Exploration of alternative splicing events in ten different grapevine cultivars

Background: The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. Results: Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44 % of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. Conclusions: Our analysis shows that similar to 70 % of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars

Regulation of flavonol content and composition in (Syrah×Pinot Noir) mature grapes: integration of transcriptional profiling and metabolic quantitative trait locus analyses

8openInternationalFlavonols are a ubiquitous class of flavonoids that accumulate preferentially in flowers and mature berries. Besides their photo-protective function, they play a fundamental role during winemaking, stabilizing the colour by co-pigmentation with anthocyanins and contributing to organoleptic characteristics. Although the general flavonol pathway has been genetically and biochemically elucidated, the genetic control of flavonol content and composition at harvest is still not clear. To this purpose, the grapes of 170 segregating F1 individuals from a ‘Syrah’×’Pinot Noir’ population were evaluated at the mature stage for the content of six flavonol aglycons in four seasons. Metabolic data in combination with genetic data enabled the identification of 16 mQTLs (metabolic quantitative trait loci). For the first time, major genetic control by the linkage group 2 (LG 2)/MYBA region on flavonol variation, in particular of tri-hydroxylated flavonols, is demonstrated. Moreover, seven regions specifically associated with the fine control of flavonol biosynthesis are identified. Gene expression profiling of two groups of individuals significantly divergent for their skin flavonol content identified a large set of differentially modulated transcripts. Among these, the transcripts coding for MYB and bZIP transcription factors, methyltranferases, and glucosyltranferases specific for flavonols, proteins, and factors belonging to the UV-B signalling pathway and co-localizing with the QTL regions are proposed as candidate genes for the fine regulation of flavonol content and composition in mature grapesopenMalacarne, G.; Costantini, L.; Coller, E.; Battilana, J.; Velasco, R.; Vrhovsek, U.; Grando, M.S.; Moser, C.Malacarne, G.; Costantini, L.; Coller, E.; Battilana, J.; Velasco, R.; Vrhovsek, U.; Grando, M.S.; Moser, C

Analysis of the PC algorithm as a tool for the inference of gene regulatory networks: evaluation of the performance, modification and application to selected case studies.

The expansion of a Gene Regulatory Network (GRN) by finding additional causally-related genes, is of great importance for our knowledge of biological systems and therefore relevant for its biomedical and biotechnological applications. Aim of the thesis work is the development and evaluation of a bioinformatic method for GRN expansion. The method, named PC-IM, is based on the PC algorithm that discovers causal relationships starting from purely observational data. PC-IM adopts an iterative approach that overcomes the limitations of previous applications of PC to GRN discovery. PC-IM takes in input the prior knowledge of a GRN (represented by nodes and re- lationships) and gene expression data. The output is a list of genes which expands the known GRN. Each gene in the list is ranked depending on the frequency it appears causally relevant, normalized to the number of times it was possible to find it. Since each frequency value is associated with precision and sensitivity values calculated using the prior knowledge of the GRN, the method provides in output those genes that are above the value of frequency that optimize precision and sensitivity (cut-off frequency). In order to investigate the characteristics and the performances of PC-IM, in this thesis work several parameters have been evaluated such as the influence of the type and size of input gene expression data, of the number of iterations and of the type of GRN. A comparative analysis of PC-IM versus another recent expansion method (GENIES) has been also performed. Finally, PC-IM has been applied to expand two real GRNs of the model plant Arabidopsis thaliana

Soil Communities: Who Responds and How Quickly to a Change in Agricultural System?

The use of conservation and sustainable practices could restore the abundance and richness of soil organisms in agroecosystems. Fitting in this context, this study aimed to highlight whether and how different soil living communities reacted to the conversion from an integrated to an organic orchard. The metataxonomic approach for fungi and bacteria and the determination of biological forms of diatoms and microarthropods were applied. Soil analyses were carried out in order to evaluate the effect of soil chemical features on four major soil living communities. Our results showed that the different taxa reacted with different speeds to the management changes. Fungi responded quickly to the changes, suggesting that modification in agricultural practices had a greater impact on fungal communities. Bacteria and microarthropods were more affected by abiotic parameters and less by the management. The diatom composition seemed to be affected by seasonality but the highest H’ (Shannon index) value was measured in the organic system. Fungi, but also diatoms, seemed to be promising for monitoring changes in the soil since they were sensitive to both the soil features and the anthropic impact. Our study showed that soil biodiversity could be affected by the conversion to sustainable management practices from the early years of an orchard onwards. Therefore, better ecological orchard management may strengthen soil sustainability and resilience in historically agricultural regions

Regulation of flavonol content and composition in (Syrah×Pinot Noir) mature grapes: integration of transcriptional profiling and metabolic quantitative trait locus analyses

Flavonols are a ubiquitous class of flavonoids that accumulate preferentially in flowers and mature berries. Besides their photo-protective function, they play a fundamental role during winemaking, stabilizing the colour by co-pigmentation with anthocyanins and contributing to organoleptic characteristics. Although the general flavonol pathway has been genetically and biochemically elucidated, the genetic control of flavonol content and composition at harvest is still not clear. To this purpose, the grapes of 170 segregating F1 individuals from a ‘Syrah’×’Pinot Noir’ population were evaluated at the mature stage for the content of six flavonol aglycons in four seasons. Metabolic data in combination with genetic data enabled the identification of 16 mQTLs (metabolic quantitative trait loci). For the first time, major genetic control by the linkage group 2 (LG 2)/MYBA region on flavonol variation, in particular of tri-hydroxylated flavonols, is demonstrated. Moreover, seven regions specifically associated with the fine control of flavonol biosynthesis are identified. Gene expression profiling of two groups of individuals significantly divergent for their skin flavonol content identified a large set of differentially modulated transcripts. Among these, the transcripts coding for MYB and bZIP transcription factors, methyltranferases, and glucosyltranferases specific for flavonols, proteins, and factors belonging to the UV-B signalling pathway and co-localizing with the QTL regions are proposed as candidate genes for the fine regulation of flavonol content and composition in mature grape

Additional file 2: of Exploration of alternative splicing events in ten different grapevine cultivars

Table with all AS predicted by FindAS software. First row (beginning with '#') hold the column labels. (ZIP 1893 kb

Induction of terpene biosynthesis in berries of microvine transformed with VvDXS1 alleles

Terpenoids, especially monoterpenes, are major aroma-impact compounds in grape and wine. Previous studies highlighted a key regulatory role for grapevine 1-deoxy-D-xylulose 5-phosphate synthase 1 (VvDXS1), the first enzyme of the methylerythritol phosphate pathway for isoprenoid precursor biosynthesis. Here, the parallel analysis of VvDXS1 genotype and terpene concentration in a germplasm collection demonstrated that VvDXS1 sequence has a very high predictive value for the accumulation of monoterpenes and also has an influence on sesquiterpene levels. A metabolic engineering approach was applied by expressing distinct VvDXS1 alleles in the grapevine model system “microvine” and assessing the effects on downstream pathways at transcriptional and metabolic level in different organs and fruit developmental stages. The underlying goal was to investigate two potential perturbation mechanisms, the former based on a significant over-expression of the wild-type (neutral) VvDXS1 allele and the latter on the ex-novo expression of an enzyme with increased catalytic efficiency from the mutated (muscat) VvDXS1 allele. The integration of the two VvDXS1 alleles in distinct microvine lines was found to alter the expression of several terpenoid biosynthetic genes, as assayed through an ad hoc developed TaqMan array based on cDNA libraries of four aromatic cultivars. In particular, enhanced transcription of monoterpene, sesquiterpene and carotenoid pathway genes was observed. The accumulation of monoterpenes in ripe berries was higher in the transformed microvines compared to control plants. This effect is predominantly attributed to the improved activity of the VvDXS1 enzyme coded by the muscat allele, whereas the up-regulation of VvDXS1 plays a secondary role in the increase of monoterpene