Plant response to environmental stresses and pathogen attacks involves several biologicalprocesses that require fine and precise regulation at transcriptional and post-transcriptional levels.MicroRNAs (miRNAs) are small RNAs (sRNAs) widely diffused in animals and plants andimplicated in post-transcriptional regulation of gene transcripts. Identification of differentiallyexpressed miRNAs, following infection, may provide an insight into the processes involved insignalling and defence of plants against pathogens.In order to characterize artichoke miRNAs differentially expressed during fungus or virusinfection, we sequenced five sRNA libraries obtained from artichoke using Illumina technology.Libraries were obtained from leaves and roots of control plants, and from plants infected withTomato spotted wilt virus (TSWV), or Verticillium dahliae.After removing low quality reads and adapter sequences, all artichoke libraries wereannotated according to small noncoding RNAs contained in Rfam, and all previously known plantmiRNAs extracted from the miRNA Registry Database (miRBase Release 18).Change in miRNA read counts between infected and non infected artichoke tissues wasrecorded and used to select putative infection-responsive miRNAs. Differential expression ofmiRNAs was validated by quantitative real-time PCR (qPCR).Artichoke miRNA precursors were identified and their fold-back structure was predictedusing Mfold program. Artichoke miRNA target genes were also identified and characterizedaccording to the homologous Arabidopsis proteins.In conclusion, miRNAs involved in the response to virus or fungus infection were detectedand validated in artichoke plant tissues.

Powdery mildew (PM) is a major fungal disease of thousands of plant species, including many cultivated Rosaceae. PM pathogenesis is associated with up-regulation of MLO genes during early stages of infection, causing down-regulation of plant defense pathways. Specific members of the MLO gene family act as PM-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance.Results: We carried out a genome-wide characterization of the MLO gene family in apple, peach and strawberry, and we isolated apricot MLO homologs through a PCR-approach. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Homologs that are candidates for being PM susceptibility genes were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen Podosphaera leucotricha.Conclusions: Genomic tools available for Rosaceae were exploited in order to characterize the MLO gene family. Candidate MLO susceptibility genes were identified. In follow-up studies it can be investigated whether silencing or a loss-of-function mutations in one or more of these candidate genes leads to PM resistance.

MicroRNAs (miRNAs) are small noncoding endogenous RNAs playing a regulatory role bynegatively affecting gene expression at the post-transcriptional level. miRNAs vary in lengthbetween 16 and 35 nucleotides with a mode of 22-nt, and are produced from longer hairpin-likeprecursor transcripts. Functional studies have demonstrated that miRNAs are involved in variousdevelopmental and physiological processes, and recent reports indicate their association with bioticand abiotic stress responses in plants.High-throughput sequencing technology has allowed the identification and profiling ofseveral conserved and non-conserved miRNAs. Additionally, deep sequencing provides quantitativeexpression information, since frequency of an individual miRNA generally reflects its relativeabundance in the sample. This strategy has been successfully applied to both model and non-modelplants.In the present study, small RNA (sRNA) libraries were generated separately from roots andleaves of globe artichoke, obtained from plants subjected or not to saline treatment. Libraries weresequenced using Illumina technology, and results were analysed by bioinformatics tools.The majority of sRNAs were 18-28 nt long with 21 nt and 24 nt sRNA as major peaks in thelength distribution graph. To identify conserved miRNAs in globe artichoke, all sRNA sequenceswere Blastn searched against the currently known miRNAs contained in miRbase(http://www.mirbase.org/). Only perfectly matched sequences were considered as putativeconserved miRNAs: this analysis highlighted the presence of at least 29 different miRNA familiesin artichoke.Comparison to artichoke EST database was performed to find potential miRNA targets andprecursors, and to predict hairpin structure. Subsequently, miRNAs were validated using a PCRapproach.Differential expression of miRNA between tissues (leaves and roots) and untreated and salttreatedsamples was analysed after sample normalization (count per million).In conclusion we have characterized the sRNA transcriptome in artichoke, in different tissues,in the presence or absence of salt stress, using deep sequencing strategy by Illumina platform.

Diet in human health is no longer simple nutrition but, in the light of recent findings, it might play a pivotal role on cell health status by modulating apoptosis, detoxification, and appropriate gene response to environmental stresses. Epidemiological studies suggest a role of fruits and vegetables in protection against several diseases, and nutrients have been demonstrated to alter gene expression by DNA methylation and histone modifications [1-2]. Diet has also been found to modulate micro RNA (miRNA) expression, leading to a subsequent regulation of the effectors genes [3]. Furthermore, recent studies demonstrate that some plant/food-derived microRNAs (miRNAs) regulate gene expression in a sequence specific manner [4]. On the basis of all these findings, we have carried out a pilot study, using a combined "in-silico and wet" approach, to investigate the potential effects, and elucidate the molecular mechanisms, of edible plant miRNAs on the expression of human genes involved in cancer onset and progression. In the present paper we report the results obtained by transfecting 2 colon cancer cell lines, p53 wild type and p53 knock-out, with selected miRNAs of G. max, Z. mais and M. truncatula, which we found, by in silico analysis, to have a putative targeting activity on human oncogenes and tumor suppressor genes.

RNA silencing (RS) is a conserved eukaryotic mechanism acting as anti-viral immune systemin plants. Small interfering RNAs (siRNAs) are RS effectors accumulating in virus-infected tissuesas immune system components, providing target specificity for post-transcriptional degradation ofinvading RNAs. Virus-derived siRNAs (vsiRNAs, 21-24 nt) are abundant in infected plants, withcertain regions ("hot spots") more represented than others.Two isolates of Potato virus Y (PVY), PVYC-to and PVY-SON41, yield very differentdisease phenotypes on tomato (Solanum lycopersicum), the former inducing leaf distortions, thelatter mild symptoms. We applied in silico and molecular approaches to identify PVY vsiRNAssuppressing host mRNAs by sequence complementarity and RS-based suppression, inducingdysfunctional processes upon infection. Aim was to explore differential expression of host-targetingvsiRNA, as responsible of isolates differences.A computational pipeline retrieved 21nt vsiRNAs from PVY isolates, complementary topredicted mRNAs (Solgenomics, ITAG2.3). Two PVYC-to genome regions showed specificsecondary structures absent in PVY-SON41, accounting for vsiRNAs accumulation. Two tomatotranscript lists were obtained, perfectly or imperfectly (one or two mismatches) complementary tovsiRNA computed for either PVY isolates, putative targets of vsiRNA-driven RS suppression.Targets common to both isolates were discarded, leaving a gene list representing only PVYC-totargets. Quantitative RT-PCR of transcription factors (e.g. NAC, MYB, TCP, HD-ZIP, MADS-boxfamilies) active in leaf development and morphogenesis showed differential expression uponPVYC-to and PVY-SON41 infections. Lower isolate-specific mRNA accumulation suggested RSdrivenpost-transcriptional regulation, confirming a link between symptoms and vsiRNAs-mediatedRS. Results from vsiRNAs deep sequencing from infected leaves are also discussed.

In the scientific biodiversity community, it is increasingly perceived the need to build a bridge between molecular and traditional biodiversity studies. We believe that the information technology could have a preeminent role in integrating the information generated by these studies with the large amount of molecular data we can find in bioinformatics public databases. This work is primarily aimed at building a bioinformatic infrastructure for the integration of public and private biodiversity data through the development of GIDL, an Intelligent Data Loader coupled with the Molecular Biodiversity Database. The system presented here organizes in an ontological way and locally stores the sequence and annotation data contained in the GenBank primary database.MethodsThe GIDL architecture consists of a relational database and of an intelligent data loader software. The relational database schema is designed to manage biodiversity information (Molecular Biodiversity Database) and it is organized in four areas: MolecularData, Experiment, Collection and Taxonomy. The MolecularData area is inspired to an established standard in Generic Model Organism Databases, the Chado relational schema. The peculiarity of Chado, and also its strength, is the adoption of an ontological schema which makes use of the Sequence Ontology.The Intelligent Data Loader (IDL) component of GIDL is an Extract, Transform and Load software able to parse data, to discover hidden information in the GenBank entries and to populate the Molecular Biodiversity Database. The IDL is composed by three main modules: the Parser, able to parse GenBank flat files; the Reasoner, which automatically builds CLIPS facts mapping the biological knowledge expressed by the Sequence Ontology; the DBFiller, which translates the CLIPS facts into ordered SQL statements used to populate the database. In GIDL Semantic Web technologies have been adopted due to their advantages in data representation, integration and processing.Results and conclusionsEntries coming from Virus (814,122), Plant (1,365,360) and Invertebrate (959,065) divisions of GenBank rel.180 have been loaded in the Molecular Biodiversity Database by GIDL. Our system, combining the Sequence Ontology and the Chado schema, allows a more powerful query expressiveness compared with the most commonly used sequence retrieval systems like Entrez or SRS.

Specific homologs of the plant Mildew Locus O (MLO) gene family act as susceptibility factors towards the powdery mildew (PM) fungal disease, causing significant economic losses in agricultural settings. Thus, in order to obtain PM resistant phenotypes, a general breeding strategy has been proposed, based on the selective inactivation of MLO susceptibility genes across cultivated species. In this study, PCR-based methodologies were used in order to isolate MLO genes from cultivated solanaceous crops that are hosts for PM fungi, namely eggplant, potato and tobacco, which were named SmMLO1, StMLO1 and NtMLO1, respectively. Based on phylogenetic analysis and sequence alignment, these genes were predicted to be orthologs of tomato SlMLO1 and pepper CaMLO2, previously shown to be required for PM pathogenesis. Full-length sequence of the tobacco homolog NtMLO1 was used for a heterologous transgenic complementation assay, resulting in its characterization as a PM susceptibility gene. The same assay showed that a single nucleotide change in a mutated NtMLO1 allele leads to complete gene loss-of-function. Results here presented, also including a complete overview of the tobacco and potato MLO gene families, are valuable to study MLO gene evolution in Solanaceae and for molecular breeding approaches aimed at introducing PM resistance using strategies of reverse genetics.

RNA silencing (RS) is a conserved mechanism in a broad range of eukaryotes. In plants, RSacts as an antiviral system and a successful virus infection requires suppression or evasion of theinduced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected withRNA and DNA viruses and provide specificity to this RNA mediated immune system.High-throughput sequencing has contributed to expanding our knowledge of siRNApopulations better describing their abundance, complexity and diversity in infected tissues. VirusderivedsiRNAs (vsiRNAs, 21-24 nt) from virus-infected plants are extraordinarily abundant anddiverse. However, certain regions of viral genomes ("hot spots") are usually more represented thanothers in sequenced vsiRNA populations.Potato virus Y (PVY) is an important pathogen of solanaceous crops belonging to the largestplant virus family, Potyviridae. The PVY genome is a single-stranded, positive-sense RNA of about10 kb. PVYC-to and PVY-SON41 are two PVY isolates that induce different disease symptoms ontomato (Solanum lycopersicum): while the former provokes severe leaf distortion, the latterproduces in the same host no visible phenotype.This study propose an innovative in silico approach, which consisted in mining genomicregions of PVY isolates and looking at possible PVY vsiRNAs putatively able, by sequencecomplementarity, to targeting and suppressing accumulation of host messenger RNA (mRNA) aspredicted by RS mechanisms, leading to dysfunctional biological processes that could explainisolate-specific disease phenotypes.A computational pipeline was implemented, which allowed the retrieval within the viralgenome of 21-nt vsiRNAs from PVYC-to and PVY-SON41 isolates complementary to tomatopredicted mRNA sequences (database Solgenomics, release ITAG2.3). The pipeline was based onfive bioinformatic algorithms and a relational database (DBMS MySQL) for management of theresults obtained in each steps. The comparative study for identifying the putative tomato targetgenes was performed with NCBI blast+ package 2.2 (option -task 'blastn-short' identity > 94%,max 2 mismatch or gap, alignment length > 19 bp). RandFold was employed for searching in thePVY genomes secondary structures containing putative vsiRNAs identified in previous steps. Withthis tool, five regions in the viral genome showing potential tRNA-like or microRNA-likesecondary structures were identified, that might account for as many "hot spots" of vsiRNAsaccumulation.

Motivations. RNA silencing, or post-transcriptional gene silencing (PTGS), is a conserved mechanism in a broad range of eukaryotes. In plants, PTGS acts as an antiviral system and a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with RNA and DNA viruses and provide specificity to this RNA-mediated immune system. High-throughput sequencing has contributed to expanding our previously knowledge of siRNA populations better describing their abundance, complexity and diversity in infected tissues. It is now known that siRNAs from virus-infected plants are extraordinarily abundant and diverse, and are widespread in near saturation at any region of either positive and negative genomic RNAs. However, certain regions of viral genomes ("hot spots") are usually more represented than others in sequenced siRNA populations. A gene involved in chlorophyll biosynthesis has been shown targeted by a siRNA derived from viral satellite RNA, revealing PTGS mechanism at basis of the symptom. Potato virus Y (PVY) is the type species of Potyvirus, a genus of agricultural importance belonging to the largest plant virus family, Potyviridae. The potyviral genome is a single-stranded, positive-sense RNA of ca. 10 kilobases (kb). PVYc-to and PVY-SON41 are two isolates of PVY that infect solanaceous hosts. While PVYc-to induces severe leaf distortion in different cultivars of tomato (Solanum lycopersicum), PVY-SON41 produces in the same host only a mild mosaic, followed by recovery. In order to elucidate the molecular mechanism underlying the different symptoms induced by PVYc-to and PVY-SON41 infections on tomato, we set up an in silico approach, mining genomic regions of PVY isolates and looking at possible RNA-based mechanisms where siRNA putatively generated from the PVY genome could target and suppress accumulation of host messenger RNA (mRNA), leading to dysfunctional biological processes that could explain different disease phenotypes.Methods. A Perl script was used to extract the complete datasets of 21-mers from isolates PVYc-to and PVY-SON41 complete genomes, leading the scanning of the complete sequence, shifted by one base at the time. MySQL was used for identification of the 21-mers shared and unique between the two viral genomes, highlighting sequence differences that could be at the base of diverse induced symptoms. The data obtained in the previous step were used to build the genomic map of identical regions, by fancyGene. A BLAST analysis was conducted with the identified 21-mer dataset, considering only the dissimilar sequences between the two isolates (blastn-short identity > 94%, max 2 mismatch or gap, alignment length 21 bp). The 21-mer were used as query and the tomato mRNA database (Solgenomics, release ITAG2.3) was used as target. The results were further used as input in a gene ontology analysis, through Blast2GO.Results. Despite the hig

MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower.ResultsTwo complete expressed sequence tags (ESTs) datasets from artichoke (3.6·104 entries) and safflower (4.2·104), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10-4) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity >= 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves.ConclusionsMature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with >= 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon.

MotivationThe family Asteraceae represents one of the largest evolutive radiation of flowering plants, including more than 1500 genera and 23000 species, and comprising economically important as well as ornamental crops (Jansen, 1987; Bremer, 2008). Among members of this dicotyledon family two edible species, Carthamus tinctorius L. and Cynara cardunculus var. scolymus L., also have a phytopharmaceutical interest. The former, known as safflower, is the only member of this genus widely cultivated for industrial oil, as a livestock feed or for use in traditional medicine (Han et al., 2009). The second species, the globe artichoke, apart of its importance as a food, is popular for its dietary and therapeutic potentials, especially for hepato-biliary dysfunctions and digestive complaints (Gebhardt, 1998; López-Molina, 2005). The discovery of microRNAs (miRNAs) in different genomes provided a new paradigm in evolution studies, due to their ancient origin and the important role played in development regulation, since the egress of multicellular organisms. This system is highly conserved in plant and animal cells. Plant miRNAs derive from long primary transcripts giving rise to mature 21-24nt RNAs products, fundamental in RNA-based gene regulation (Bartell, 2004). In plants, miRNAs control messengers degradation or restrain translation, affecting somatic development and the response to biotic and abiotic stresses (Jones-Rhoades, 2006). A feature of miRNAs is their imperfect but extensive complementarity to corresponding mRNA targets, thus making their computational prediction possible. This approach is useful when data mining is performed on the basis of miRNA:mRNA targets conservation, among different species, and was herein applied to the study of Carthamus and Cynara spp.MethodsA comparative approach was used to identify both evolutionary and functionally conserved miRNAs as well as their targets, by detection of common expressed sequence tags (ESTs) in Carthamus and Cynara spp. A bioinformatic pipeline was developed at this regard to analyze publicly available ESTs dataset and identify miRNA candidates in these two related species. Complete EST datasets from Carthamus and Cynara (36323 and 42011 sequences, respectively) were screened looking for putative miRNA targets by means of RNAhybrid (Rehmsmeier, et al., 2004) software and using the mirBase mature miRNA sequences from Arabidopsis as a reference set. Afterward the NCBI Blast algorithm (Altschul et al., 1990) embedded in a Perl script was used to identify the homologous region in Carthamus and Cynara EST datasets. The RNAhybrid results and the homologous region calculated, have been downloaded into a relational database (DBMS MySql) ad hoc developed. We generate the "mapping" of the miRNA target on the "EST homologous region" by means SQL queries. Finally, the signal-to-noise ratio and specificity were assessed using two approaches: in the first one, for each Arabidopsis mature miRNA, the

Specific members of the plant Mildew Locus O (MLO) protein family act as susceptibility factors towards powdery mildew (PM), a worldwide-spread fungal disease threatening many cultivated species. Previous studies indicated that monocot and dicot MLO susceptibility proteins are phylogenetically divergent. Methods: A bioinformatic approach was followed to study the type of evolution of Angiosperm MLO susceptibility proteins. Transgenic complementation tests were performed for functional analysis. Results: Our results show that monocot and dicot MLO susceptibility proteins evolved class-specific conservation patterns. Many of them appear to be the result of negative selection and thus are likely to provide an adaptive value. We also tested whether different molecular features between monocot and dicot MLO proteins are specifically required by PM fungal species to cause pathogenesis. To this aim, we transformed a tomato mutant impaired for the endogenous SlMLO1 gene, and therefore resistant to the tomato PM species Oidium neolycopersici, with heterologous MLO susceptibility genes from the monocot barley and the dicot pea. In both cases, we observed restoration of PM symptoms. Finally, through histological observations, we demonstrate that both monocot and dicot susceptibility alleles of the MLO genes predispose to penetration of a non-adapted PM fungal species in plant epidermal cells. Conclusions: With this study, we provide insights on the evolution and function of MLO genes involved in the interaction with PM fungi. With respect to breeding research, we show that transgenic complementation assays involving phylogenetically distant plant species can be used for the characterization of novel MLO susceptibility genes. Moreover, we provide an overview of MLO protein molecular features predicted to play a major role in PM susceptibility. These represent ideal targets for future approaches of reverse genetics, addressed to the selection of loss-of-function resistant mutants in cultivated species.

MotivationBiodiversity research concerns with data coming from many different domains (e.g., Biology, Geography, Evolutionary Studies, Genomics, Taxonomy, Environmental Sciences, etc.) which need to be integrated for leading to valuable Biodiversity knowledge. Collecting and integrating data from so many heterogeneous resources is not a trivial task. Data are extremely scattered, heterogeneous in format and purpose, and protected in repositories of several research institutes. Driven by the widely diffused trend of the web of sharing information through aggregation of people with the same interests (social networks), and by the new type of database architecture defined as dynamic distributed federated database, we are proposing a new paradigm of data integration in the Biodiversity domain. Here we present a new approach for the development of a Knowledge Base aiming to the collection, integration and analysis of biodiversity data implemented as a product of the MBLab project.MethodsThe implementation of the Biodiversity Knowledge Base is based on the integration of several components: a robust Database Management System (IBM DB2) managing the large volume of information from public databases like GenBank, a set of GaianDB nodes [1] to manage remote private collections of biodiversity data; the IBM Federator Server to implement the general conceptual schema integrating all biodiversity databases available across remote nodes of MBLab project partners.ResultsGaianDB is a Dynamic Distributed Federated Database of sources whose growth is regulated by biologically inspired principles and graph theoretic methods. By means of the GaianDB network architecture data remains on the remote research group servers, and each database owner is responsible for its integrity, availability and sharing. Each vertex of this network is a suitable entry point receiving the user query and responding with an output aggregating different pieces of information retrieved from the different data sources spanned all over the network. To integrate GenBank molecular data in the MBLabDB we built an efficient and reliable ETL (Extraction, Transformation and Load) module, implemented with CLIPS Rule Based Programming Language. The ETL extracts information from the feature- based GenBank entries and fits them in the MBLabDB schema. Molecular data collections are structured following a Chado-like model [2], using Sequence Ontology entities and relations. This allows to retrieve data using the biological concepts expressed by the Sequence Ontology [3]. The main result of this work is the development of a standard conceptual schema and a knowledge base architecture tailored to biodiversity data collection, integration and analysis. The database is modeled on six main sections: Taxonomic, Individual, Collection, Supply chain, Experimental molecular data. Currently two biodiversity data collections have been integrated by using GaianDB: the ITEM Collection [4] located at the I

The powdery mildew disease affects thousands of plant species and arguably represents the major fungal threat for many Cucurbitaceae crops, including melon (Cucumis melo L.), watermelon (Citrullus lanatus L.) and zucchini (Cucurbita pepo L.). Several studies revealed that specific members of the Mildew Locus O (MLO) gene family act as powdery mildew susceptibility factors. Indeed, their inactivation, as the result of gene knock-out or knock-down, is associated with a peculiar form of resistance, referred to as mlo resistance. Results: We exploited recently available genomic information to provide a comprehensive overview of the MLO gene family in Cucurbitaceae. We report the identification of 16 MLO homologs in C. melo, 14 in C. lanatus and 18 in C. pepo genomes. Bioinformatic treatment of data allowed phylogenetic inference and the prediction of several ortholog pairs and groups. Comparison with functionally characterized MLO genes and, in C. lanatus, gene expression analysis, resulted in the detection of candidate powdery mildew susceptibility factors. We identified a series of conserved amino acid residues and motifs that are likely to play a major role for the function of MLO proteins. Finally, we performed a codon-based evolutionary analysis indicating a general high level of purifying selection in the three Cucurbitaceae MLO gene families, and the occurrence of regions under diversifying selection in candidate susceptibility factors. Conclusions: Results of this study may help to address further biological questions concerning the evolution and function of MLO genes. Moreover, data reported here could be conveniently used by breeding research, aiming to select powdery mildew resistant cultivars in Cucurbitaceae.

Tetraploid wheat germplasm with A and B genomes is preserved at the seed-bank of the National Research Council - lnstitute of Plant Genetics (CNR-IGV, fomerly Germplasm Institute) since the early 1970's. Nowadays, the bank preserves over 5876 seed samples of this germplasm, which were directly collected by exploration teams of the Institute or acquired through exchange with Intemational lnstitutions. Germplasm safeguarding is carried out within the frame of the European Cooperative Programme for Plant Genetic Resource (ECP/GR). Moreover, the CNR-IGV has contributed all its wheat samples to the Multilateral System of the lnternational Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA). The management of tetraploid wheat genetic resources is one of the key activities of the seed-bank. lt involves the ex situ and in situ conservation of genetic entities under threat, regeneration, characterization, evaluation, monitoring of seed viability and genetic integrity during storage, and storing of all data in appropriate data banks. The durun wheat collection is also safety duplicated at other three sites (USA, Japan and Russia). Sources of tetraploid wheat germplasm important for plant breeding were identified in all natural resources, wild and cultivated, landraces, primitive, obsolete, commercial varieties, genetic stocks. In particular species and subspecies of tetraploid wheats, with A and B genomes, have been evaluated recording specific traits, following the prescribed guidelines of ECPGR, and the genetic profiles traced by biochemical and molecular techniques.Within the years of activity of the Institute, useful genes were identified and successfully used for the development of improved varieties and breeding lines.

RNA silencing (RS) is a conserved eukaryotic mechanism acting in plants also as antiviral immune system. A successful virus infection requires suppression or evasion of host RS. Small interfering RNAs (siRNAs) are potent RS effectors, and accumulate in plants infected by RNA and DNA viruses as components of this plant immune system, providing targer specificity for pathogen RNA post-transcriptional degradation. Virus-derived siRNAs (vsiRNAs, 21-24 nt) are abundant and diverse in infected plants. Although any viral genomic regions can potentially be targeted to generating vsiRNAs, certain regions (also referred to as "hot spots") are more represented than others in vsiRNA sequenced libraries.Potato virus Y (PVY, Potyviridae) is an important plant pathogen on solanaceous hosts. The two PVY isolates PVYC-to and PVY-SON41 induce very different disease phenotypes on tomato (Solanum lycopersicum), the former being responsible of leaf distortions and the latter inducing to mild symptoms. In this study, we applied combined in silico and molecular biological approaches to identify PVY vsiRNAs putatively able to suppress host mRNAs by sequence complementarity and deriving RS-based suppression, a mechanisms that would induce dysfunctional processes in infected host plants. As the final aim of the study, we explored differential expression of specific host-targeting vsiRNA by the two isolates as one of the possible sources of diverse disease phenotype expression.A computational pipeline was implemented to retrieve 21nt vsiRNAs from PVY isolates, complementary to tomato predicted mRNAs (Solgenomics, ITAG2.3). The comparative study used NCBI blast+ package 2.2. RandFold searched secondary structures in the PVY RNA genome containing putative vsiRNAs sequences identified in previous steps.Two PVYC-to regions showed potential tRNA-like or microRNA-like secondary structures, not present in the other isolate, possibly accounting for vsiRNAs accumulation "hot spots". Moreover, we obtained two lists of tomato transcripts perfectly or imperfectly (one or two mismatches were allowed, according to typical microRNA recognition sites) complementary to vsiRNA computed for either of the two PVY isolates, and therefore putative target of vsiRNA-driven RS suppression. Putative targets common to the two isolates were discarded, so to identify a list of genes that could represent targets specific to the aggressive isolate PVYC-to only. Some host transcription factors (e.g. NAC, MYB, TCP, HD-ZIP, MADS-box families) active in vegetative development and leaf morphogenesis were selected for further investigation. Quantitative RT-PCR showed differential expression levels of selected host transcripts upon PVYC-to and PVY-SON41 infections, differing also from healthy plants. For some genes, lower isolate-specific mRNA accumulation suggested RS-driven post-transcriptional regulation, thus apparently confirming the starting hypothesis of a correlation between symptoms and RS