 'Malbec'. Several clones could be distinguished by agronomic traits like berry number or cluster weight, and mainly by phenology characteristics like the length of the phase between flowering and veraison, which reached more than 16 days between early and late clones. These results support the approach of exploring grapevine clone collections in searching for genotypes with delayed phenology, and thus with the potential to maintain some expected quality characteristics under warm conditions. Fil: van Houten, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Muñoz, Claudio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Bree, Laura. Vivero Mercier; Argentina Fil: Bergamín, Daniel. Vivero Mercier; Argentina Fil: Sola, Cristobal. Vivero Mercier; Argentina Fil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina

In grapevine, proanthocyanins (PAs) and anthocyanins are obtained via the phenylpropanoid pathway. These metabolites are synthesized in specific tissues of the berry and accumulate in different developmental stages. The transcriptional control of the route is affected by transcription factors (TFs), mainly R2R3- Myb type, which can act as activators or repressors. Here we studied the fine regulation of the pathway by analysing the expression profiles of nine TFs operating as activators of the route as well as two main structural genes. In addition, we analysed the profiles of six R2R3-Myb subgroup 4 TFs described as repressors of the route (VviMybC2-L1, VviMybC2-L2, VviMybC2-L3, VviMyb4a, VviMyb4b and VviMyb4-like). The analysis of gene expression was carried out in five different development stages of the berry and in two berry tissues (skin and flesh) of cultivar ?Malbec?. On the other hand, the putative R2R3-Myb repressors were analysed and compared in the tenturier cultivar ?Alicante Bouschet?, in the same tissues and stages of development as ?Malbec?. Concerning the R2R3-Myb repressors, our results indicate that in the ?Malbec? skins, VviMyb4a and VviMyb4b would act on the early genes of the route while VviMybC2-L2 and VviMyb4-like are co-expressed towards the end of the anthocyanin biosynthesis. In addition, the VviMybC2-L1 and VviMybC2-L3 are induced at two stages of development, first towards the end of the green phase of the berry, indicating the possible synergistic interaction by repressing the synthesis of PAs, then they are induced again towards maturation to inhibit the synthesis of anthocyanins. On the other hand, our results showed that the expression profiles of Myb repressor genes may explain the presence of colour in the flesh of Alicante Bouschet variety. Fil: Muñoz, Claudio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Fanzone, Martín Leandro. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria Mendoza. Laboratorio de Aromas y Sustancias Naturales; Argentina Fil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina

The analyses of the grapevine (Vitis vinifera L.) genome have revealed an unusually large and closely related stilbene synthase (VvSTS) gene family. Interestingly, despite the high sequence similarity among those genes, several studies have observed clear differences between their expression patterns. Here, we studied the transcriptional responses to different elicitors of several VvSTSs in cellular suspension cultures. Primarily, we performed the in silico analysis of the VvSTS regulatory sequences and found the presence of several putative cis-regulatory elements. Then, we evaluated the effect of three treatments—naphtalene acetic acid, methyl jasmonate (MeJA), and ethylene—over the gene expression and found that the genes follow expression patterns probably specific to their sequences. According to this, we focused our study on their regulatory regions and adopted a novel and efficient transient expression assay to determine the activity of these promoters. The results demonstrated that variation in gene expression could be assessed through the analysis of VvSTS regulatory sequences under the effect of different stimuli such as MeJA and cyclodextrins. Furthermore, taking advantage of the lower sequence identity at the promoter level, this strategy accomplished a more accurate alternative to differentiate the members of a large multi-gene family such as STS. Fil: Chialva, Constanza Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Muñoz, Claudio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Miccono, María. Instituto de Investigaciones Agropecuarias; Chile Fil: Eichler, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Calderón, Pablo Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Prieto, Humberto. Instituto de Investigaciones Agropecuarias; Chile Fil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina

Grape (Vitis vinifera) color somatic variants that can be used to develop new grapevine cultivars occasionally appear associated with deletion events of uncertain origin. To understand the mutational mechanisms generating somatic structural variation in grapevine, we compared the Tempranillo Blanco (TB) white berry somatic variant with its black berry ancestor, Tempranillo Tinto. Whole-genome sequencing uncovered a catastrophic genome rearrangement in TB that caused the hemizygous deletion of 313 genes, including the loss of the functional copy for the MYB transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species., This work was partially supported by the project GrapeReSeq from the Spanish Ministry of Education and Sciences (EUI2008-03752), by the European Union Seventh Framework Programme (KBBE Contract 311775 Innovine), and by the Spanish Ministry of Economy (MINECO) (BIO2014-59324-R). This work also benefited from the networking activities within the funded European Cooperation in Science and Technology (COST) Action FA1106 QualityFruit.

The publication of the genome sequences of inbred grapevine plant PN40024 and the cultivar Pinot Noir has provided a new generation of molecular tools and has opened the way to functional genomics in grapevine. Establishing gene biological function is now a major challenge requiring the parallel development of molecular and genetic information. New massive pyrosequencing technologies will ensure no shortage of nucleotide sequence information. However, genetic analysis and genetic tools in grapevine still require additional development. Exploiting the existing natural genetic variation in Vitis vinifera L. and other inter-fertile Vitis species should be a priority to focus functional analyses on genes contributing to phenotypic variation because their genetic variation constitutes the basis for genetic improvement of classical cultivars and for the development of new ones. In this review, we discussed the current molecular and genetic tools available in grapevine and considered those that need to be developed to exploit natural genetic variation in the analyses of gene function. We also reviewed the scarce information on the genetic and molecular structure of relevant grapevine traits and proposed future directions, Research activity in the author's laboratories is funded by grants from the Spanish Ministry of Science and innovation BIO2008-03892 and GEN2006-27782-C2-1-E/VEG.

Sunlight exposure has multiple effect on fruits, as it affects the light climate perceived by fruit photoreceptors and fruit tissue temperature. In grapes (Vitis vinifera L.), light exposure can have a strong effect on fruit quality and commercial value; however, the mechanisms of light action are not well understood. The role of fruit-localized photoreceptors in the control of berry quality traits was evaluated under field conditions in a commercial vineyard in Mendoza (Argentina). Characterization of the diurnal dynamics of the fruit light environment in a vertical trellis system indicated that clusters were shaded by leaves during most of the photoperiod. Supplementation of the fruit light environment from 20 days before veraison until technological harvest showed that red (R, 660 nm) and blue (B, 470 nm) light strongly increased total phenolic compound levels at harvest in the berry skins without affecting sugar content, acidity or berry size. Far-red (FR, 730 nm) and green (G, 560 nm) light supplementation had relatively small effects. The stimulation of berry phytochromes and cryptochromes favored accumulation of flavonoid and non-flavonoid compounds, including anthocyanins, flavonols, flavanols, phenolic acids and stilbenes. These results demonstrate that the chemical composition of grape berries is modulated by the light quality received by the clusters under field conditions, and that fruit photoreceptors are not saturated even in areas of high insolation and under management systems that are considered to result in a relatively high exposure of fruits to solar radiation. Therefore, manipulation of the light environment or the light sensitivity of fruits could have significant effects on critical grape quality traits. Fil: Gonzalez, Carina Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fanzone, Martín Leandro. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria Mendoza. Laboratorio de Aromas y Sustancias Naturales; Argentina Fil: Cortés, Leandro Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bottini, Ambrosio Ruben. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina Fil: Ballare, Carlos Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - la Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martin. Instituto de Investigaciones Biotecnologicas; Argentina Fil: Boccalandro, Hernan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina

Rust resistance genes Lr37, Sr38, and Yr17 are located within a segment of Triticum ventricosum (Tausch) Cess. chromosome 2NS translocated to the short arm of bread wheat chromosome 2AS. Characterization of this chromosome segment by 13 restriction fragment length polymorphism (RFLP) markers indicated that the 2NS translocation replaced approximately half of the short arm of chromosome 2A (distal 25-38 centimorgans, cM). The objective of this study was to develop polymerase chain reaction (PCR) assays based on RFLP marker cMWG682 to facilitate the transfer of this cluster of rust resistance genes into commercial wheat (Triticum aestivum L.) cultivats. DNA sequence was obtained from the A-, B-, D-, and N-alleles of cMWG682 and was used to design N-allele specific primers. The 2NS fragment amplified by PCR primers cosegregated with the presence of the RFLP-2NS band in all backcross populations. A cleaved amplified polymorphic sequence (CAPS) was used to develop a marker for the 2A-allele. This marker can be used to differentiate homozygous and heterozygous plants carrying the 2NS translocation in the final cycle of backcross introgression or in screenings for homozygous plants in segregating populations. Finally, a third PCR assay was developed by means of TaqMan technology as a high-throughput alternative for selection of the 2NS/2AS translocation in large segregating populations in breeding programs that have access to real time PCR equipment. These molecular markers were used to develop four hard red spring isogenic lines homozygous for the 2NS chromosome segment. One of the isogenic lines, derived from 'Anza,' did not show the expected resistance in spite of the presence of all the RFLP markers for the 2NS chromosome segment. Analysis of F1 hybrids suggested that a suppressor of the Lr37 gene is present in Anza. These isogenic lines will provide a valuable tool to test the effects of the large 2NS translocation on quality and agronomic performance.

The plant vp1 gene encodes a transcription factor originally identified in maize that participates in the control of the transition from embryogenesis to seed germination. Different lines of evidence suggest that vp1 participates in preharvest sprouting (PHS) resistance in cereals, but the genetic relationship is not yet established. Thus, the aim of this study was to investigate if there is a connection between vp1 and the formerly documented quantitative trait loci for PHS in Sorghum bicolor. This gene was mapped in sorghum using two well-studied inbred lines displaying contrasting PHS phenotypes. Linkage analysis revealed that the S. bicolor vp1 (Sbvp1) locus is linked to markers located on chromosomes 3 and 8 in maize. Analysis indicated that this gene is not correlated with PHS at a statistically significant level. Structural analysis of the Sbvp1 gene from both inbred lines showed a high degree of overall conservation (low polymorphism), even in the promoter region. These findings, taken together with the previously observed correlation between the pattern of expression of this gene and PHS performance, suggest that Sbvp1 might act downstream in the signalling pathway that leads to seed germination, or even be differentially regulated as a consequence rather than as a cause of the phenotypic behaviour.Key words: viviparous-1, Sbvp1, dormancy, quantitative trait loci (QTL), pre-harvest sprouting, genetic mapping.

Anthocyanin profiles are commonly used for grapevine cultivar identification because it is currently accepted that this trait is closely related to their genetic characteristics. Nevertheless, the extent of the variation for the anthocyanin profiles among clones of the same cultivar has not yet been studied in depth. The relative concentration of anthocyanins of 131 Malbec clones grown in the same vineyard was investigated by HPLC-DAD and the use of comprehensive statistic procedures. Complementarily, the expression level of structural and regulatory genes was studied via real time polymerase chain reaction. Significant variation was identified among the profiles of the clones, mainly due to variations in the amounts of malvidin derivatives. Finally, the differential expression in F3′5′H, OMT1 and AM2 genes seems to be related to the malvidin content variation. This work shows the existence of variation for the anthocyanin profiles among clones from the same grapevine cultivar and the putative involvement of genes related to hydroxylation, methylation, and transport of anthocyanins on the basis of such variation. © 2014 American Chemical Society., This work was funded by ANPCyT (PICT-2008-00270 and PAE-PICT-2007-02360) and SECTyP-UNCuyo (A504A/11) grants to D.L. and MINCyT-MICINN (Argentine−Spain) bilateral collaboration grant to D.L. and J.M.M.-Z. (ES/09/07). We also received partial funding from Bodegas Esmeralda S.A.

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively. Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete. Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.

One of the most important agronomic problems in the production of sorghum [Sorghum bicolor (L.) Moench] in humid climates is pre-harvest sprouting (PHS). A molecular linkage map was developed using 112molecular markers in an F2 mapping population derived from a cross between IS 9530 (high resistance to PHS) and Redland B2 (susceptible to PHS). Two year phenotypic data was obtained. By means of interval mapping analysis, two significant QTL were detected in two different linkage groups with LOD scores of 8.77and 4.39. Each of these two QTL individually explained approximately 53% of the phenotypic variance, but together, in a two-QTL model, they explained 83% of the phenotypic variance with a LOD score of 12.37.These results were corroborated by a one-way ANOVA in which the four flanking markers of the most likely QTL positions displayed highly significant values in theF-test, and significant variation in trait expression was associated with marker genotypic classes. The four markers with highest effect in the one-way ANOVA were also detected in the second year replication of the F2 population, and significant genotype × environment interactions was observed. The putative relationship between PHS resistance in sorghum and the maize Vp1 gene is also discussed.

Endosperm texture, i.e. the hardness or softness of the grain, is an important quality criterion in cereals because it determines many grain end-use properties. Grain softness is the dominant trait and is mainly controlled by the Ha locus on the short arm of chromosome 5D in hexaploid bread wheat. Genes for puroindoline a (Pina-D1), puroindoline b (Pinb-D1), and grain softness related protein (Gsp-D1) have been shown to be linked to the Ha locus in different mapping populations and have been associated with the expression of grain softness. The study of the linkage relationships among these genes has been limited by the low level of polymorphism in the D genome of hexaploid Triticum aestivum. In the present study, a highly polymorphic Triticum monococcum mapping population was used to analyze linkage relationships among these three genes. Gsp-Am1 and Pina-Am1 were found to be completely linked and lie 0.14 cM distal to Pinb-Am1 in the distal region of the short arm of chromosome 5Am. The tight genetic linkage among these three genes was paralleled by their physical proximity within a single 105-kb clone isolated from a T. monococcum bacterial artificial chromosome (BAC) library. A restriction map of this BAC clone showed that Pina-Am1 is located between Pinb-Am1 and Gsp-Am1. Partial sequences of the T. monococcum genes showed a high degree of similarity with their T. aestivum counterparts (> or =94%). Marker-assisted selection strategies based on the tight linkage among Ha-related genes are discussed.

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. © 2013 The Author., This study was supported by Genoma España [within a collaborative agreement with Genome Canada (Grapegen Project)]; the Spanish Ministry of Science and Innovation [grant Nos. BIO2008-03892, BIO2011-26229]; a bilateral collaborative grant with the University of Cuyo (Mendoza, Argentina) [grant No. AR2009-0021].

The following reference was erroneously omitted from the published manuscript: Hemert J, Hong L, Wise RP, Dickerson JA. PLEXdb: gene expression resources for plants and plant pathogens. Nucleic Acids Res. 40(D1):D1194-D1201)

Grapevine (Vitis vinifera L.) is an economically important fruit crop. The berry components that determine the final quality traits of both table and wine grapes, such as sugars, acids, flavours, anthocyanins, tannins, etc., are synthesized or accumulated along the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes known to occur in the grape berries along development is of paramount importance to advance in the understanding the ripening process. We report the developmental analysis of V. vinifera cv. Muscat d’Hamburg berries at protein level. We undertook a top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry to determine differentially accumulated proteins. The green development (from fruit set to pre-veraison) and the ripening (from veraison to full ripening) were analysed as independent experiments. We have identified and quantified 154 and 61 deregulated proteins in green and ripening phases, respectively. Two key points in development at the protein change level have been determined: end of green development and beginning of ripening. An additional DIGE experiment was performed comparing fully ripe berries from two vintages in order to detect seasonal changes. As few spots changed we have concluded that protein changes detected in this study are developmental-specific. The role of amount of identified proteins is discussed in relation to grape berry development and to quality traits. The well known shift of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch between sucrose synthase and soluble acid invertase. Fil: Martínez Esteso, María José. Universidad de Alicante; España Fil: Sellés Marchart, Susana. Universidad de Alicante; España Fil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentina. Consejo Superior de Investigaciones Científicas; España Fil: Pedreño, María Ángeles. Universidad de Murcia; España Fil: Bru Martínez, Roque. Universidad de Alicante; España

[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., This study was financially supported by Grapegen and the 14322 Agreement Projects from Genoma España as well as the VIN01-025 Project from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria from MICINN (Spanish Ministry for Science and Innovation) and in part by CSIRO Plant Industry and the Grape and Wine Research and Development Corporation (GWRDC). We also thank MICINN for a bilateral collaborative grant with Argentina (AR2009-0021), Applied Biosystems for their support in the design of the 48 SNPlex set and the Centro Nacional de Genotipado http://www.cegen.org for SNPlex genotyping. The research group participates in COST Action FA1003.

13 páginas, 5 figuras, 2 tablas., [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., This study was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PID 5028 to RB), Secretaría de Ciencia y Técnica de la Nación (SECyT, PICT 08-12398 and PICT 20-20093 to RB) and Genoma España (Project GrapeGen to JMMZ). M. Pontin was recipient of a CONICET postdoctoral scholarship.

16 pages, 5 figures.-- PMID: 18997115 [PubMed].-- PMCID: PMC2613716.-- First published online Nov 7, 2008., Supporting information (Suppl. fig. S1-S4, tables S1-S2) available at: http://www.plantphysiol.org/cgi/content/full/pp.108.131052/DC1, Full-text article available Open Access at the publisher's site., MIKC(C)-type MADS box genes encode transcription factors that play crucial roles in plant growth and development. Analysis of the grapevine (Vitis vinifera) genome revealed up to 38 MIKC(C)-type genes. We report here a complete analysis of this gene family regarding their phylogenetic relationships with homologous genes identified in other sequenced dicot genomes, their genome location, and gene structure and expression. The grapevine genes cluster in 13 subfamilies with their Arabidopsis (Arabidopsis thaliana) and poplar (Populus trichocarpa) counterparts. The lack of recent whole genome duplications in grapevine allows assigning the gene diversification processes observed within each subfamily either to an ancestral polyploidization event predating the divergence of those three species or to later duplication events within each lineage. Expression profiles of MIKC(C)-type genes in vegetative and reproductive organs as well as during flower and tendril development show conserved expression domains for specific subfamilies but also reflect characteristic features of grapevine development. Expression analyses in latent buds and during flower development reveal common features previously described in other plant systems as well as possible new roles for members of some subfamilies during flowering transition. The analysis of MIKC(C)-type genes in grapevine helps in understanding the origin of gene diversification within each subfamily and provides the basis for functional analyses to uncover the role of these MADS box genes in grapevine development., This work was supported by the Ministerio de Educación y Ciencia (grant no. BIO2005–7612 and a predoctoral fellowship to J.D.-R.).

8 pages, 3 figures, 1 table.-- PMID: 19102745 [PubMed].-- PMCID: PMC2628674., Supplementary information (additional files 1, 2, 3) available at: http://www.biomedcentral.com/1756-0500/1/132/additional/, [Background] Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the trans-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in Vitis vinifera cv Monastrell albino cell suspension cultures., [Findings] MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes., [Conclusion] The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway., SBN and LA hold grants from the Fundación Séneca. This work has been partially supported by the MEC and FEDER (BIO2005-00332), Consejería de Educación, Ciencia e Investigación de la Región de Murcia (2I05SU0020 and BIO-BVA 07/01-0003) and the GrapeGen project (a joint international project funded by Genoma España and Genome Canada).

Fred Y. Peng et al., We report the generation and analysis of a total of 77,583 expressed sequence tags (ESTs) from two grapevine (Vitis vinifera L.) cultivars, Cabernet Sauvignon (wine grape) and Muscat Hamburg (table grape) with a focus on EST sequence quality and assembly optimization. The majority of the ESTs were derived from normalized cDNA libraries representing berry pericarp and seed developmental series, pooled non-berry tissues including root, flower, and leaf in Cabernet Sauvignon, and pooled tissues of berry, seed, and flower in Muscat Hamburg. EST and unigene sequence quality were determined by computational filtering coupled with small-scale contig reassembly, manual review, and BLAST analyses. EST assembly was optimized to better discriminate among closely related paralogs using two independent grape sequence sets, a previously published set of Vitis spp. gene families and our EST dataset derived from pooled leaf, flower, and root tissues of Cabernet Sauvignon. Sequence assembly within individual libraries indicated that those prepared from pooled tissues contributed the most to gene discovery. Annotations based upon searches against multiple databases including tomato and strawberry sequences helped to identify putative functions of ESTs and unigenes, particularly with respect to fleshy fruit development. Sequence comparison among the three wine grape libraries identified a number of genes preferentially expressed in the pericarp tissue, including transcription factors, receptor-like protein kinases, and hexose transporters. Gene ontology (GO) classification in the biological process aspect showed that GO categories corresponding to ‘transport’ and ‘cell organization and biogenesis’, which are associated with metabolite movement and cell wall structural changes during berry ripening, were higher in pericarp than in other tissues in the wine grape studied. The sequence data were used to characterize potential roles of new genes in berry development and composition., he authors gratefully acknowledge funding from Genome Canada, Genoma España, as well as project management support from Genome British Columbia as part of the Genome Canada-Genoma España collaborative research and development initiative. JB is an NSERC EWR Steacie Fellow.

Diego Lijavetzky et al.. -Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article., The genetics and biochemistry of anthocyanins and flavonol biosynthesis and their role in plant organ pigmentation is well established in model species. However, the genetic basis of colour variation is species specific and understanding this variation is very relevant in many fruit and flower crop species. Among grape cultivars, there is a wide genetic variation for berry colour ranging from yellow-green (“white” cultivars) to dark blue berries. Berry colour results from the synthesis and accumulation of anthocyanins in the berry skin, which in plants is commonly regulated by transcription factors belonging to the MYB and bHLH families. In this work, we aimed to identify the major genetic determinants of berry colour variation in a large collection of table grape cultivars and somatic variants. The genetic analyses of berry colour in a few grape segregating progenies had previously identified a single locus on linkage group 2 responsible for colour variation. Furthermore, somatic variation for berry skin colour in cultivar Italia had been associated with the presence of a Gret1 retrotransposon in the promoter region of VvmybA1, a Myb gene whose expression is associated to skin colouration. The results show that VvmybA1 is the gene underlying the mapped locus controlling berry colour in grape. Additionally, the molecular analyses indicate that genetic and somatic berry colour variation can be associated to molecular variation at VvmybA1 in more than 95% of the analyzed cultivars. Thus, VvmybA1 is a major determinant of berry colour variation in table grape and its instability is the major cause of somatic variation for this trait., This work was funded by a grant from the CDTI (Spanish Ministry of Science and Technology).

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720 384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.Key words: bacterial artificial chromosome, BAC library, Triticum monococcum, wheat.

This article is available from: http://www.biomedcentral.com/1471-2164/8/424, [Background] Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes., [Results] In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (π = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlex™ genotyping technology in a sample of grapevine genotypes and segregating progenies., [Conclusion] These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlex™ genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlex™ high throughput genotyping technology provide a powerful tool for grapevine genetic analysis., This work was funded by GRAPEGEN, a collaborative project funded by Genoma España and Genome Canada.

Dof proteins are a family of plant-specific transcription factors that contain a particular class of zinc-finger DNA-binding domain. Members of this family have been found to play diverse roles in gene regulation of processes restricted to the plants. The completed genome sequences of rice and Arabidopsis constitute a valuable resource for comparative genomic analyses, since they are representatives of the two major evolutionary lineages within the angiosperms. In this framework, the identification of phylogenetic relationships among Dof proteins in these species is a fundamental step to unravel functionality of new and yet uncharacterised genes belonging to this group. We identified 30 different Dof genes in the rice Oryza sativa genome and performed a phylogenetic analysis of a complete collection of the 36-reported Arabidopsis thaliana and the rice Dof transcription factors identified herein. This analysis led to a classification into four major clusters of orthologous genes and showed gene loss and duplication events in Arabidopsis and rice, that occurred before and after the last common ancestor of the two species. According to our analysis, the Dof gene family in angiosperms is organized in four major clusters of orthologous genes or subfamilies. The proposed clusters of orthology and their further analysis suggest the existence of monocot specific genes and invite to explore their functionality in relation to the distinct physiological characteristics of these evolutionary groups.

The adaptability of Triticum aestivum to a large range of environments is partially due to genetic differences in sensitivity to vernalization. The most potent gene reducing the vernalization requirement in hexaploid wheat is Vrn-A1. An orthologous vernalization gene, designated Vrn-A m 1, was mapped in the diploid wheat Triticum monococcum between RFLP markers Xwg908 and Xabg702 on the long arm of chromosome 5AmL. The orthology of VrnA m 1 with Vrn-A1 (5A wheat, originally Vrn1), Vrn-D1 (5D wheat, originally Vrn3), Vrn-R1 (5R rye, originally Sp1) and Vrn-H1 (5H barley, originally Sh2) was shown by mapping RFLP markers linked to these vernalization genes on the T. monococcum linkage map. A second vernalization gene, designated Vrn-A m 2, was found in the distal region of chromosome 5AmL within a segment translocated from homoeologous group 4. This gene is completely linked to RFLP marker Xbcd402 and located between the same RFLP markers (Xβ-Amy-1 and Xmwg616) as the Vrn-H2 (originally Sh) locus in Hordeum vulgare.

Vp1 is a seed-specific gene involved in the control of dormancy and germination. We here present the complete sequence of the sorghum vp1 promoter/enhancer region highlighting its main features, especially the lack of canonical TATA and CAAT boxes and the presence of elements responsive to abscisic acid and light. The region closest to the start of transcription is highly homologous to the partial proximal sequence reported for the maize vp1 promoter. This region is interrupted by a 57-nt stretch containing 14 CT microsatellite repeats. We observed a poor overall homology to the promoter from abi3 gene, the Arabidopsis counterpart bearing a similar coding sequence. However, there exists a high degree of homology (89%) between a TATA-rich 103-bp stretch of the sorghum vp1 promoter located about 700 nt upstream of the startpoint and miniature inverted transposable elements (MITEs) interspersed within the sorghum seed-specific kafirin cluster. This sorghum MITE-like element displays considerable homology (68%) to the TATA-less promoter from the sorghum NADP-malate dehydrogenase gene and lesser similarity to the Tourist, Pilgrim and Batuta MITEs previously identified within the promoter from the maize Abp1 (auxin-binding protein) gene.