Development and Use of Biotechnology Tools for Grape Functional Analysis

The aim of this chapter is to provide a description of the latest scientific advances in the field of gene functional analysis in grapevine. It provides general information about the studies conducted during the past decade to understand the natural variation of this plant and how this information has been exploited for the understanding of traits of interest. Likewise, it is exposed how the use of biotechnology tools have helped to characterize the mechanisms of gene expression and its regulation, as well as the subcellular localization of proteins and their interactions with other molecules. Finally, an approximation to the new technologies of gene editing and their potential application in the functional study of grapevine has been carried out

Malbec, genética y cambio climático

La evolución de la temperatura en los últimos 20 años muestra una tendencia de calentamiento global en el hemisferio Norte que se acentuará en los próximos 50 años.Según una publicación de Hans R. Schultz and Gregory Jones (2010) en Journal of Wine Research, esto cambiaría los márgenes de adecuación para el crecimiento de la uva, afectando los límites del cultivo de la vid y la distribución de las variedades. En el hemisferio Sur, adicionalmente, es posible se que sumen progresivos cambios hacia un clima de carácter subtropical. En áreas de bajo riego como Cuyo y otros valles la escasez de agua sería el escenario más probable.El aumento de la temperatura acelera el desarrollo de la vid, alterando la interacción entre los ciclos vegetativos y reproductivos. Tiene efectos en el desarrollo de la uva,acelera la caída de ácidos orgánicos e induce un incremento en la concentración de azúcares, desacoplando ambos procesos, de la acumulación de antocianos y de la maduración fenólica, que no responden de igual manera al incremento de temperatura.Como consecuencia, estas uvas dan lugar a vinos que pueden presentar un grado alcohólico excesivo, baja acidez, baja intensidad de color y caracteres fenólicos verdes.Mientras que las estrategias de producción o elaboración de vinos pueden permitir una aplicación a corto plazo, para revertir esta situación en el medio plazo resulta más atractivo generar soluciones con un mayor recorrido de su aplicación y que no impliquen un incremento en los costes de producción. En este sentido, la selección de variantes somáticas (clones) puede suponer una posibilidad de identificar genotipos más adaptados para las nuevas condiciones ambientales manteniendo el genotipo básico de la variedad, sus características productivas y enológicas y su denominación. Todo ello resulta muy importante en la elaboración de vinos de calidad al mantener el mismo producto final que es reconocido por productores, entes reguladores y consumidores.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; Argentin

Identification and functional prediction of anthocyanin biosynthesis regulatory long non-coding RNAs (lncRNAs) in carrot

Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We identified 639 differentially expressed lncRNAs between genotypes, and certain were specifically associated with a particular tissue. We then established regulatory correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript (NAT) linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.Competing Interest StatementThe authors have declared no competing interest.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; ArgentinaFil: Blein, Thomas. Université de Paris; FranciaFil: Crespi, Martin. Université de Paris; FranciaFil: 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; Argentin

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology

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 ( = 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. © 2007 Lijavetzky et al; licensee BioMed Central Ltd

Natural genetic variation for grapevine phenology as a tool for climate change adaptation

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; ArgentinaFil: 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; ArgentinaFil: Bree, Laura. Vivero Mercier; ArgentinaFil: Bergamín, Daniel. Vivero Mercier; ArgentinaFil: Sola, Cristobal. Vivero Mercier; ArgentinaFil: 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; Argentin

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

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro </it>cultured <it>Vitis vinifera </it>cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m^-2d^-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^®.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Our results show the UV-B radiation effects on the leaf transcriptome of grapevine (<it>Vitis vinifera </it>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.</p

Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures

<p>Abstract</p> <p>Background</p> <p>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 <it>trans</it>-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 <it>Vitis vinifera </it>cv Monastrell albino cell suspension cultures.</p> <p>Findings</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Characterization of a cv. Tempranillo Tinto variant exhibiting a male-like flower phenotype

Domesticated grapevine (Vitis vinifera L.) is used for wine, fresh fruit, raisins and juice production. Two subspecies can be identified within this species: V. vinifera ssp. vinifera, the cultivated form comprising mostly hermaphrodite and some female cultivars and V. vinifera ssp. sylvestris, the suggested wild dioecious ancestor. Studies dealing with this trait identified a major QTL on chromosome 2 as the grapevine Sex Determining Region (SDR), which harbours several proposed candidate genes. The aim of this work is the genetic and molecular characterization of a Tempranillo Tinto somatic variant that shows an androgenized flower phenotype. Whilst flowers in this somatic variant develop normal stamens, they present a reduced gynoecium that, unlike canonical male flowers of V. vinifera ssp. sylvestris, still enable fruit setting and ripening. Phenotyping results of a self-cross progeny of this variant line (more than 100 offspring) indicated that the mutant flower phenotype is inheritable. Furthermore, genotyping results of the microsatellite marker VVIB23, linked to the SDR, showed that the putative mutation co-localizes with this locus. One of the proposed female development inhibitor genes underlying the SDR locus is VviAPT3, which encodes an adenine phosphoribosyl transferase that may inactivate cytokinins by using them as substrate. The inactivation of these hormones, which promote gynoecium development in wild male vines if applied exogenously, could explain the mutant phenotype. RT- qPCR and RNA-seq expression analyses during flower development demonstrated the overexpression of VviAPT3 in the mutant line compared to a normal flower Tempranillo Tinto line used as control. Several experiments are ongoing to identify the genetic variation that causes this male-like phenotype, such as the comparison of the whole genome sequences of the variant and a control Tempranillo line, or the genotyping of VviAPT3 and other candidate genes through Sanger sequencing.Fil: Alañón, Noelia. Instituto de Ciencias de la Vid y del Vino; EspañaFil: Carbonell Bejerano, Pablo. Max Planck Institute for Developmental Biology; AlemaniaFil: Mauri, Nuria. Centre for Research in Agricultural Genomic; EspañaFil: Ferradás, Yolanda. Instituto de Ciencias de la Vid y del Vino; EspañaFil: 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; ArgentinaFil: Martinez-Zapater, José Miguel. Instituto de Ciencias de la Vid y del Vino; EspañaFil: Ibañez, Javier. Instituto de Ciencias de la Vid y del Vino; EspañaXIth International Symposium on Grapevine Physiology and BiotechnologyStellenboschSudáfricaInternational Society for Horticultural Scienc