The LATERAL ORGAN BOUNDARIES Domain gene family in grapevine: Genome-wide characterization and expression analyses during developmental processes and stress responses

LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) constitute a family of plant-specific transcription factors with key roles in the regulation of plant organ development, pollen development, plant regeneration, pathogen response, and anthocyanin and nitrogen metabolisms. However, the role of LBDs in fruit ripening and in grapevine (Vitis vinifera L.) development and stress responses is poorly documented. By performing a model curation of LBDs in the latest genome annotation 50 genes were identified. Phylogenetic analysis showed that LBD genes can be grouped into two classes mapping on 16 out of the 19 V. vinifera chromosomes. New gene subclasses were identified that have not been characterized in other species. Segmental and tandem duplications contributed significantly to the expansion and evolution of the LBD gene family in grapevine as noticed for other species. The analysis of cis-regulatory elements and transcription factor binding sites in the VviLBD promoter regions suggests the involvement of several hormones in the regulation of LBDs expression. Expression profiling suggest the involvement of LBD transcription factors in grapevine development, berry ripening and stress responses. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming to clarify mechanisms responsible for the onset of fruit ripening and fruit defense strategies. © 2017 The Author(s)

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

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

Comparative Analysis of Grapevine Whole-genome Gene Predictions, Functional Annotation, Categorization and Integration of the Predicted Gene Sequences

Background: The first draft assembly and gene prediction of the grapevine genome (8X base coverage) was made available to the scientific community in 2007, and functional annotation was developed on this gene prediction. Since then additional Sanger sequences were added to the 8X sequences pool and a new version of the genomic sequence with superior base coverage (12X) was produced. Results: In order to more efficiently annotate the function of the genes predicted in the new assembly, it is important to build on as much of the previous work as possible, by transferring 8X annotation of the genome to the 12X version. The 8X and 12X assemblies and gene predictions of the grapevine genome were compared to answer the question, “Can we uniquely map 8X predicted genes to 12X predicted genes?” The results show that while the assemblies and gene structure predictions are too different to make a complete mapping between them, most genes (18,725) showed a one-to-one relationship between 8X predicted genes and the last version of 12X predicted genes. In addition, reshuffled genomic sequence structures appeared. These highlight regions of the genome where the gene predictions need to be taken with caution. Based on the new grapevine gene functional annotation and in-depth functional categorization, twenty eight new molecular networks have been created for VitisNet while the existing networks were updated. Conclusions: The outcomes of this study provide a functional annotation of the 12X genes, an update of VitisNet, the system of the grapevine molecular networks, and a new functional categorization of genes

A completely-phased diploid genome assembly for ‘Malbec’ cultivar (Vitis vinifera L.)

Poster. Publicado en: BAG Journal of Basic and Applied Genetics, 32 (1 suppl), 2021Most grapevine cultivars originated from the outcrossing of two genetically diverse parents, and are clonally propagated to preserve phenotypes of productive interest. Hence, cultivars are first filial generations (F1) with highly heterozygous diploid genomes, that turn challenging to assemble. ‘Malbec’ is the main cultivar for the Argentine wine industry and it originated in France, from the outcrossing of ‘Magdeleine Noir des Charentes’ and ‘Prunelard’ cultivars. Based on that mother-father-offspring relationship, here we followed the algorithm implemented in the software CanuTrio to produce a phased assembly of ‘Malbec’ genome. For this aim, parental cultivars’ Illumina short-reads were used to sort ‘Malbec’ PacBio long-reads into its haploid complements, to be assembled separately. Postassembly, bioinformatic procedures were employed to reduce the number of duplicated regions and perform sequence error corrections (using ‘Malbec’ Illumina short-reads). We obtained two highly complete and contiguous haploid assemblies for ‘Malbec’, Haplotype-Prunelard (482.4 Mb size; contig N50=7.7 Mb) and Haplotype-Magdeleine (479.4 Mb size; contig N50=6.6 Mb), with 96.1 and 95.8% of BUSCO genes, respectively. We tested for the composition of both haplophases with the tool Merqury, and observed 15% of both assemblies affected by structural variations, along with 3.2 million SNPs and 0.6 million InDels. Our results indicate that this is a valid approach to assemble highly heterozygous and complex diploid genomes in a completely-phased way.EEA MendozaFil: Calderón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Carbonell-Bejerano, P. Max Planck Institute for Developmental Biology; AlemaniaFil: Mauri, Nuria. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Muñoz, C. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; ArgentinaFil: Bree, Laura. Vivero Mercier; ArgentinaFil: Sola, Cristóbal. Vivero Mercier; ArgentinaFil: Bergamin, Daniel. Vivero Mercier; ArgentinaFil: Gomez Talquenca, Gonzalo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Martínez Zapater, José Miguel. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Weigel, D. Max Planck Institute for Developmental Biology; AlemaniaFil: Lijavetzky, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentin

A completely phased diploid genome assembly for "Malbec" cultivar (Vitis vinifera L.)

Most grapevine cultivars originated from the outcrossing of two genetically diverse parents, and are clonally propagated to preserve phenotypes of productive interest. Hence, cultivars are first filial generations (F1) with highly heterozygous diploid genomes, that turn challenging to assemble. "Malbec" is the main cultivar for the Argentine wine industry and it originated in France, from the outcrossing of Magdeleine Noir des Charentes and Prunelard cultivars. Based on that mother-father-offspring relationship, here we followed the algorithm implemented in the software CanuTrio to produce a phased assembly of Malbec genome. For this aim, parental cultivars? Illumina short-reads were used to sort ?Malbec? PacBio long-reads into its haploid complements, to be assembled separately. Post- assembly, bioinformatic procedures were employed to reduce the number of duplicated regions and perform sequence error corrections (using Malbec Illumina short-reads). We obtained two highly complete and contiguous haploid assemblies for Malbec, Haplotype- Prunelard (482.4 Mb size; contig N50=7.7 Mb) and Haplotype-Magdeleine (479.4 Mb size; contig N50=6.6 Mb), with 96.1 and 95.8% of BUSCO genes, respectively. We tested for the composition of both haplophases with the tool Merqury, and observed 15% of both assemblies affected by structural variations, along with 3.2 million SNPs and 0.6 million InDels. Our results indicate that this is a valid approach to assemble highly heterozygous and complex diploid genomes in a completely-phased way.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; ArgentinaFil: Carbonell Bejerano, P.. Max Planck Institute for Developmental Biology; AlemaniaFil: Mauri, N. Instituto de Ciencias de la Vid y del Vino; EspañaFil: 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. No especifíca;Fil: Sola, C. No especifíca;Fil: Bergamin, D. No especifíca;Fil: Gómez Talquenca, Sebastián. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Ibañez, J. Instituto de Ciencias de la Vid y del Vino; EspañaFil: Martinez-Zapater, JM. 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; ArgentinaXVIII Congreso Latinoamericano de Genética; LIV Reunión Anual de la Sociedad de Genética de Chile; XLIX Congreso Argentino de Genética; VIII Congreso de la Sociedad Uruguaya de Genética; I Congreso Paraguayo de Genética y V Congreso Latinoamericano de Genética HumanaChileSociedad Argentina de Genétic

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

<p>Abstract</p> <p>Background</p> <p>Grapes (<it>Vitis vinifera </it>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.</p> <p>Results</p> <p>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 <it>véraison </it>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.</p> <p>Conclusions</p> <p>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.</p

Clonal propagation history shapes the intra-cultivar genetic diversity in Malbec grapevines

Grapevine (Vitis vinifera L.) cultivars are clonally propagated to preserve their varietal 26 attributes. However, novel genetic variation still accumulates due to somatic mutations. Aiming 27 to study the potential impact of clonal propagation history on grapevines intra-cultivar genetic 28 diversity, we have focused on ‘Malbec’. This cultivar is appreciated for red wines elaboration, 29 it was originated in Southwestern France and introduced into Argentina during the 1850s. Here, 30 we generated whole-genome resequencing data for four ‘Malbec’ clones with different 31 historical backgrounds. A stringent variant calling procedure was established to identify reliable 32 clonal polymorphisms, additionally corroborated by Sanger sequencing. This analysis retrieved 33 941 single nucleotide variants (SNVs), occurring among the analyzed clones. Based on a set of 34 validated SNVs, a genotyping experiment was custom-designed to survey ‘Malbec’ genetic 35 diversity. We successfully genotyped 214 samples and identified 14 different clonal genotypes, 36 that clustered into two genetically divergent groups. Group-Ar was driven by clones with a long 37 history of clonal propagation in Argentina, while Group-Fr was driven by clones that have 38 longer remained in Europe. Findings show the ability of such approaches for clonal genotypes 39 identification in grapevines. In particular, we provide evidence on how human actions may have 40 shaped ‘Malbec’ extant genetic diversity pattern.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; ArgentinaFil: Mauri, Nuria. Consejo Superior de Investigaciones Científicas; EspañaFil: 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: Carbonell Bejerano, Pablo. Max Planck Institute for Biology of Ageing; AlemaniaFil: Bree, Laura. No especifíca;Fil: Sola, Cristobal. No especifíca;Fil: Gómez Talquenca, Sebastián. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Royo, Carolina. Consejo Superior de Investigaciones Científicas; EspañaFil: Ibañez, Javier. Consejo Superior de Investigaciones Científicas; EspañaFil: Martinez-Zapater, José Miguel. Consejo Superior de Investigaciones Científicas; 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; Argentin