Ethylene is involved in pistil fate by modulating the onset of ovule senescence and the GA-mediated fruit set in Arabidopsis

Background: Ovule lifespan is an important factor in determining the ability to set fruits and produce seeds. Once ovule senescence is established, fruit set capacity in response to gibberellins (GAs) is lost. We aimed to elucidate whether ethylene plays a role in controlling ovule senescence and the fruit set response in Arabidopsis. Results: Ethylene response inhibitors, silver thiosulphate (STS) and 1-methylcyclopropene (1-MCP), were able to delay the loss of pistil response to GA3 . In addition, ethylene insensitive mutants ein2-5 and ein3-1 showed delayed loss of pistil response, as in plants treated with STS and 1-MCP, while constitutive mutant ctr1-1 displayed premature loss of response. The analysis of the expression of ethylene biosynthesis genes suggests that ethylene is synthesised in ovules at the onset of ovule senescence, while a transcriptional meta-analysis also supports an activated ethylene-dependent senescence upon the establishment of ovule senescence. Finally, a SAG12:GUS reporter line proved useful to monitor ovule senescence and to directly demonstrate that ethylene specifically modulates ovule senescence. Conclusions: We have shown that ethylene is involved in both the control of the ovule lifespan and the determination of the pistil/fruit fate. Our data support a role of the ovule in modulating the GA response during fruit set in Arabidopsis. A possible mechanism that links the ethylene modulation of the ovule senescence and the GA3 -induced fruit set response is discussed.The authors wish to thank Drs. Alonso and Amasino for their gifts of seeds; Drs. Alonso, Alabadi, and Blazquez for critically reading the manuscript, and Ms. Argomaniz and Ms. Fuster for technical assistance in the lab. This work has been supported by grants BIO2005-07156-C02-01 and BIO2008-01039 from the Spanish Ministry of Science and Innovation, Plan Nacional de I+D. PCB received a PhD fellowship from the Spanish Ministry of Science and Innovation.Carbonell Bejerano, P.; Urbez Lagunas, C.; Granell Richart, A.; Carbonell Gisbert, J.; Perez Amador, MA. (2011). Ethylene is involved in pistil fate by modulating the onset of ovule senescence and the GA-mediated fruit set in Arabidopsis. BMC Plant Biology. 11:84-84. https://doi.org/10.1186/1471-2229-11-84S84841

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

Solar ultraviolet radiation is necessary to enhance grapevine fruit ripening transcriptional and phenolic responses

Background: Ultraviolet (UV) radiation modulates secondary metabolism in the skin of Vitis vinifera L. berries, which affects the final composition of both grapes and wines. The expression of several phenylpropanoid biosynthesis-related genes is regulated by UV radiation in grape berries. However, the complete portion of transcriptome and ripening processes influenced by solar UV radiation in grapes remains unknown.Results: Whole genome arrays were used to identify the berry skin transcriptome modulated by the UV radiation received naturally in a mid-altitude Tempranillo vineyard. UV radiation-blocking and transmitting filters were used to generate the experimental conditions. The expression of 121 genes was significantly altered by solar UV radiation. Functional enrichment analysis of altered transcripts mainly pointed out that secondary metabolism-related transcripts were induced by UV radiation including VvFLS1, VvGT5 and VvGT6 flavonol biosynthetic genes and monoterpenoid biosynthetic genes. Berry skin phenolic composition was also analysed to search for correlation with gene expression changes and UV-increased flavonols accumulation was the most evident impact. Among regulatory genes, novel UV radiation-responsive transcription factors including VvMYB24 and three bHLH, together with known grapevine UV-responsive genes such as VvMYBF1, were identified. A transcriptomic meta-analysis revealed that genes up-regulated by UV radiation in the berry skin were also enriched in homologs of Arabidopsis UVR8 UV-B photoreceptor-dependent UV-B -responsive genes. Indeed, a search of the grapevine reference genomic sequence identified UV-B signalling pathway homologs and among them, VvHY5-1, VvHY5-2 and VvRUP were up-regulated by UV radiation in the berry skin.Conclusions: Results suggest that the UV-B radiation-specific signalling pathway is activated in the skin of grapes grown at mid-altitudes. The biosynthesis and accumulation of secondary metabolites, which are appreciated in winemaking and potentially confer cross-tolerance, were almost specifically triggered. This draws attention to viticultural practices that increase solar UV radiation on vineyards as they may improve grape features. © 2014 Carbonell-Bejerano et al.; licensee BioMed Central Ltd.ENO and JMA are grateful to the Ministerio de Economía y Competitividad of Spain and the Fondo Europeo de Desarrollo Regional (FEDER) for financial support (Project CGL2011-26937). This study was funded in part by Project BIO2011-026229 from the Spanish MINECO. Microarray hybridizations were carried out at the Genomics Unit of the National Centre for Biotechnology, CNB-CSIC, Madrid, Spain. The present work is integrated in the COST (European Cooperation in Science and Technology) Action FA0906 of the European Union “UV-B radiation: a specific regulator of plant growth and food quality in a changing climate” as well as COST Action FA1106 “Quality fruit”.Peer Reviewe

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

Analysis of the origin of parthenocarpy in grapevine cultivar Corinto Bianco

Póster presentado en la 2nd Annual ConferenceFleshy Fruit Development & Ripening (COST Action FA1106 QualityFruit), celebrada en Chania (Grecia) del 22 al 25 de septiembre de 2013.Seedless fruits appear spontaneously in grapevine (Vitis vinifera L.) as a result of somatic variation. Stenospermocarpic and parthenocarpic seedlesness are known. The first type is widely used in table grapes production because seed development aborts after fertilization giving rise to seed traces and almost normal size berries. In contrast, small berries without seed traces develop in absence of fertilization in parthenocarpic cultivars that are appreciated for raisin production. The cultivar Corinto Bianco is a parthenocarpic somatic variant of the Spanish seeded cultivar Pedro Ximénez. Morphological and molecular comparison of flower development and gametogenesis between both genotypes were directed to understand the genetic and molecular basis of this parthenocarpic phenotype. Histological analyses showed that ovules developed similarly in both genotypes. However, macrogametogenesis was altered in Corinto Bianco showing disorders during mitosis after mother cell meiotic reduction. Microgametogenesis was also altered in Corinto Bianco, which pollen was 100% sterile in 2012 and 2013, compared with Pedro Ximénez pollen that showed only 8.47 and 21.64% of sterility in those years. The average number of seeds per berry was 1.35 in Pedro Ximénez berries, which weight was six times higher than in Corinto Bianco parthenocarpic berries with zero seeds. In addition, we observed partial phenotypic reversion in 2.6% of Corinto Bianco berries that carried one seed and displayed a comparable size to that of Pedro Ximénez berries. These seeds were unable to germinate in soil under normal conditions. Fortunately, following an in vitro germination protocol we were able to rescue 41 seedlings derived from revertant berries for further analyses. Gene expression alterations between Pedro Ximénez and Corinto Bianco, that could lead to the identification of candidate genes responsible for the phenotypic change, were analyzed by comparing closed flowers of both genotypes at 50% bloom time using the NimbleGen Vitis HX12 microarray. We identified 441 genes upregulated and 949 downregulated in the parthenocarpic mutant (¿2-fold change and 5% FDR). Interestingly, genes related with cell cycle and gametogenesis were downregulated in Corinto Bianco, including a cyclin (CYB1;2- like) and a MADS-box gene (AGL66-like), whose function in Arabidopsis is related with mitosis and pollen development, respectively. The expression of these genes could be related with defects in gametogenesis in Corinto Bianco. These preliminary results suggest the presence of defects in the meiotic mechanisms central to the process of gametogenesis in Corinto Bianco, providing clues for further characterization of the origin of parthenocarpy in this cultivar.Peer Reviewe

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

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

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

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

Caracterización genética y de caracteres reproductivos en variedades de vid sin semilla de Armenia

Trabajo presentado en las IV Jornadas del Grupo de Viticultura de la SECH (Sociedad Española de Ciencias Hortícolas), celebrada en Pamplona (España), los días 26 y 28 de octubre de 2022La apirenia de la mayoría de las variedades comerciales de la vid (Vitis vinifera L.) procede de 'Sultanina', una variedad con origen en Asia Menor. El principal objetivo de este trabajo ha sido la caracterización de posibles fuentes alternativas de apirenia en el germoplasma armenio. Se han estudiado 40 accesiones apirenas de las colecciones armenias de vid en Echmiadzin (ARM006) y en Nalbandyan (ARM011), así como de explotaciones privadas de la región de Armavir (Armenia). El análisis fenotípico de bayas se realizó de acuerdo con los descriptores de la OIV, y el análisis genético mediante el estudio de la mutación causal de apirenia en Sultanina en el gen VviAGL11 y del marcador VviAPT3 ligado al locus del sexo. El análisis de viabilidad y morfología de los granos de polen se visualizó por microscopía óptica y electrónica de barrido. El análisis fenotípico de bayas reveló una amplia variación en el peso de las mismas, así como en la formación de rudimentos seminales. Las flores de nueve cultivares son hermafroditas con un alto nivel de viabilidad del polen. La accesión 'Karmir kishmish' se caracterizó por tener flores funcionalmente femeninas con baja viabilidad de polen y se confirmó genéticamente con VviAPT3. El análisis de microscopía mostró que los granos de polen de las flores hermafroditas tienen forma esferoidal con 3 colporaciones y numerosas perforaciones, mientras que el de la variedad 'Karmir kishmish' es también esferoidal, pero acolporado y con menos perforaciones. El análisis genético reveló que todas las accesiones portan la mutación puntual dominante en VviAGL11 que causa la estenospermocarpia en 'Sultanina'. De hecho, el análisis de 7 marcadores SSR y 48 SNPs demostró que todas son descendientes de la misma. Este estudio confirma que las variedades apirenas armenias descienden de 'Sultanina', y motiva la búsqueda de otros determinantes genéticos que causen variación en el contenido de semillas de las uvas para utilizar como fuentes alternativas en programas de mejora de uva de mesa