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

An overview about the impacts of agricultural practices on grape nitrogen composition: Current research approaches

Nitrogen is a structural component of proteins, nucleic acids, chlorophyll, hormones and amino acids. The last one and ammonium are important primary metabolites in grapes and are key compounds in winemaking since they are primary sources for yeast fermentation. Currently, grape quality has been affected due to the negative impacts of global warming and anthropogenic activity. Certain studies have reported a significant decrease in the free amino acids content and an increase in berry soluble solids and in proline biosynthesis in grapes in some grapevine varieties cultivated under warm climate conditions and water restriction. Proline is not metabolized by yeasts and stuck and sluggish fermentations can occur when the content of yeast assimilable nitrogen is low. Nitrogen composition of grape is mainly affected by variety, edaphoclimatic conditions of the vineyard and agricultural practices performed to the grapevines. This review summarized the most current research carried out to modify the nitrogen composition of the grape and give an overview of the technical and scientific aspects that should be considered for future research in this field.Peer reviewe

Characterization of a flower sex mutant in Vitis vinifera

Resumen del trabajo presentado en el EMBO/EMBL Symposia on The Molecular Basis and Evolution of Sexual Dimorphism, celebrado de forma virtual del 14 al 16 de septiembre de 2020Grapevine, Vitis vinifera L., is one of the first domesticated fruit crops. Currently it is used for the production of wine (mostly), table grapes, raisins and juice. In 2016, almost 8 million hectares were dedicated to growing grapes worldwide, with a total wine production of more than 270 million liters. Vitis vinifera is divided into two subspecies: the relict wild form (sylvestris), that still grows near river basins, and the cultivated one (sativa or vinifera). It is generally accepted that sativa varieties, normally developing hermaphrodite and less often female flowers, come from the domestication of sylvestris, a dioecious subspecies. While sex determinism in grapevines is not well-known, a QTL in chromosome 2 has been described as a sex locus with several suggested candidate genes. The aim of this work is the genetic and molecular characterization of a somatic variant line of Tempranillo Tinto variety bearing a mutant flower phenotype. The variant line presents male-like flowers, with normal stamens and a reduced gynoecium that is still functional to set fruits. Phenotyping of flower sex in a self-progeny of the variant line showed that the mutant phenotype was inheritable, while genotyping of a sex locus-linked microsatellite marker indicated that the putative mutation is located at the sex locus. VviAPT3 is a plausible candidate gene within this locus as it encodes an adenine-phosphoribosyl transferase enzyme that can inactivate cytokinins, which are plant hormones known to promote gynoecium development in wild male vines. Gene expression analysis through RT-qPCR during flower development showed overexpression of VviAPT3 in the mutant compared to a control Tempranillo Tinto line. Whole genome sequence comparison and APT3 genotyping aimed at identifying the causal genetic variation in this Tempranillo variant are ongoing. Understanding this mutant phenotype could help providing molecular genetic evidence on the control of sexual dimorphism in grapevine

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

Trabajo presentado en el International XI Symposium on Grapevine Physiology and Biotechnology, celebrado en Stellenbosch (Sudáfrica), del 31 de octubre al 5 de noviembre de 2021Domesticated 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. RTqPCR 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

Reduced bunch compactness in somatic variants of 'Tempranillo' relate to genome structural variation and the sex locus

Trabajo presentado en el XIII International Symposium on Grapevine Breeding and Genetics, celebrado en Landau in der Pfalz (Alemania), del 10 al 17 de julio de 2022Grapevine cultivars are vegetatively propagated to keep their varietal attributes. Still, spontaneous somatic variation that emerges during long cycles of vegetative growth provides the opportunity for natural improvement of traditional grape cultivars. A lower bunch compactness is an advantageous trait for winegrowing as it decreases susceptibility to pests and fungal diseases and enables better adaptation to climate change by enabling a more homogeneous berry ripening. To understand the genetic and molecular mechanisms generating variation in bunch compactness, we studied here two somatic variants of Tempranillo Tinto cultivar that produce looser bunches. One of the somatic variants exhibits a male-like flower phenotype, with an underdeveloped but functional gynoecium (flower sex note 2 according to OIV 151 descriptor), instead of the hermaphroditic flowers regularly developed in this cultivar (note 3). Histological analyses revealed a reduced development of the style and stigma, as well as a remarkable thinning of the gynoecium septum. Genetic analyses of its selfprogeny revealed the co-segregation of the male-like phenotype with the hermaphrodite allele of the grape sex locus (SDR). A treatment of flower clusters in stages prior to flowering with cytokinins, hormones involved in the gynoecium development, reversed the phenotype in the somatic variant. These findings suggest that some somatic mutation of the hermaphrodite SDR allele into male-like could hinder the correct fertilization of the ovules, leading to lower fruit set and looser bunches. The other somatic variant of Tempranilllo Tinto with looser bunches presents a notable reduction in pollen viability, around 50%, compared to the >90% regularly observed in Tempranillo clones. Pollen sterility also segregated in the self-progeny of the somatic variant, suggesting the inheritance of this trait in the progeny. Whole genome DNAseq identifed structural variation specific of this clone, concretely a translocation breakpoint between chromosomes 1 and 3 that was confirmed by PCR and Sanger sequencing and was observed also in individuals of the self-progeny with low pollen viability. These findings together with previous reports in other somatic variants such as Tempranillo Blanco suggest that genome structural variation is a recurrent source of low bunch compactness phenotypes by causing deleterious effects on haploid gametes and, ultimately, limiting fruit set

Identificación de variación clonal funcional en Tempranillo y Garnacha mediante el uso de ensamblajes de genomas de referencia varietales

Las variedades de vid (Vitis vinifera L.) se caracterizan por su alta heterocigosidad(variación entre sus copias genómicas de herencia materna y paterna), lo que obliga a lapropagación vegetativa como única vía posible para reproducir su genotipo y sus característicasvarietales. Sin embargo, la propagación vegetativa prolongada da lugar a la acumulación demutaciones somáticas que en ocasiones pueden producir fenotipos de interés que son la basepara la mejora mediante selección clonal. En este trabajo hemos producido ensamblajes de losgenomas de Tempranillo y Garnacha. Usando los ensamblajes como referencia, pudimosidentificar variación genómica responsable de caracteres de interés en clones o variantessomáticas seleccionadas de cada variedad. En Tempranillo se identificó una deleción de grantamaño en un clon con color de baya más oscuro de lo normal, relacionada con una reducciónen la acumulación de ceras en la cutícula de la baya. En Garnacha se identificaron dos patronesde deleción distintos que causan la pérdida de los genes MYB necesarios para la síntesis deantocianinas en el hollejo, que suponen dos orígenes independientes de la variedad GarnachaBlanca. Estos resultados muestran como el ensamblaje de genoma puede ayudar a conocer elorigen de las características varietales y posibilitar la trazabilidad de variación genética clonalútil para la innovación intravarietal

Clonal improvement-associated somatic mutations identified from diploid genome assembly in Tempranillo grapevine cultivar

Trabajo presentado en el International XI Symposium on Grapevine Physiology and Biotechnology, celebrado en Stellenbosch (Sudáfrica), del 31 de octubre al 5 de noviembre de 2021Tempranillo is the third most widely grown red wine cultivar worldwide and the most relevant in the Iberian Peninsula. Here we produced a diploid genome assembly of Tempranillo to study the origin of clonal improvement in this cultivar. After PacBio and Oxford Nanopore sequencing, a trio binning approach produced chromosome-arm level and complete assemblies for the two haplo-phases. Based on the de novo assembly, we searched for genetic variation in ten re-sequenced Tempranillo clones selected for variation in agronomically relevant traits including fruit colour, low fruit sugar accumulation adaptable to warmer climates or loose bunches that are less prone to fungal diseases. Thousands of somatic mutations were detected in the ten clones, which, combined with gene annotations based on PacBio Iso-seq, identified candidate dominant missense variants related with selected phenotypes. We could also phase inter-chromosomal structural variation (SV) in the origin of white gape and loose cluster phenotypes. Our results provide a quality genome assembly for inter- and intra-cultivar comparisons and release candidate missense and SV mutations that could be responsible for clonal diversification and improvement

Identificación de variación clonal funcional en Tempranillo y Garnacha mediante el uso de ensamblajes de genomas de referencia varietales

Trabajo presentado en las IV Jornadas del Grupo de Viticultura de la SECH (Sociedad Española de Ciencias Hortícolas), celebrada en Pamplona (España), del 26 al 28 de octubre de 2022Las variedades de vid (Vitis vinifera L.) se caracterizan por su alta heterocigosidad (variación entre sus copias genómicas de herencia materna y paterna), lo que obliga a la propagación vegetativa como única vía posible para reproducir su genotipo y sus características varietales. Sin embargo, la propagación vegetativa prolongada da lugar a la acumulación de mutaciones somáticas que en ocasiones pueden producir fenotipos de interés que son la base para la mejora mediante selección clonal. En este trabajo hemos producido ensamblajes de los genomas de Tempranillo y Garnacha. Usando los ensamblajes como referencia, pudimos identificar variación genómica responsable de caracteres de interés en clones o variantes somáticas seleccionadas de cada variedad. En Tempranillo se identificó una deleción de gran tamaño en un clon con color de baya más oscuro de lo normal, relacionada con una reducción en la acumulación de ceras en la cutícula de la baya. En Garnacha se identificaron dos patrones de deleción distintos que causan la pérdida de los genes MYB necesarios para la síntesis de antocianinas en el hollejo, que suponen dos orígenes independientes de la variedad Garnacha Blanca. Estos resultados muestran como el ensamblaje de genoma puede ayudar a conocer el origen de las características varietales y posibilitar la trazabilidad de variación genética clonal útil para la innovación intravarietal.Esta investigación ha sido financiada con fondos EU de la Marie Skłodowska-Curie Individual Fellowship (SomaGrapeGenome 79460) y de los proyectos del Ministerio de Economía (SOMAVID, BIO2014-59324) y del Ministerio de Ciencia e Innovación (DIGEVIDA, PID2020-120183RB-I00), así como con fondos de la ‘Max Planck Society