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

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

Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9

Producción Científica.Ly-9 is a mouse cell-surface glycoprotein that is selectively expressed on thymocytes and on mature T and B lymphocytes. Ly-9 belongs to the CD2 subset of the immunoglobulin superfamily, an emerging family of cell signaling receptors. Recently, a partial human Ly-9 complementary DNA (cDNA) sequence has been described. Full-length cDNA clones were isolated that included the initiation codon, the sequence encoding the full signal peptide, and 14 amino acids more in the cytoplasmic domain than in the previously reported clone. The predicted extracellular domain of human Ly-9 contains 4 immunoglobulinlike domains, similar to those in mouse Ly-9. Northern blot analysis revealed that the human Ly-9 messenger RNA (2.6 kb) is expressed predominantly in lymph node, spleen, thymus, and peripheral blood leukocytes. Four monoclonal antibodies (mAbs) were raised against human Ly-9 by immunizing mice with the pre-B-cell line 300.19 stably transfected with human Ly-9 full-length cDNA. These mAbs strongly stained the surfaces of cells transfected with human Ly-9 cDNA but not of untransfected cells. Human Ly-9 expression was restricted to T and B lymphocytes and thymocytes, with the highest levels of expression on CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. Monocytes, granulocytes, platelets, and red blood cells were uniformly negative for Ly-9. These mAbs immunoprecipitated major polypeptides of 120 kd from the transfected cells and 120 kd and 100 kd from B-cell line Daudi, probably because of the cell-surface-expressed isoforms. These data demonstrate that human Ly-9 is a new marker for the study of normal and malignant leukocyte

CD229 (Ly9) lymphocyte cell surface receptor Interacts homophilically through Its N-Terminal domain and relocalizes to the immunological synapse

Producción CientíficaCD229 is a member of the CD150 family of the Ig superfamily expressed on T and B cells. Receptors of this family regulate cytokine production and cytotoxicity of lymphocytes and NK cells. The cytoplasmic tail of CD229 binds to SAP, a protein that is defective in X-linked lymphoproliferative syndrome. To identify the CD229 ligand, we generated a soluble Ig fusion protein containing the two N-terminal extracellular domains of human CD229 (CD229-Ig). CD229-Ig bound to CD229-transfected cells, whereas no binding was detected on cells expressing other CD150 family receptors, showing that CD229 binds homophilically. Both human and mouse CD229 interacted with itself. Domain deletion mutants showed that the N-terminal Ig-domain mediates homophilic adhesion. CD229-CD229 binding was severely compromised when the charged amino acids E27 and E29 on the predicted B-C loop and R89 on the F-G loop of the N-terminal domain were mutated to alanine. In contrast, one mutation, R44A, enhanced the homophilic interaction. Confocal microscopy image analysis revealed relocalization of CD229 to the contact area of T and B cells during Ag-dependent immune synapse formation. Thus, CD229 is its own ligand and participates in the immunological synapse

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

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.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. 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 Developmental Biology; AlemaniaFil: Bree, Laura. No especifíca;Fil: Bergamin, Daniel. 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: Ibáñez, Javier. Consejo Superior de Investigaciones Científicas; EspañaFil: Martínez 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