GEM, una proteína con dominio GRAM, es un regulador negativo de la señalización por ABA durante la germinación en Arabidopsis thaliana

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-03-201

Somatic Variation and Cultivar Innovation in Grapevine

Paradoxically, continuous vegetative multiplication of traditional grapevine cultivars aimed to maintain cultivar attributes in this highly heterozygous species ends in the accumulation of considerable somatic variation. This variation has long contributed to cultivar adaptation and evolution under changing environmental and cultivation conditions and has also been a source of novel traits. Understanding how this somatic variation originates provides tools for genetics-assisted tracking of selected variants and breeding. Potentially, the identification of the mutations causing the observed phenotypic variation can now help to direct genome editing approaches to improve the genotype of elite traditional cultivars. Molecular characterization of somatic variants can also generate basic information helping to understand gene biological function. In this chapter, we review the state of the art on somatic variation in grapevine at phenotypic and genome sequence levels, present possible strategies for the study of this variation, and describe a few examples in which the genetic and molecular basis or very relevant grapevine traits were successfully identified

A genome-wide 20 K citrus microarray for gene expression analysis

<p>Abstract</p> <p>Background</p> <p>Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant.</p> <p>Results</p> <p>We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability.</p> <p>Conclusion</p> <p>This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos.</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

Microbiological study of patients hospitalized for acute exacerbation of chronic obstructive pulmonary disease (AE-COPD) and the usefulness of analytical and clinical parameters in its identification (VIRAE study)

Ramon Boixeda,1,5 Nuria Rabella,2 Goretti Sauca,3 Maria Delgado,1 Xavier Martinez-Costa1, Montserrat Mauri,1 Vanessa Vicente,1 Elisabet Palomera,4 Mateu Serra-Prat,4 Josep Ant&amp;oacute;n Capdevila11Department of Internal Medicine, Hospital of Matar&amp;oacute;, Barcelona, Spain; 2Department of Microbiology, Hospital of Santa Creu and Sant Pau, Barcelona, Spain; 3Department of Microbiology, Hospital of Matar&amp;oacute;, Barcelona, Spain; 4Department of Research, Hospital of Matar&amp;oacute;, Barcelona, Spain; 5Department of Medicine, Autonomous University of Barcelona, Barcelona, SpainPurpose: Respiratory infection is the most common cause for acute exacerbation of chronic obstructive pulmonary disease (AE-COPD). The aim of this work was to study the etiology of the respiratory infection in order to assess the usefulness of the clinical and analytical parameters used for COPD identification.Patients and methods: We included 132 patients over a period of 2 years. The etiology of the respiratory infection was studied by conventional sputum, paired serology tests for atypical bacteria, and viral diagnostic techniques (immunochromatography, immunofluorescence, cell culture, and molecular biology techniques). We grouped the patients into four groups based on the pathogens isolated (bacterial versus. viral, known etiology versus unknown etiology) and compared the groups.Results: A pathogen was identified in 48 patients. The pathogen was identified through sputum culture in 34 patients, seroconversion in three patients, and a positive result from viral techniques in 14 patients. No significant differences in identifying etiology were observed in the clinical and analytical parameters within the different groups. The most cost-effective tests were the sputum test and the polymerase chain reaction.Conclusion: Based on our experience, clinical and analytical parameters are not useful for the etiological identification of COPD exacerbations. Diagnosing COPD exacerbation is difficult, with the conventional sputum test for bacterial etiology and molecular biology techniques for viral etiology providing the most profitability. Further studies are necessary to identify respiratory syndromes or analytical parameters that can be used to identify the etiology of new AE-COPD cases without the laborious diagnostic techniques.Keywords: respiratory viruses, chronic obstructive pulmonary disease, exacerbation, diagnostic tests, hospitalizatio

Genomic variation and clone genotyping in Vitis vinifera L. Malbec

Somatic mutations are a major force introducing novel genetic variation; this role becomes enhanced in systems lacking of sexual reproduction. The later is the case of grapevines used in the wine industry. Even though clonal propagation is a normal practice in this industry, a remarkable phenotypic variation has been reported at the intra-cultivar level. However, less is known about the genetic variability among clones. Malbec is the main cultivar for the Argentinean viticulture, showing a notorious phenotypic variation on many traits of technological interest, for example the biochemical composition of berries. Therefore, it turns relevant to develop a formal protocol to discriminate among clones exhibiting different properties. Here we performed a genomic analysis in order to test if the genetic variability is in agreement with the phenotypic variability, and also to develop a genetic-based protocol for clones? discrimination. For this aim we obtained Illumina reads at a 35x depth for four different Malbec clones (MB53, MB59, Cot143 and Cot225). Bioinformatic tools were employed to align these reads to the Pinot noir reference genome (PN40024) and to perform variant calling analysis for single nucleotide variants (SNVs) discovery. Afterwards, strict quality and frequency filters were applied to obtain a set of reliable SNVs. We discovered 2 million of shared SNVs (i.e. all clones shared the same allele); these variants allow distinguishing Malbec from the reference genome. On the other hand, we identified 458 non-shared SNVs (i.e. at least one of the clones has the same allele than the reference); these were of particular interest to us because they allow for clone discrimination. From the latter set we picked 48 SNVs to validate them through Sanger sequencing. After validation these same 48 SNVs were employ to build a chip for the high throughput genotyping platform FLUIDIGM. We genotyped 221 plants, including clones of known origin as well as plants belonging to five different mass selections. We were able to classify all genotyped plants in 10 different haplo-groups; showing that with a small but informative number of SNVs it is possible to discriminate among clones of the same cultivar in an efficient manner.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 Panadero, Nuria. Instituto de Ciencias de la Vid y el 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. Instituto de Ciencias de la Vid y el Vino; EspañaFil: Carbonell Bejerano, Pablo. No especifíca;Fil: Royo, Carolina. Instituto de Ciencias de la Vid y el Vino; EspañaFil: Sola, Cristobal. No especifíca;Fil: Martínez Zapater, José M.. Instituto de Ciencias de la Vid y el 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; Argentina63rd Italian Society of Agricultural Genetics Annual CongressNapoliItaliaItalian Society of Agricultural Genetic

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

Whole genome resequencing and custom genotyping unveil clonal lineages in ‘Malbec’ grapevines (Vitis vinifera L.)

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.EEA MendozaFil: Calderón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Calderón, Luciano. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Mauri, Nuria. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Muñoz, Claudio. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; ArgentinaFil: Carbonell Bejerano, Pablo. Max Planck Institute for Developmental Biology; AlemaniaFil: Bree, Laura. Vivero Mercier; ArgentinaFil: Bergamin, Daniel. Vivero Mercier; ArgentinaFil: Sola, Cristóbal. Vivero Mercier; ArgentinaFil: Gomez Talquenca, Gonzalo. Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria Mendoza; ArgentinaFil: Royo, Carolina. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Ibáñez, Javier. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Martínez Zapater, José Miguel. Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja). Finca La Grajera; ArgentinaFil: Lijavetzky, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Lijavetzky, Diego. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; Argentin

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

Identification of a missense mutation in the MADS-box gene VviAGL11 responsible for table grape seedlessness

Trabajo presentado a la XXIII Reunión Bianual de la Sociedad Española de Fisiología Vegetal y al XVI Congreso Hispano-Luso de Fisiología Vegetal, celebrados en Pamplona (España) del 26 al 28 de junio de 2019