A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis

<p>Abstract</p> <p>Background</p> <p><it>Arabidopsis thaliana </it>is the main model species for plant molecular genetics studies and world-wide efforts are devoted to identify the function of all its genes. To this end, reverse genetics by TILLING (Targeting Induced Local Lesions IN Genomes) in a permanent collection of chemically induced mutants is providing a unique resource in Columbia genetic background. In this work, we aim to extend TILLING resources available in <it>A. thaliana </it>by developing a new population of ethyl methanesulphonate (EMS) induced mutants in the second commonest reference strain. In addition, we pursue to saturate the number of EMS induced mutations that can be tolerated by viable and fertile plants.</p> <p>Results</p> <p>By mutagenizing with different EMS concentrations we have developed a permanent collection of 3712 M₂/M₃independent mutant lines in the reference strain Landsberg <it>erecta </it>(L<it>er</it>) of <it>A. thaliana</it>. This population has been named as the Arabidopsis TILL<it>er </it>collection. The frequency of mutations per line was maximized by using M₁plants with low but sufficient seed fertility. Application of TILLING to search for mutants in 14 genes identified 21 to 46 mutations per gene, which correspond to a total of 450 mutations. Missense mutations were found for all genes while truncations were selected for all except one. We estimated that, on average, these lines carry one mutation every 89 kb, L<it>er </it>population providing a total of more than five million induced mutations. It is estimated that TILL<it>er </it>collection shows a two to three fold higher EMS mutation density per individual than previously reported <it>A. thaliana </it>population.</p> <p>Conclusions</p> <p>Analysis of TILL<it>er </it>collection demonstrates its usefulness for large scale TILLING reverse genetics in another reference genetic background of <it>A. thaliana</it>. Comparisons with TILLING populations in other organisms indicate that this new <it>A. thaliana </it>collection carries the highest chemically induced mutation density per individual known in diploid species.</p

Morphological and Molecular Characterization of Grapevine Accessions Known as Albillo

Twenty-eight accessions identified under the name of Albillo or cited as a synonym of this ancient Spanish grapevine variety were characterized using 43 morphological descriptors, four different isozyme systems (CO, ACPH, GOT, and SOD) and two Amplified Fragment Length Polymorphism (AFLP) primer combinations. Genetic similarity data resulting from these analyses allowed three different groups of Albillo to be distinguished that corresponded with geographical and historical origin of representative varieties: Turruntés, Malvasía de El Bierzo, and the classical Albillo de Madrid, related to the oldest Albillo ampelographical descriptions. In addition, homonyms as well as varieties incorrectly cited as Albillo synonyms were identified. To ensure accurate variety identification, association among historical descriptions based on morphological descriptors and molecular fingerprints was required. Isozymes proved to be useful markers for varietal identification. However, only AFLP allowed distinction among closely related varieties as well as the analysis of intravarietal variation.M.T.C. was funded by a Ministerio de Educación y Cultura contract; I.R., J.A.C., and J.C. were funded by predoctoral fellowships from the European Union, Instituto Nacional de Investigaciones Agrarias y Agroalimentarias, and Agencia Española de Cooperación Internacional, respectively. This research was funded in part by projects INIA SC96-010, SC94-092, and CM 07B-0010-1997. Support for research activity at the Centro Nacional de Biotecnología is provided by a CSIC-INIA specific agreement.Peer reviewe

Genetic Origins of Cultivated and Wild Grapevines from Morocco

Nuclear and chloroplast microsatellite loci were used to genotype Moroccan grapevine accessions (Vitis vinifera L.) from germplasm collections and from plants cultivated in farm fields or found under wild conditions. The genetic diversity of the global Moroccan sample was equivalent to what has been described for cultivated grapevines from other regions around the Mediterranean basin. The predominant chlorotypes among the analyzed samples were A and C, which are detected at higher frequencies in western and eastern regions of the V. vinifera range. Comparison of Moroccan accession genotypes with published genotypes for other grapevine cultivars shows the existence of multiple synonyms, especially between cultivars grown in Morocco and Spain, as expected given the long history of interaction in the region. Analysis of genetic relationships among Moroccan samples distinguished groups of cultivated and wild samples. The wild samples had chlorotype A, which could represent remnants of natural populations of V. vinifera ssp. sylvestris (Gmelin) Hegi, or chlorotype C, which could represent cultivar derivatives or ancient cultivars generated by spontaneous hybridization among cultivated grapevines. Together the results point out the diverse genetic origin of cultivated and wild samples of grapevine in Morocco and emphasize the need to further collect and characterize its cultivated and wild grapevine germplasm.Peer reviewe

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

Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures

<p>Abstract</p> <p>Background</p> <p>Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the <it>trans</it>-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in <it>Vitis vinifera </it>cv Monastrell albino cell suspension cultures.</p> <p>Findings</p> <p>MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes.</p> <p>Conclusion</p> <p>The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway.</p

Pedigree analysis of the Spanish grapevine cultivar Heben

Trabajo presentado en la Final Conference Progress in Vitis Vinifera diversity evaluation and use, celebrada en Lisboa del 7 al 9 de octubre de 2014.Cost action FA1003 - GRAPENET. East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding.Several studies have shown that some varieties have had a large contribution in the production of varieties in certain regions, like ‘Pinot’ and ‘Heunisch Weiss’ in France. Previous results had pointed out that the old Spanish female variety ‘Hebén’, known as ‘Mourisco Branco’ in Portugal, could be one of those varieties with a remarkable role in the Iberian Peninsula. This ancient winegrape cultivar was already described in Spain in the 16th century as a white variety of grapevine producing large and sparse bunches of grape with big seeds.Peer Reviewe

Pedigree analysis of the Spanish grapevine cultivar 'Hebén'

The use of Single Nucleotide Polymorphism (SNP) markers allows genetic identification and parentage analysis of grapevine cultivars. Ancient cultivars like 'Hebén' were widely cultivated in the Iberian Peninsula along its history and likely contributed to the origin of varieties which are still cultivated today. The goal of our study was to search for possible first-degree relationships between the female variety 'Hebén' and other Iberian and Mediterranean cultivars. For this purpose, we used a set of 261 SNP markers which allowed identifying 23 trios and 37 parent-offspring relationships supported by high overall likelihood ratios (LOD scores). The results shed light on the relationships among several cultivars from Spain and Portugal, and demonstrated that 'Hebén' is a major founder of current varieties in the Iberian Peninsula viticulture, and contributed to spread the chlorotype A in this region.L. H. ZINELABIDINE was supported by a fellowship from the Agencia Española de Cooperación Internacional. J. CUNHA was supported by a Post-doctoral fellowship “Fundação para a Ciência e Tecnologia” (SFRH/BPD/ 74895/2010). LHZ and JC were supported by COST action FA1003 “East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding”, from European Cooperation in the Field of Scientific and Technical Research, through the funding of short scientific missions at the ICVV.Peer Reviewe

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

Grapevine cultivar "Alfrocheiro" or "Bruñal" plays a primary role in the relationship among iberian grapevines

The grapevine cultivar known in Portugal as 'Alfrocheiro', and in Spain as 'Bastardo Negro', 'Bruñal' or 'Baboso Negro', plays a central role in the genetic network of the Iberian Peninsula grapevine cultivars. Three sets of different molecular markers, SNPs, nSSRs and cpSSRs, revealed more than twenty parent-offspring links with this cultivar. 'Alfrocheiro' chlorotype is definitely Western European but their parents are still unknown. The distribution of the cultivar, their offspring as well as the two main co-parents direct to a geographic origin around the Portuguese and Spanish border. This cultivar and their progenies represent about 15 % of the total grapevine acreage in Portugal, stressing the importance of 'Alfrocheiro' in the Portuguese wine character.This research was supported by: IVV – Instituto da Vinha e do Vinho (Ministério da Agricultura e do Mar-MAM); Programa de Desenvolvimento Rural (PRODER - Ação 2.2.3.1. - PA 18621 - MAM) and J. CUNHA was supported by a Post-doctoral fellowship >Fundação para a Ciência e Tecnologia> (SFRH/BPD/74895/2010) and a short scientific mission in the ICVV was part of COST action, FA 1003 from European Cooperation in the Field of Scientific and Technical Research. L. H. ZINELABIDINE was supported by a fellowship from the Agencia Española de Cooperación Internacional and a short-term scientific mission from COST FA1003 >East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding>.Peer Reviewe