The accurate description of plant biodiversity is of utmost importance to efficiently addressefforts in conservation genetics and breeding. Herein, we report the successful application of agenotyping-by-sequencing (GBS) approach in chickpea (Cicer arietinum L.), resulting in thecharacterization of a cultivated germplasm collection with 3,187 high-quality singlenucleotide polymorphism (SNP) markers. Genetic structure inference, principal componentanalysis and hierarchical clustering all together indicated the identification of a genetic clustercorresponding to black-seeded genotypes traditionally cultivated in Southern Italy.Remarkably, this cluster was clearly distinct at both genetic and phenotypic levels fromgermplasm groups reflecting the commercial chickpea classification in desi and kabuli seedtypes. Fixation index estimates for individual polymorphisms pointed out loci and genomicregions that might be of significance for the diversification of agronomic and commercialtraits. Overall, our findings provide information on genetic relationships within cultivatedchickpea and highlight a gene pool of great interest for the scientific community and chickpeabreeding, which is limited by the low genetic diversity available in the primary gene pool

Wheat is one of the most important crops worldwide and, as for other crops, production is highly dependent on the input of fertilizers. Among them, nitrogen is particularly important because it is a key component of many macromolecules, including proteins and nucleic acids and is essential for normal growth and development of plants. Crop yield and many quality aspects of wheat derived products (e.g. bread, pasta) are related to the protein composition, and therefore to nitrogen availability. Modern breeding is particularly interested in understanding the mechanisms regulating the assimilation, utilization, and remobilization of nitrogen and the way how plants can sense and translate environmental stimuli, such as nitrogen starvation, and activate subsequent adaptive responses. In the present work, two durum wheat genotypes, Svevo and Ciccio, were grown in a hydroponic system in nitrogen starvation conditions (0mM of NO 3 ) and in normal conditions (2mM of NO 3 ). Phenotypic analysis performed on both varieties at different growth stages (Z14, and Z77) revealed in all stages and for both varieties significant differences for some parameters directly involved in plant yield such as primary roots length, number of culms per plant, plant height, flag leaf area, number of spike per plant, and number of spikelets per spike. Moreover roots, leaves and spikes of both varieties at different growth stages were collected and the microRNAs involved in the nitrogen regulatory pathway and their function will be analyzed.

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.

Wheat is one of the most widely growncereal crops based on the amount of calories itprovides in the human diet. Durum wheat (Triticumturgidum ssp. durum) is largely used for production ofpasta and other products. In order to use geneticknowledge to improve the understanding of N-useefficiency, we carried out, for the first time in durumwheat, the isolation and the characterization of fourmembers of the asparagine synthetase (AsnS) genefamily. Phylogenetic inference clustered the Ttu-AsnS1 (1.1 and 1.2) and Ttu-AsnS2 (2.1 and 2.2)genes in AsnS gene class I, which is present inmonocots and dicots. Class I genes underwent asubsequent duplication leading to the formation of twosubgroups. Plants of Svevo cultivar were grown underN-stress conditions and expression of the four AsnSgenes was investigated at three developmental stages(seedling, booting, and late milk development), crucialfor N absorption, assimilation and remobilization.AsnS1 genes were down-regulated in N-stressed roots,stems and leaves during seedling growth and booting,but seemed to play a role in N remobilization in flagleaves during grain filling. AsnS2 genes were scarcelyexpressed in roots, stems, and leaves. In N-stressedspikes there was no differential expression in any ofthe genes. The genes were mapped in silico using adurum wheat SNP map, assigning Ttu-AsnS1 genes tochromosome 5 and Ttu-AsnS2 to chromosome 3.These findings provide a better understanding of therole of ASN genes in response to N stress in durumwheat.

La biodiversità rappresenta la variabilità di tutti gli esseri viventi e può essere riferita agli ecosistemi, alle specie, o a livellidi rango tassonomico infraspecifico, quali le varietà. In particolare, la biodiversità vegetale di interesse agrario è riferita allespecie coltivate e spontanee utilizzate dall"uomo a scopi alimentari o per altri usi e ai relativi progenitori selvatici.Nell"ambito dei Progetti Integrati per la Biodiversità, PSR Puglia 2007-2013 (Misura 214/4 sub-azione a), la Regione Pugliaha finanziato il progetto BiodiverSO - Biodiversità delle specie orticole della Puglia - con l"obiettivo primario disalvaguardare le risorse genetiche orticole della regione. Infatti, a tutt"oggi, sul territorio pugliese non è stata effettuataun"indagine capillare e sistematica delle risorse genetiche ortive presenti. Le azioni di conservazione del germoplasma sonospesso affidate alle iniziative di singoli agricoltori che tramandano semi o altre parti di pianta di generazione in generazione(conservazione on farm), e alla conservazione ex situ che viene normalmente praticata da enti pubblici, quali CNR edUniversità. La rivalutazione di genotipi di varietà locali e/o il loro risanamento possono ampliare la base genetica delcomparto orticolo consentendo migliore tolleranza agli stress biotici e abiotici, in un periodo in cui si assiste a notevolicambiamenti climatici, per salvaguardare la salute del consumatore e l"ambiente, nonché per valorizzare alcune produzionitipiche pugliesi. L"Italia meridionale rappresenta il centro di domesticazione e/o diversificazione di alcune colture, quali adesempio alcune Brassicacee, il carciofo, il melone, e questo significa che le nostre regioni sono estremamente ricche dibiodiversità di interesse agrario. La Puglia produce numerose varietà coltivate di ortaggi come la carota di Polignano e diSant"Ippazio; la cipolla di Acquaviva e di Margherita; il cavolfiore, cavolo broccolo (cima nera, cavolo riccio, mugnoli) ecima di rapa; il melone immaturo (carosello e barattiere) e d"inverno; la cicoria (di Molfetta, di Galatina, di Otranto); ilcarciofo (di Mola, Centofoglie, Bianco tarantino, nero di Ostuni, ecc.); il pomodoro Regina ed il pomodoro di Manduria.Alcune di queste varietà locali sono a rischio di estinzione e potrebbero scomparire per sempre per essere soppiantate davarietà moderne. La principale finalità del progetto integrato BiodiverSO è quella di contribuire a raggiungere unasignificativa riduzione del tasso attuale di erosione della biodiversità delle specie orticole pugliesi. Dopo aver reperito sulterritorio pugliese le risorse genetiche orticole a rischio, esse saranno catalogate mediante strumenti informatizzati ecaratterizzate sia dal punto di vista morfo-agronomico che nutrizionale, che genetico-molecolare. Il materiale geneticoraccolto verrà conservato presso le banche del germoplasma del CNR-IBBR di Bari e dell"Università di Bari. Laconservazione ex si

Bowman-Birk inhibitors and their variants (BBI) from legumes, such as soybean, pea, lentil and chickpea, are a class of naturally-occurring protease inhibitors which have potential health-promoting properties within the gastrointestinal tract. BBI can resist both acidic conditions and the action of proteolytic enzymes, and transit through the stomach and small intestine without major degradation, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. These potential pharmacological benefits have been linked recently to the intrinsic ability of BBI to inhibit serine proteases, and the data suggest that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI. However, the therapeutic targets and the action mechanisms of BBI remain unknown. Their elucidation will provide insights into the properties of these plant protease inhibitors as colorectal chemopreventive agents, providing a strong base for the development of legume crops and their products as pro-nutritional, health-promoting food. The deployment of modern genomic tools and genome sequence information are underpinning studies of natural and induced polymorphism in BBI. Genetic markers for BBI variants with improved properties can be exploited ultimately in legume breeding programmes to assist the introgression of such variant genes and the development of superior genotypes for human nutrition.

A full-length cDNA, encoding a Bowman-Birk protease inhibitor (BBI), was isolated from lentil immature seeds. The deduced amino acid sequence was longer than that of the BBI extracted from lentil seeds and contained two binding sites; the first inhibitory site inhibits trypsin whereas the second one inhibits chymotrypsin. In order to characterize this lentil BBI, a longer (complete) and its C-terminally processed (mature) form were heterologously expressed in the yeast Pichia pastoris. The recombinant BBI proteins proved to be active against trypsin and chymotrypsin, showing Ki values at nanomolar levels. Mass spectrometry analysis revealed that complete BBI was composed of an array of molecular masses, whereas mature BBI showed the presence of a major peak of the expected size. The effects of mature BBI on the growth of human colon adenocarcinoma HT29 and colonic fibroblast CCD-18Co cells were evaluated. Lentil BBI was able to inhibit the growth of such cells at concentrations higher than 19 ¼M, in a concentration-dependent manner; by contrast, the CCD18-Co cells were unaffected. These data broaden our knowledge of the beneficial biological activities of naturally-occurring BBI proteins and address the need for systematic evaluation of natural variants in order to design novel strategies in preventive medicine.

È difficile parlare della Puglia senza subire il fascino dei suoi profumi mediterranei, delle distese di ulivi e di grano, dei colori e della varietà dei suoi frutti: la regione è tra le più ricche della Penisola in termini di biodiversità, con 1.150 varietà orticole e 440 possibili antiche coltivazioni che mancano ancora all'appello.

The globe artichoke (Cynara cardunculus var. scolymus; n=x=17 and genome size of ~1.08 Gbp/1C) is a member of the Asteraceae family that contributes significantly to the agricultural economy of the Mediterranean basin. Despite its commercial importance, relatively little is known about its genome. Here, we report on the characterization of a bacterial artificial chromosome (BAC) library of artichoke and the preliminary effort to integrate the artichoke linkage groups with specific chromosomes. The artichoke BAC library consisted of 57,600 clones, representing approximately 5 haploid genome equivalents. We employed a multidimensional BAC pooling strategy for PCR-screening. The BAC-pools were screened with various PCR-based markers, including map-anchored SSR markers and other PCR-markers for genes most of which involved in the biosynthesis of phenylpropanoids. Marker screening was carried out by using High Resolution Melt (HRM) analysis on BAC library pools. We identified some 50 BAC clones, and their sequencing is in progress. In addition, to provide a cytogenetic map of the artichoke genome, we are localizing these map-anchored BACs onto the artichoke chromosomes using FISH (fluorescence in situ hybridization). This artichoke BAC library will serve as an important resource to build an artichoke physical map, integrate the available linkage data to specific chromosomes and for comparative genome analyses.

Genetic relationships among 13 grasspea (Lathyrus sativus L.) landraces mainly collected in Southern Italy were assessed using agronomic traits, biochemical and molecular markers. Field trials were carried out in two locations and revealed a high influence of field locations on yield, but a low genotype 9 environment interaction. Despite this, the agronomic data obtained provided useful information for the choice of the best grasspea landraces for southern Italian marginal areas. Seed storage proteins utilised as biochemical markers were not able to detect polymorphisms, on the contrary both classes of molecular markers used i.e. AFLP and SSR, provided useful information on genetic variation and relationships among landraces. Even though the number of polymorphic fragments detected by AFLP technique was low, it was sufficient to discriminate all the accessions. The use of SSR to detect polymorphic sites in grasspea showed that most landraces were clearly grouped in two sub-clusters. One comprised two landraces from most northern localities, while all the other landraces were clustered together at a very narrow genetic distance.

Genetic relationships, agronomic, nutritional and technological traits of ten Italian landraces, two improved lines and two cultivars of lentil (Lens culinaris Medik.) were investigated using a multi-disciplinary approach. Seed storage proteins, used as biochemical markers, were able to detect polymorphisms with variability mainly related to the polypeptide abundance. Microsatellite (SSR) molecular markers provided very useful information on genetic variation and relationships among landraces, with polymorphic fragments able to discriminate all the accessions. Lentil landraces were grouped in different clusters and sub-clusters principally on the basis of their geographical origin. The highest levels of genetic diversity were observed for lentils from ‘Castelluccio di Norcia’, ‘Colliano’ and ‘Villalba’. Field trials, performed in two locations of Southern Italy, revealed a high influence of location on yield. Comparing performances at both tested locations, the best landraces were ‘Linosa’ and ‘Valle di Nevola’ suggesting that these have the highest adaptability. Technological and nutritional data together with the agronomic ones evidenced that ‘Linosa’ lentil is the best landrace, however also ‘San Gerardo’ deserves some attention.

Characterization of 15 chickpea (Cicer arietinum L.) accessions (6 cultivars, 3 selected lines and 6 landraces) was performed using morphological, agronomic, and technological traits, together with biochemical (seed storage proteins) and molecular markers. Field trials, performed in two different geographical areas of Southern Italy, one in a plain area near Tyrrhenian sea and one in an hilly internal area, revealed a high influence of field location on yield. A wide variation was also registered for technological traits and molecular markers. Microsatellite (SSR) markers grouped chickpea accessions in different clusters, providing useful information on genetic variation and relationships among them, with polymorphic fragments useful to discriminate among all accessions. On the contrary, seed storage protein pattern showed scarce variation resulting very similar among all the accessions considered.

Considering the Cynara cardunculus germplasm there is a miss of information: most populations have not been characterised and many of them, taking their name from the cultivation area, are synonymous. In the frame of the European project CYNARES, sponsored by the AGRGENRES programme, European C. cardunculus accessions have been characterized at the morphological, biochemical and molecular level. Moreover, disease resistance, post-harvest behaviour and industrial-food processes are studied with regard to quality and safety objectives. Morphological characterisation was carried out in different fields/countries since C. cardunculus germplasm, belonging to different typologies, has different environmental requirements. The descriptors developed by IPGRI and UPOV were used with a view to identifying and validating the most useful field descriptors.The most promising material is also being tested for fresh handling and minimal processing. The ready-to-eat artichoke products, with high organoleptic quality of raw material, must be accompanied by a long-enough postharvest shelf-life of the fresh and ready-to-eat commodity. In the core collection, biochemical characterization of the different artichoke/cardoon genotypes is being undertaken by evaluating the flowerhead content of polyphenols (mono-and di-caffeoylquinic acids and flavonoids), sugars (glucose, fructose, sucrose) and fructans (inulin).Molecular characterization was conducted on a European core collection constituted by 556 genotypes, utilizing markers of different typologies (AFLP, ISSR, SSR). Molecular data fingerprinting of single lines have made it easier to register/patent socio-economically important varieties.This characterization is fundamental for rationalizing the European germplasm. Further cluster analysis obtained with different characterization data will be utilized to define an European core collection for artichoke.

The high-throughput technologies have helped promote sequencing of many plant genomes. This showed to be the fastest approach for developing molecular resources in agriculture and ecology and revolutioned the way in which the research products are generated. In the present work, 22 samples from Cynara L. genus were analysed, including 18 genotypes belonging to the three taxa of C. cardunculus L., along with other four wild species (C. syriaca, C. cornigera, C. baetica, and C. humilis).The main aim was to isolate the entire chloroplast (cp) genome sequences from this germplasm, in order to retrieve the most useful resources for phylogenetic and population studies in the Cynara genus. Firstly, reads obtained by whole-genome and BAC clone high-throughput sequencing of the "Brindisino" globe artichoke were used to assemble the cp genome. This genome sized 152529 bp and consisted of two single-copy regions separated by a pair of inverted repeats (IRs) of 25155 bp. The large (LSC) and the small (SSC) single-copy regions spanned 83578 bp and 18641 bp, respectively.For the other genotypes, a different approach was used. The Cynara cp genome was virtually divided into 15 regions and long-range PCR was employed for producing the same number of partially overlapping amplicons. Subsequently, all sets of amplicons were sequenced with an Illumina platform. Sequences were then assembled for each genotype using de novo and reference-guided methods. For reference-based assembly we used the cp sequence of cultivar "Brindisino" (KM035764) as a reference. No structural differences regarding gene content and order were highlighted among all analysed genotypes. Cynara cp genome consists of 114 unique genes, including 30 tRNA, 4 rRNA, and 80 predicted protein-coding genes. The comparative analysis among all newly sequenced genomes allowed the discovery of a large number of informative loci, in terms of variable microsatellites, INDELS, and SNPs. We developed a new set of primer pairs directed to the amplification of SSR markers, which could be used for exploring the diversity of Cynara populations. Furthermore, the Cynara complete cp sequence allowed the placing of this genus in the Asteraceae family tree.

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for "specific barcode" purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants

In this study, new chloroplast (cp) resources were developed for the genus Cynara, using whole cp genomes from 20 genotypes, by means of high-throughput sequencing technologies. Our target species included seven globe artichokes, two cultivated cardoons, eight wild artichokes, and three other wild Cynara species (C. baetica, C. cornigera and C. syriaca). One complete cp genome was isolated using short reads from a whole-genome sequencing project, while the others were obtained by means of long-range PCR, for which primer pairs are provided here. A de novo assembly strategy combined with a reference-based assembly allowed us to reconstruct each cp genome. Comparative analyses among the newly sequenced genotypes and two additional Cynara cp genomes ('Brindisino' artichoke and C. humilis) retrieved from public databases revealed 126 parsimony informative characters and 258 singletons in Cynara, for a total of 384 variable characters. Thirty-nine SSR loci and 34 other INDEL events were detected. After data analysis, 37 primer pairs for SSR amplification were designed, and these molecular markers were subsequently validated in our Cynara genotypes. Phylogenetic analysis based on all cp variable characters provided the best resolution when compared to what was observed using only parsimony informative characters, or only short 'variable' cp regions. The evaluation of the molecular resources obtained from this study led us to support the 'super-barcode' theory and consider the total cp sequence of Cynara as a reliable and valuable molecular marker for exploring species diversity and examining variation below the species level.

Durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) is an economically important crop used for the production of semolina, which is the basis of pasta and other food products. Its grains provide proteins and starch for human consumption. Grain development is a key process in wheat physiology; it is highly affected by a number of enzymes that control the metabolic processes governing accumulation of starch and storage proteins and ultimately grain weight. Most of these enzymes are present in the albumin/globulin grain fraction, which represents about a quarter of total seed proteins. With the aim to describe the dynamic profile of the albumin/globulin fraction during durum wheat grain development, we performed a proteomic analysis of this subproteome using a two-dimensional differential gel electrophoresis (2D-DIGE)-based approach and compared six developmental stages. A total of 285 differentially (237 over- and 48 under-) represented spots was identified by nanoLC-ESI-LIT-MS/MS, which were associated with 217 non-redundant Triticum sequence entries. Quantitative protein dynamics demonstrated that carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division functional categories were highly affected during grain development, concomitantly with progressive grain size increase and starch/protein reserve accumulation. Bioinformatic interaction prediction revealed a complex network of differentially represented proteins mainly centered at enzymes involved in carbon and protein metabolism. A description of 18 proteins associated with wheat flour human allergies was also obtained; these components showed augmented levels at the last developmental stages. By providing a comprehensive understanding of the molecular basis of durum wheat grain development, yield and quality formation, this study provides the foundation and reveals potential biomarkers for further investigations of durum wheat breeding and semolina quality. Biological significance: A 2D-DIGE-based comparative analysis of the albumin/globulin fraction from durum wheat caryopses at six developmental stages was performed to describe the dynamic subproteomic changes associated with grain development. Quantitative variations of 217 differentially proteins demonstrated that highly affected are the functional categories of carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division, which displayed a general over-representation, consistently with concomitant occurrence of grain size increase and starch/protein reserve accumulation. Bioinformatics revealed a complex protein network centered mainly at enzymes involved in carbon and protein metabolism. Differentially represented proteins and corresponding functional categories highly resembled those previously identified as variable in developing bread wheat grain. This suggests that the main differences in kernel hardness between durum and bread wheat probably do not depen

With over 20,000 species, Asteraceae is the second largest plant family. In the last decade the advent of Next Generation Sequencing (NGS) allowed insights directly on the nuclear, chloroplastic and mithocondrial genomes giving the opportunity to better understand the evolutionary relationships within this family. Cynara cardunculus is a complex species including two crops (globe artichoke and cardoon) and a wild taxon (wild cardoon). We isolated chloroplast Illumina reads as a contamination of nuclear genome sequencing for an artichoke and a wild cardoon sample. After mapping all the reads on Lactuca sativa chloroplast (cp) genome , the closest cp genome present in public databases, we obtained a very reliable coverage (about 130x). Contigs and supercontigs generated by assembling the raw sequencing data were aligned onto the reference genome showing a 98% coverage of the full cp sequence, with five gaps which were filled by Sanger sequencing. The typical cp quadripartite architecture comprising a large single copy (LSC), a small single copy (SSC) and two inverted repeats (IR1 and IR2) was checked by Sanger sequencing of the relative junctions in order to avoid contingently assembling artifacts. The full sequence was then nnotated with bioinformatic softwares whose errors were corrected by hand. Gene content and order showed to be well conserved within the Asteraceae family and promising regions were used for further investigations in molecular phylogeny of Asteraceae. Moreover, sequencing of various taxa within Cynara provide useful informations to develop molecular markers suitable for performing evolutionary studiesand barcoding applications within this genus.

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.

Lo sviluppo delle tecnologie Next Generation Sequencing (NGS) negli ultimi anni sta consentendo la produzione di grandivolumi di dati di sequenza a costi sempre minori e sta ridimensionando man mano la lunghezza dei passi della ricercascientifica. Nell"ambito di un progetto di sequenziamento parziale del genoma nucleare di carciofo [Cynara cardunculus L.var. scolymus (L.) Fiori] mediante tecnologia Illumina, sono state ottenute oltre 33 milioni di sequenze paired-end da 75 pb,rappresentando una copertura di circa 2.3x del genoma totale di carciofo. Questa grande quantità di dati ha consentitol"estrapolazione delle sequenze appartenenti al genoma cloroplastico presenti come "contaminanti" tra le sequenze delgenoma nucleare. A tal fine tutte le sequenze ottenute dal sequenziamento sono state utilizzate per l"allineamento contro ilgenoma cloroplastico di Lactuca sativa, per un assemblaggio su genoma di riferimento. In totale, sono state mappate edassemblate 1.3 M di sequenze, corrispondenti al 3.92% delle reads. È stato così possibile ottenere la sequenza completa delgenoma cloroplastico di carciofo, che presentava cinque gap, i quali sono stati facilmente colmati tramite sequenziamentoSanger. In questo modo è stato ottenuto il genoma cloroplastico di riferimento per il genere Cynara. Al fine di valutare ladiversità del genoma cloroplastico nella specie C. cardunculus e nel genere Cynara, sono stati amplificati e sequenziati igenomi cloroplastici di 20 altri genotipi. Questi comprendevano sette carciofi appartenenti ai principali gruppi morfoagronomici,due cardi coltivati (C. cardunculus var. altilis DC) e sette cardi selvatici (C. cardunculus L. var. sylvestrisLam.). Sono state incluse nell"esperimento anche altre quattro specie del genere Cynara: C. baetica (Spreng.) Pau, C.humilis L., C. syriaca Boiss e C. cornigera Lindley. Ognuno dei 20 genomi cloroplastici, amplificati tramite Long-RangePCR e marcati singolarmente mediante barcode, è stato sequenziato utilizzando la tecnologia Illumina-Miseq. Ilsequenziamento ha prodotto una copertura media di 950x ed ogni genoma cloroplastico è stato assemblato sul genoma diriferimento ottenuto in precedenza. In conclusione, i due diversi approcci sperimentali basati su tecnologie NGS hannopermesso l"ottenimento di 21 sequenze complete di genomi cloroplastici appartenenti al genere Cynara. Questi sono statiutilizzati per studi di genomica comparativa ed hanno portato all"individuazione di marcatori molecolari del tipo SNP, Indeled SSR, utili per analisi filogenetiche, evolutive ed applicabili alla tracciabilità alimentare dei prodotti derivati del carciofo.

The Leonforte broad bean is a grain legume known since ancient times that, besides enriching of nitrogen the soil, is an important component of traditional dishes.The Leonforte broad bean is characterized by large sized seeds and short cooking time. It is used both fresh and dry consumption. In order to valorise this important material, genetic diversity was evaluated in selected accessions of Fava larga of Leonforte. The agronomic test was carried out at the experimental farm of the Faculty of Agriculture of Catania. At harvest, yield and other main traits were evaluated. For DNA extraction, leaves were used from 10 individuals for each selection. AFLPs (Amplified Fragment Length Polymorphism) were used as molecular markers to assess genetic variation. The agronomic results showed remarkable differences between local accessions and the commercial cultivar Superaguadulce. From the genetic perspective, the selections appeared well differentiated from the commercial cultivar and confirmed the remarkable variability present among them.

Il frumento duro (Triticum durum Desf.) è una tra le piante di più antica domesticazione, rientrando tra i cereali piùimportanti per l"alimentazione umana. Gli stress abiotici influenzano negativamente l"adattabilita delle cultivar di frumentoduro all"ambiente in cui vengono coltivate. Pertanto, in conseguenza dello stress, generalmente si rileva una riduzione dellaresa e un peggioramento della salubrità e della qualità delle proteine, con le relative ripercussioni sull"attitudinepastificatoria. L"azoto è l"elemento nutritivo più importante per lo sviluppo delle piante poiché è indispensabile per lacostituzione delle proteine, degli acidi nucleici e di altri costituenti cellulari. L"utilizzazione di fattori regolatori della viabiosintetica dell"azoto potrebbe essere una strategia efficace per la modificazione del metabolismo azotato con l"obiettivo digenerare colture più efficienti nell"assorbimento e trasporto di azoto. In questo lavoro sono state analizzate le differenze dirisposte nel metabolismo dell"azoto di due genotipi di frumento duro con diversi gradi di resistenza allo stress per mancanzadi azoto. I geni codificanti per i fattori di trascrizione DOF1, GLB1 e l"enzima asparagina sintetasi (AsnS), coinvolti nelmetabolismo azotato, sono stati analizzati in diversi tessuti. L"analisi della diversità genetica e di espressione di tali geni ingermoplasma di frumento duro e le caratterizzazioni funzionali offrono un importante contributo ai programmi dimiglioramento genetico che hanno lo scopo di produrre piante di frumento duro resistenti alla mancanza di questo elementoessenziale. Ponendo le basi per un incremento della resa del frumento duro e per una maggiore conservazione delle risorsenaturali, i risultati di questo lavoro avranno notevole utilità per l"industria sementiera e per il settore agroalimentare. Questolavoro è stato finanziato dal progetto PON-ISCOCEM (PON01_01145).

Durum wheat highly depends on nitrogen for seed development and yield, and the obtainmentof varieties with a better nitrogen use efficiency is crucial to reduce production costsand environmental pollution. In this study, sequencing of two small RNA libraries obtainedfrom tissues of Ciccio and Svevo cultivars grown under nitrogen starvation conditions produced84 novel, and 161 conserved miRNAs. Of these, 7 novel and 13 known miRNAs werenewly identified in this work. Quantitative PCR analysis of selected miRNAs highlighted thatthe expression levels of some of them depends on the tissue and on the cultivar, Svevobeing the most responsive to nitrogen starvation. A number of target genes were predictedto be involved in nitrogen metabolism. An inverse correlation for the qPCR expression dataof miRNA/target pairs miR399b/PHO2, miR393c/AFB2, ttu-novel-61/CCAAT-TF wasobserved in specific tissues or cultivar. Especially, ttu-novel-61 was down-regulated and itstarget CCAAT-TF up-regulated in almost all tissues both in Svevo and in Ciccio. Moreover,CCAAT-TF was confirmed to be cleaved by ttu-novel-61 at the expected site. The discoveryof miRNAs involved in the response to nitrogen stress represents an important step towardsfunctional analyses, with the final aim to design strategies for improving nitrogen use efficiencyin durum wheat.

This paper refers on the yields and productive aspects of mineral and foliarnutrition of globe artichoke plants (cv. 'Opal' and 'Madrigal'). Marketableyields were investigated in response to different nitrogen rates and foliarapplications of biostimulating products. Field experiments were conductedover two seasons on an alluvial salty-clay soil in sub-humid climateconditions. Analyzed traits differed between years because of the age ofplants while the lowest nitrogen rate and foliar spraying of biostimulantsinfluenced significantly head weight, dry matter, and Soil Plant AnalysisDivision (SPAD) index. Biostimulants induced longer cultural cycle and headslighter in weight. Cultivar 'Madrigal' was most productive than 'Opal', both innumber (26.28% higher) and in weight (28.51%) of total heads. The two-yearinvestigation showed that the synergic action was effective in improvingsome of the investigated productive traits of globe artichoke.

Previous studies have shown that alexithymia is associated with gene polymorphisms that regulate the availability of serotonin (5-HT) in the brain. Since the 5-HT network is involved in interferon (IFN)-induced depression, this paper aimed to investigate the role of alexithymia and the functional gene variants of the 5-HT1A receptor (HTR1A) and the 5-HT transporter (5-HTTLPR) in induction of depression during antiviral treatment. Methods: The depressive symptoms of 130 consecutive patients with chronic hepatitis C and no current psychopathology were measured during treatment with IFN and ribavirin (6-12 months) and at a 6-month follow-up. At baseline, alexithymia and 2 genotypes (5-HTTLPR and HTR1A) were also assessed. Results: Patients with homozygosity for HTR1A-G and 5-HTTLPR long alleles had significantly higher levels of alexithymia. After controlling for sociodemographic and disease-related factors, alexithymia and HTR1A-G polymorphism, both separately (20-22%) and jointly (14-16%), significantly and independently predicted the development of IFN-induced depression. Conclusions: Subjects carrying HTR1A-G and 5-HTTLRP double long alleles are more vulnerable to alexithymia. Also patients with a higher level of alexithymia and the HTR1A-G gene variant are more vulnerable to experiencing IFN-induced depressive symptoms. The clinical implications of targeting alexithymia and HTR1A receptors as a possible treatment option for mood disorders should be investigated in further studies. (C) 2015 S. Karger AG, Basel

Understanding the distribution of genetic variations and accession structures is an important factor for managing genetic resources, but also for using proper germplasm in association map analyses and breeding programs. The globe artichoke is the fourth most important horticultural crop in Europe. Here, we report the results of a molecular analysis of a collection including globe artichoke and leafy cardoon germplasm present in the Italian, French and Spanish gene banks. The aims of this study were to: (i) assess the diversity present in European collections, (ii) determine the population structure, (iii) measure the genetic distance between accessions; (iv) cluster the accessions; (v) properly distinguish accessions present in the different national collections carrying the same name; and (vi) understand the diversity distribution in relation to the gene bank and the geographic origin of the germplasm. A total of 556 individuals grouped into 174 accessions of distinct typologies were analyzed by different types of molecular markers, i.e. dominant (ISSR and AFLP) and co-dominant (SSR). The data of the two crops (globe artichoke and leafy cardoon) were analyzed jointly and separately to compute, among other aims, the gene diversity, heterozygosity (He, Ho), fixation indexes, AMOVA, genetic distance and structure. The findings underline the huge diversity present in the analyzed material, and the existence of alleles that are able to discriminate among accessions. The accessions were clustered not only on the basis of their typology, but also on the basis of the gene bank they come from. Probably, the environmental conditions of the different field gene banks affected germplasm conservation. These outcomes will be useful in plant breeding to select accessions and to fingerprint varieties. Moreover, the results highlight the particular attention that should be paid to the method used to conserve the Cynara cardunculus germplasm and suggest to the preference of using accessions from different gene banks to run an association map.

Ten accessions of wild Borago officinaliswere collected during missions in the course of aresearch activity carried out on plants producingnutraceutical compounds. The aim was to identifypopulations that produce a higher content ofc-linolenic acid, as this compound has importanttherapeutic properties and has been used in medicaltests to treat rheumatoid arthritis, eczema, and psoriasiswith good therapeutical results. The present studyreports on the characterization of both the polyunsaturatedfatty acid composition and on genetic diversityestimated by means of AFLP markers in severalB. officinalis accessions collected in Southern Italyand grown in isolation by means of screen housesspecifically designed for each single population.Polyunsaturated fatty acid were found in differentamounts in all investigated accessions. The populationswere very variable at the molecular level, edthat similarity based on Jaccard's index rangedbetween 0.330 and 0.742. Our investigations allowedus to identify a population that may be considered as agood source of GL

An integrated consensus linkage map is proposed for globe artichoke. Maternal and paternal genetic maps were constructed on the basis of an F(1) progeny derived from crossing an artichoke genotype (Mola) with its progenitor, the wild cardoon (Tolfa), using EST-derived SSRs, genomic SSRs, AFLPs, ten genes, and two morphological traits. For most genes, mainly belonging to the chlorogenic acid pathway, new markers were developed. Five of these were SNP markers analyzed through high-resolution melt technology. From the maternal (Mola) and paternal (Tolfa) maps, an integrated map was obtained, containing 337 molecular and one morphological markers ordered in 17 linkage groups (LGs), linked between Mola and Tolfa. The integrated map covers 1,488.8 cM, with an average distance of 4.4 cM between markers. The map was aligned with already existing maps for artichoke, and 12 LGs were linked via 31 bridge markers. LG numbering has been proposed. A total of 124 EST-SSRs and two genes were mapped here for the first time, providing a framework for the construction of a functional map in artichoke. The establishment of a consensus map represents a necessary condition to plan a complete sequencing of the globe artichoke genome.

An integrated consensus linkage map is proposed for globe artichoke. Maternal and paternal genetic maps were constructed on the basis of an F(1) progeny derived from crossing an artichoke genotype (Mola) with its progenitor, the wild cardoon (Tolfa), using EST-derived SSRs, genomic SSRs, AFLPs, ten genes, and two morphological traits. For most genes, mainly belonging to the chlorogenic acid pathway, new markers were developed. Five of these were SNP markers analyzed through high-resolution melt technology. From the maternal (Mola) and paternal (Tolfa) maps, an integrated map was obtained, containing 337 molecular and one morphological markers ordered in 17 linkage groups (LGs), linked between Mola and Tolfa. The integrated map covers 1,488.8 cM, with an average distance of 4.4 cM between markers. The map was aligned with already existing maps for artichoke, and 12 LGs were linked via 31 bridge markers. LG numbering has been proposed. A total of 124 EST-SSRs and two genes were mapped here for the first time, providing a framework for the construction of a functional map in artichoke. The establishment of a consensus map represents a necessary condition to plan a complete sequencing of the globe artichoke genome.

This research aims at characterizing germplasm diversity of wild cardoon (Cynara cardunculus var.sylvestris) for future breeding. Six populations were identified in in the north of Tunisia and 20 individ-uals per population were evaluated using UPOV descriptors, and a large variability was revealed betweenpopulations for their leaf characteristics. The genetic diversity was also assessed using SSR markers atseventeen loci, which were all polymorphic, and a high genetic variation was found out. A low differenti-ation (Fst = 0.104) coupled with a high level of gene flow (Nm = 2.25) were observed among populations.Only 10.4% of total genetic variability is shared between populations in comparison to 89.6% within pop-ulation. Both morphological and molecular characterization showed that population grouping was notclearly related to their geographical distribution and this could be the result of the gene flow betweenregions. These results highlight a high level of genetic variation in Tunisian wild cardoon germplasm,which represents an interesting material to be conserved and exploited in breeding programs ofartichoke.

Cynara cardunculus L. of the Asteraceae family is a diploid, cross-pollinated species complex, originated in the Mediterranean Basin area. This species contains three different taxa: the wild perennial cardoon [var. sylvestris (Lam.) Fiori], the globe artichoke [var. scolymus (L.) Fiori] and the leafy or cultivated cardoon (var. altilis DC). In order to assess genetic variation and population structure in Cynara cardunculus , 801 individuals representing 60 populations of wild cardoon (from across the Mediterranean Region) and the two cultigens, were genotyped at 35 microsatellite (SSR) loci evenly distributed on all the linkage groups of an artichoke x wild cardoon genetic map previously developed by our group. Genetic diversity parameters, including polymorphic information content, total number of alleles, mean number of alleles, allelic richness, observed (Ho) and expected (He) heterozygosity, were calculated. Genetic diversity was assessed for the whole germplasm data set and for four main groups: i) eastern wild cardoon, originated from Italy, Greece, Tunisia, and Malta; ii) western wild cardoon from Spain and Portugal; iii) cultivated cardoon; iv) artichoke. A more detailed analysis was carried out for eastern wild cardoon samples, which was the bigger group. The highest variation was observed in wild populations, particularly for the eastern germplasm, and especially for the Italian material. An excess in heterozigosity was observed for artichoke, suggesting a heterotic advantage for this cultigen. The wild material appeared to be well structured, although the western (from Iberian Peninsula) wild cardoon seemed to be very closely related to the cultivated cardoon. Wild populations from Tunisia formed a defined group and the Italian wild material was also structured. Both UPGMA tree and principal coordinate analysis (PCA) depicted a clear picture of the relative distribution of wild and cultivated material, providing evidence for the origin of artichoke and cardoon.

Exploiting the biodiversity of crops and their wild relatives is fundamental for maintaining andincreasing food security. The species Cynara cardunculus includes three taxa: the globe artichoke,one of the most important Mediterranean vegetables, the leafy cardoon, and the wildcardoon. In this study, genotyping by sequencing (GBS) was successfully applied to revealthousands of polymorphisms in a C. cardunculus germplasm collection, including 65 globeartichoke, 9 leafy cardoon, and 21 wild cardoon samples. The collection showed a strong populationstructure at K = 2, separating the globe artichoke from the leafy and wild cardoon. Athigher K values, further substructures were observed, in which the wild cardoon was separatedfrom the leafy cardoon, and the latter included the Spanish wild cardoons, while the wildsample from Portugal was admixed. Moreover, subpopulations within the globe artichoke setwere highlighted. Structure analysis restricted to the globe artichoke dataset pointed outgenetic differentiation between the ?Catanesi? typology and all the other samples (K = 2). Athigher values of K, the separation of the ?Catanesi? group still held true, and green headedlandraces from Apulia region, Italy (?Green Apulian?) formed a distinct subpopulation. ?Romaneschi?artichoke types fell in a variable group with admixed samples, indicating that theyshould not be considered as a genetically uniform typology. The results of principal componentanalysis and Neighbor-Joining hierarchical clustering were consistent with structureresults, and in addition provided a measure of genetic relationships among individual genotypes.Both analyses attributed the wild material from Spain and Portugal to the cultivated cardoongroup, supporting the idea that this might be indeed a feral form of the leafy cardoon.Different reproductive habit and possibly selective pressure led to a slower LD decay in artichokecompared to cardoon. Genotyping by sequencing has proven a reliable methodology toobtain valuable SNPs and assess population genetics in C. cardunculus

Cowpea (Vigna unguiculata (L.) Walp.) is a major tropical pulse in a number of countries and especially in sub-Saharan Africa where this species was domesticated. Although as niche product, a remarkable number of cowpea landraces are cultivated in the Mediterranean basin, primarily for human consumption of the grains and fresh pods.In this study, within the frame of a regional project devoted to the safeguard and management of local horticultural genetic resources, some germplasm explorations were carried out in Apulia region (southern Italy) to collect, among other crops, cowpea landraces still present in the area and promote on-farm conservation of this traditional material. A second objective was to investigate the efficiency of SSR markers in detecting genetic polymorphism in cowpea landraces from a restricted geographical area. In total, 13 samples of V. unguiculata were gathered, 12 of cultivar group unguiculata and 1 of cultivar group sesquipedalis. Genetic diversity and phylogenetic relationships among 13 Apulian accessions, 9 samples from various provenances and 1 var. spontanea accession were evaluated using SSR molecular markers. A set of 19 SSR primer pairs successfully identified closely related accessions and were able to separate the different cultivar groups of V. unguiculata.

Notwithstanding the arrival of "third-generation sequencing," Sanger sequencing, developed in 1980, is still the most accurate and used method for sequencing, although on a smaller scale. It is a powerful resource for studying sequences and discovering polymorphisms and genes, as well as regulatory elements. There has already been described a wide range of possible problems with this very sensitive and accurate technology. Here, we show that a specific event, related to genomes rich in repetitive sequences, can mislead operators working with Sanger sequencing.

Polyphenols are important constituents of food products of plant origin and represent major antioxidants in our diet, since endogenous defense mechanisms are inadequate for the complete prevention of oxidative damage. The most abundant types of polyphenols found in the human diet are flavonoids, most often conjugated as glycosides.Among different sources of dietary antioxidants, artichoke extracts are particularly rich in bioavailable poliphenolic compounds as flavonoids and are important for their marked antioxidative potential and cancer chemopreventive properties. The understanding of flavonoids biosynthesis and its regulation in artichoke is essential to enhance the levels of these bioactive molecules in plants used as food.The biosynthetic pathway leading to the accumulation of flavonoids has been elucidated using genetic and biochemical information from many plant species (parsley, maize, petunia, Antirrhinum species, Arabidopsis, apple and grapevine) and has been recently studied in artichoke, where several enzymes directly involved in the early steps of the biosynthesis of flavonoids have been isolated and characterized, but little is still known on the regulation of these genes.The fine regulation of flavonoids accumulation is achieved by combinatorial actions of transcription factors (TF) belonging to various classes, among which MYB TF. Two putative artichoke MYB TF genes were isolated from a BAC library and were sequenced using Illumina technology. Sequencing allowed the structural characterization of coding and promoter regions of both genes, which showed a high similarity to MYB TF of other plant species (e.g. Arabidopsis, tomato). A phylogenetic analysis of putative MYB factors showed that the two artichoke sequences cluster together in a group including MYB factors from other species involved in the pathway of flavonols and proanthocyanidin biosynthesis. Heterologous expression in bacteria to study protein/DNA interactions and quantitative real-time PCR have been performed to gain insights into the molecular mechanism of polyphenols regulation in this edible plant, contributing to future progress in the study of polyphenols accumulation.

It is well known that artichoke possesses a number of beneficial properties, mainly attributable to polyphenols, a class of compounds with beneficial effects on human health. These are secondary metabolites including hydroxycinnamates, particularly caffeic acid, chlorogenic acid (CGA), di-caffeoylquinic acids, and flavonoids (e.g. luteolin). Chlorogenic acid, in particular, is a potent antioxidant very abundant in some vegetables, included artichoke. In artichoke the synthesis of CGA has not been completely elucidated yet. There are three proposed routes for the synthesis of CGA in plants, where the first common enzymatic step is the deamination of phenylalanine by means of the enzyme phenylalanine ammonia-liase (PAL), while the enzyme hydroxycynnamoyl-quinate transferase (HQT), belonging to the BAHD family is reported to be involved in the last step of the biosinthesys of CGA in some plant species. Recently, four members of the artichoke pal gene family have been isolated (De Paolis et al., 2008); in the present contribution, we report on the relative expression of three PAL isoforms in different plant parts, at different developmental stages and under stress simulation by mechanical wounding, and also on the isolation and characterization of two artichoke full-length hqt and hqt2 cDNA sequences. The HQT sequences showed a high level of similarity to homologous genes from other plant species, particularly tobacco and tomato. The transcripts of the two artichoke genes were heterologously expressed and biochemical assays were performed, in order to assess specificity of the enzymes for various substrates.

A pool of twelve cDNA sequences coding for Bowman Birk inhibitors (BBIs) was identified in the legume grass pea (Lathyrus sativus L.). The corresponding amino acid sequences showed a canonical first antitrypsin domain, predicted according to the identity of the determinant residue P-1. A more variable second binding loop was observed allowing to identify three groups based on the identity of residue P-1: two groups (Ls_BBI_1 and Ls_BBI_2) carried a second reactive site specific for chymotrypsin, while a third group (Ls_BB1_3) was predicted to inhibit elastase. A fourth variant carrying an Asp in the P-1 position of the second reactive site was identified only from genomic DNA. A phylogenetic tree constructed using grass pea BBIs with their homologs from other legume species revealed grouping based on taxonomy and on specificity of the reactive sites.

Flavonoids are a well-studied group of secondary metabolites, belonging to the phenylpropanoid pathway. Flavonoids are known to exhibit health promoting effects such as antioxidant capacities, anti-cancer and anti-inflammatory activity. Globe artichoke is an important source of bioactive phenolic compounds, including flavonoids. To study the regulation of their biosynthesis, a R2R3-MYB transcription factor, CcMYB12, was isolated from artichoke leaves. Phylogenetic analysis showed that this protein belongs to the MYB subgroup 7 (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. CcMYB12 transcripts were detected specifically in artichoke immature inflorescence and young leaves and overlapped with the profiles of flavonol biosynthetic genes. Electrophoretic mobility shift assays (EMSAs) revealed that recombinant CcMYB12 protein is able to bind to ACII element, a DNA binding site ubiquitously present in the promoters of genes encoding flavonol biosynthetic enzymes. In transgenic Arabidopsis plants, the overexpression of CcMYB12 activated the expression of endogenous flavonol biosynthesis genes, leading to an increase of flavonol accumulation and a decrease of anthocyanins in leaves. Likewise, in transgenic tobacco petals and leaves, the overexpression of CcMYB12 decreased anthocyanin levels and increased flavonols.

The known nutraceutical properties ascribed to artichoke are mainly attributable to the presence of phenylpropanoid compounds, in particular of flavonoids, caffeic acid and its derivatives (caffeoylquinic acids). These metabolites play central roles in plant biology, as they are involved in the protection against various biotic and abiotic stresses, in the regulation of plant reproduction and development, and they act as signalling molecules. Phenylpropanoids have also significant beneficial effects on human health if consumed as part of the diet, as they act as antioxidants, reducing the incidence of cardiovascular disease, certain cancers and age-related degenerative diseases. Hence, there is considerable interest in improving our understanding of phenylpropanoid biosynthesis and its regulation, to enhance the levels of these bioactive molecules in plants used as food. The biosynthetic pathway leading to the accumulation of phenylpropanoids has been elucidated using genetic and biochemical information from many plant species. The fine regulation of this pathway is achieved by combinatorial actions of transcription factors (TF) belonging to various classes, among which MYB TF.The metabolic route for the biosynthesis of caffeoylquinic acids, particularly chlorogenic acid (CGA) and its derivatives has been recently studied in artichoke, where several enzymes directly involved in this biosynthetic pathway have been isolated and characterized. However, little is known on the regulation of these genes. We have previously studied the genes coding for phenylalanine ammonio lyase (PAL) and two hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferases (HQT) enzymes in artichoke, where we detected different binding sites recognized by MYB TF in their promoter regions, suggesting that, as in other plant species, this class of transcription factors might be involved in the regulation of these artichoke genes.In order to isolate one or more MYB TF from artichoke, sequences of the ortholog MYB12 from other species (e.g. Arabidopsis, tomato) were used to screen artichoke EST database. Primers were designed to amplify a fragment of putative MYB TF in artichoke cDNA. Sequence completion was performed by means of 5'-RACE and 3'-RACE technology. This strategy allowed us to isolate two putative transcription factor genes belonging to the MYB family. Further analyses are being performed to assess their role in phenylpropanoid accumulation in artichoke. Heterologous expression in bacteria, transient expression assays, quantitative real-time PCR may help elucidate their function and interaction with phenylpropanoid biosynthetic genes.

Diverse specie di leguminose si prestano bene alla coltivazione come piante da orto e al consumo fresco. Tra queste, nell'ambito del progetto PSR PugliaBiodiverSO (http://biodiversitapuglia.it/) sono state reperite varietà locali di fava (es. Fava grande di Castellana) e di fagiolino (es. il Fagiolino di Deliceto). Una particolare attenzione merita il fagiolino dall'occhio (Vigna unguiculata (L.) Walp.), specie di origine africana, presente in Italia meridionale con due tipologie: il fagiolino pinto (gruppo unguiculata) e il fagiolino a metro (gruppo sesquipedalis). Nonostante considerato raro e sempre più sostituito dal comune fagiolino di origine americana (Phaseolus vulgaris L.), il fagiolino pinto è ancora largamente apprezzato e spunta prezzi più alti nei piccoli e grandi mercati ortofrutticoli dell'Italia meridionale. In Puglia, il fagiolino dall'occhio viene consumato esclusivamente sotto forma di baccello fresco, al contrario di quanto avviene nel centro-nord Italia, in Africa, e in altri Paesi, dove viene impiegato soprattutto come seme. Il monitoraggio del territorio regionale ha consentito la raccolta di varietà locali definendo l'areale di diffusione, le pratiche agricole, gli usi e le tradizioni legate al consumo. La coltivazione del fagiolino pinto è per la gran parte limitata a piccoli orti e destinata all'autoconsumo e alla vendita nei mercati locali, in particolare nella provincia di Bari, in Salento e in misura minore nelle altre province pugliesi. Il fagiolino a metro risulta molto meno diffuso ed è stato rinvenuto in pochissimi siti nelle province di Bari e Brindisi. Questa coltura ha mostrato una discreta variabilità genetica tra le accessioni raccolte in diversi siti e più raramente anche all'interno della stessa accessione, dove possono essere presenti forme differenti volutamente conservate dagli agricoltori. Il fagiolino pinto presenta interessanti potenzialità di sviluppo sul mercato nazionale.

MicroRNAs (miRNAs) are small non-coding RNA molecules which down-regulate theexpression of their target genes. MiRNAs play an important role in a number of biologicalprocesses such as plant development and plant adaptive responses to nutrient deprivation. Nitrogenis the most important nutrient for the growth of plants since it is required for protein and nucleicacids synthesis, and for the production of several compounds involved in a variety of functions.The tetraploid durum wheat is one of the oldest domesticated plants, falling among one of themost important cereals for human nutrition in the Mediterranean region. In spite of this, theidentification of miRNAs and their function in this crop is still largely unknown. A limitation ofnitrogen in durum wheat plants generally results in a reduction of the yield and of protein quality.In order to elucidate the role of miRNAs in durum wheat development and nitrogenmetabolism, twelve small RNA libraries were generated from different tissues and developmentalstages of Ciccio and Svevo cultivars growing under varying nitrogen supply conditions. Followinghigh throughput sequencing, conserved and novel miRNAs were identified using custom script andtheir target transcripts were detected and annotated according to GO terms. Several miRNAs wereexpressed at different levels based on their abundance in Illumina libraries. Selected conserved andnovel miRNAs were subjected to quantitative PCR, and for some of them significant differences inexpression profiles were confirmed. Additionally, expression levels for the target genes of theseregulated miRNAs were evaluated.Some miRNAs were predicted to regulate genes involved in plant development, in importantbiochemical processes such as photosynthesis, and in nitrogen metabolism.The knowledge about the role of miRNAs in plant development and nitrogen assimilation andstorage may be used in breeding programs to improve nitrogen use efficiency in durum wheat.Financial support: Project PON01_01145 "Sviluppo tecnologico e innovazione per lasostenibilità e competitività della cerealicoltura meridionale - ISCOCEM"; Project PRIN 2010-2011 "Identificazione e caratterizzazione di geni utili ad incrementare la produttività e sostenibilitàdel frumento duro

Nitrogen is an essential macronutrient for plant growth and reproduction. In durum wheat, an appropriate nitrogen soil availability is essential for an optimal seed development. miRNAs contribute to the environmental change adaptation of plants through the regulation of important genes involved in stress processes. In this work, nitrogen stress response was evaluated in durum wheat seedlings of Ciccio and Svevo cultivars. Eight small RNA libraries from leaves and roots of chronically stressed plants were sequenced to detect conserved and novel miRNAs. A total of 294 miRNAs were identified, 7 of which were described here for the first time. The expression level of selected miRNAs and target genes was analyzed by qPCR in seedlings subjected to chronic (Ciccio and Svevo, leaves and roots) or short-term (Svevo roots) stress conditions. Some miRNAs showed an immediate stress response, and their level of expression was either maintained or returned to a basal level during a long-term stress. Other miRNAs showed a gradual up- or downregulation during the short-term stress. The newly identified miRNA ttu-novel-106 showed an immediate strongly downregulation after nitrogen stress, which was negatively correlated with the expression of MYB-A, its putative target gene. PHO2 gene was significantly upregulated after 24-48-h stress, corresponding to a downregulation of miR399b. Ttu-miR399b putative binding sites in the 5? UTR region of the Svevo PHO2 gene were identified in the A and B genomes. Both MYB-A and PHO2 genes were validated for their cleavage site using 5? RACE assay.

Ptilostemon is a fine example of the representatives of the eastern groups of the Cardueae that have diversified in the western Mediterranean. Relationships to Cynara, which exhibits a similar distribution, and Lamyropsis, which is morphologically closer according to previous studies, are investigated using Bayesian analysis of DNA sequences of the plastid intergenic spacer ycf3-trnS and two nuclear regions, the ETS and ITS spacers. The sectional classification and biogeography of Ptilostemon are also revised in the light of the molecular phylogeny Our results suggest that Cynara is the most plausible sister genus to Ptilostemon. Some paralogous copies of the ETS region found among species of the three genera by cloning are interpreted as incomplete lineage sorting of ancestral polymorphisms. The current sectional classification of Ptilostemon shows excessive fragmentation, which does not agree with our phylogeny, and therefore a more synthetic classification is proposed. The present distribution of Ptilostemon indicates that there were two colonization events in the western Mediterranean region, paralleling a similar pattern of successive waves already suggested for Cynara

MicroRNAs (miRNAs) are small non-coding RNA molecules which down-regulate gene expression by mRNA degradation or translational repression. miRNAs play critical roles in diverse biological processes such as plant development and plant adaptive responses to nutrient deprivation. Nitrogen is the most important nutrient for the growth of plants since it is essential for protein and nucleic acids synthesis, and for other cellular constituents. A limitation of nitrogen in durum wheat plants generally results in a reduction of the yield and of protein quality, with negative effects on the attitude of pasta making.In order to elucidate the role of miRNAs in durum wheat development and nitrogen metabolism, twelve small RNA libraries were generated from different tissues and developmental stages of Ciccio and Svevo cultivars growing under different nitrogen supply conditions. Following high throughput sequencing, conserved and novel miRNAs and their target genes were identified. Several miRNAs were expressed at different levels based on their abundance in Illumina libraries. Selected conserved and novel miRNAs and their corresponding target genes were subjected to quantitative PCR, and for some of them significant differences in expression profiles were confirmed. Some miRNAs were predicted to regulate genes involved in plant development, in important biochemical processes such as photosynthesis, and in nitrogen metabolism.Financial support: Project PON01_01145 "Sviluppo tecnologico e innovazione per la sostenibilità e competitività della cerealicoltura meridionale - ISCOCEM"; Project PRIN 2010-2011 "Identificazione e caratterizzazione di geni utili ad incrementare la produttività e sostenibilità del frumento duro".

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

Artichoke has beneficial effects on human health, since it possesses anticarcinogenic, anti-HIV, antioxidative, cholesterol-lowering, bile expelling, hepatoprotective, and diuretic properties. These nutraceutical qualities are mainly due to polyphenolic compounds, particularly mono- and dicaffeoylquinic acids (e.g. chlorogenic acid, cynarin), caffeic acid and flavonoids (e.g. luteolin-7-O-glucoside, naringerin), with chlorogenic acid (CGA) being the most abundant. The content of polyphenol compounds, especially chlorogenic acid and cynarin, was measured in various tissues (leaves, bracts and receptacles) and physiological stages of artichoke plants, and in different artichoke genotypes belonging to the CNR-IGV Cynara world collection. A variation among tissues and genotypes was observed, and these variations were quite reproducible among years.On the same tissues, transcript levels of key genes for the synthesis of chlorogenic acid were measured by means of real time PCR. In particular, hqt genes, coding for HQT enzymes (acyltransferases of the BAHD family), have been shown to play a fundamental role in the synthesis of CGA in some plant species. Recently, hqt1 and hqt2 genes, possessing two exons and one intron each, have been isolated from artichoke, starting from Asteraceae EST sequences. Coding sequences were heterologously expressed in E. coli and the crude extract was used for enzyme chatracterization and substrate specificity. Both HQT1 and HQT2 were able to synthesize CGA in vitro and showed a remarkable preference towards quinate over shikimate, which distinguishes HQT enzyme class from HCT class (preferring shikimate). Based on the available crystallized structures of two BAHD enzymes, modeling and docking analyses were used to assume structural models for our HQT enzymes and to predict their potential binding sites.The content of CGA in the various tissues and genotypes analysed was more directly correlated with hqt1 expression levels. Moreover, transient and stable expression of HQT1 in Nicotiana benthamiana and in N. tabacum respectively, produced an increase in the content of CGA and cynarin, a derivative of CGA. Our findings indicate that both hqt1 and hqt2 are involved in the synthesis of CGA, but possibly at different steps of the metabolic pathway, and according to the plant exigencies. Moreover, the synthesis of cynarin (which is still unclear) might take place starting from CGA.

Polyphenol compounds, particularly caffeoylquinic acids and flavonoids, were measured in different tissues anddevelopmental stages of 6 artichoke varietal types diffused in the Mediterranean region. Flower heads were subdivided intoexternal, intermediate, internal bracts, and receptacle, while leaves were collected at the vegetative and productive stages.The main polyphenols detected were chlorogenic acid, cynarin, luteolin 7-O-rutinoside, and luteolin 7-O-glucoside."Violet de Provence" artichoke proved to retain the highest content of total phenols. Single polyphenols accumulatedpreferentially in specific parts of capitula. In leaves, most polyphenols were detected in the productive stage of the plant.Altogether, results provide useful indications for the promotion of artichoke as nutraceutical food and for the extractionof health-promoting substances in particular tissues/stages of the artichoke plant

Polyphenols are widespread constituents of plants that have been used in the treatment ofdiseases for centuries. The current growing interest in dietary plants has led to renewed attention onartichoke, because of its high polyphenolic content. Polyphenolic artichoke extracts have surprisingpharmacological and biochemical effects, such as a marked antioxidative potential and cancerchemopreventive properties. Hence, there is considerable interest in improving our understanding ofpolyphenol biosynthesis and its regulation in artichoke, to enhance the levels of these bioactivemolecules in plants used as food, starting from the evaluation of phenolic content of differentgenotypes. A combination of genetic and biochemical analyses was used to help identifying genesinvolved in the accumulation of polyphenols and characterizing different artichoke varietal typesdiffused in the Mediterranean region by polyphenolic content.Six traditionally cultivated varieties of artichoke (Mola, Tondo di Paestum, Sant'Erasmo,Bianco di Ostuni, Blanca de Tudela and Violet de Provence) were selected, and qualitative andquantitative evaluations of phenolic profile were carried out on different artichoke tissues (threebract orders and receptacle of flower heads and leaves) in several developmental stages, by HPLCand mass spectrometry analyses. Our results show that polyphenols content mainly depend on thegenotype and part of the plants. Total polyphenols were more abundant in leaves than in heads,while cynarin and chlorogenic acid (CGA) were the most abundant hydroxycinnammates (mainly incapitula).The fine regulation of biosynthetic pathway leading to the accumulation of phenylpropanoidsis achieved by combinatorial actions of transcription factors (TF) belonging to various classes,among which MYB TF. Two putative MYB TF genes were isolated from artichoke (Tondo diPaestum and Locale di Mola), which showed a high similarity to MYB12 TF of other plant species(e.g. Arabidopsis, tomato). These genes were over-expressed under UV-light stress conditions.Total polyphenols content will be measured in artichoke tissues to possibly confirm correlation ofthe artichoke MYB factors with accumulation of these compounds. Heterologous expression inbacteria and quantitative real-time PCR are being performed to gain insights into the molecularmechanism of polyphenols regulation in this edible plant, contributing to future progress in thestudy of polyphenols accumulation.

Globe artichoke and leafy cardoon, two crops within the same species Cynara cardunculus, are traditionally cultivated in the Mediterranean region and play a significant role in the agricultural economy of this area. The two cultigens have different reproductive systems: artichoke is generally vegetatively propagated, while leafy cardoon is seed propagated. The domestication events underlying the origin of both artichoke and cultivated cardoon from their wild relative and the area of occurrence are not yet fully understood. The aim of this study was to investigate population structure in wild cardoon, globe artichoke and leafy cardoon material and infer domestication events.+ Methods Thirty-five microsatellite (simple sequence repeat) markers, distributed in the C. cardunculus genome, and a large geographical and numerical sampling in southern Europe and North Africawere used to assess population structure and iversity.+ Key Results The results suggest the presence of two distinct domestication events for artichoke and leafy cardoon, and also suggest a new possible scenario, with western wild cardoon having originated from cultivated cardoon escaped from cultivation. Evidence was found for a demographic bottleneck in the past history of globe artichoke.+Conclusions The results shed newlight on the relationships between the three taxa of C. cardunculus and highlight relevant aspects on the evolution of domestication of two crops with a different reproductive system within the samespecies. It is proposed that the probable centre of origin of artichoke is located in southern Italy, probably Sicily.

A cationic soluble peroxidase isoenzyme (CysPrx) has been purified and characterized from artichoke (Cynara cardunculus subsp. scolymus (L.) Hegi) leaves by combination of aqueous two phase extraction, ion exchange chromatography, and gel filtration. The purification fold was 149 and the activity recovery 5.5%. CysPrx was stable from 5 to 45 °C with a pH optimum around 5.5; the pI was 8.3 and the MW of 37.7 ± 1.5 kDa. MALDI-TOF MS analysis provided partial peptide sequences and resolved CysPrx isoenzyme into two putative isoforms. The presence of these isoforms was confirmed by the isolation of full-length cDNA encoding CysPrx that generate two slightly different sequences coding for two putative CysPrx: CysPrx1 and CysPrx2. The obtained MS peptides showed a 35% coverage with 100% identity with the two CysPrx deduced protein sequences. A molecular modeling analysis was carried out to predict in silico the protein structure and compare it with other plant Prx structures. Considering that CysPrx is quite stable, the study carried out in this paper will offer new insights for the production of the recombinant protein for utilization of CysPrx as an alternative Prx for food technology, biomedical analysis and bioremediation.

IFN-induced depression is a suitable model for investigating vulnerability to depression. Weaimed at investigating the role of two vulnerability factors, life time mood disorder (LMD) and 5-HT-related gene polymorphisms in treated patients with infection by Hepatitis C Virus (HCV).Methods: Depressive symptoms of 130 consecutive HCV patients with no current psychopathology weremeasured during treatment with interferon and ribavirin. At baseline, LMD and 3 genotypes (5-HTTLPR, HTR1A, and TPH2) were also assessed.Results: Subgroups of43 patients with LMD, 96 with HTR1A-G allele, and 12 with both LMD and HTR1A-G homozygosity scored significantly higher to depression compared to the remaining patients duringAntiviral therapy. At the multiple regression analysis, LMD and HTR1A-G, whether separately or combined together, explained a similar amount of 10-22% of depression score variance, after controllingfor the associated variables(age and gender).Limitations: HCV patients referred to a tertiary care center are not representative of all patients withchronic hepatitis C. Mediating factors, including pro inflammatory cytokines and other potentiallyrelevant gene polymorphisms, could not be evaluated. Patients were not stratified by degree of liverinflammation. LMD diagnoses were not cross-checked with medical records and IFN-induced depressionwas measured with a self-report scale only.Conclusions: History of mood disorders and HTR1AG allele variation, the C-1019G polymorphism of thetranscriptional control region of the 5-HT1A receptor, independently predicted the incidence of IFN-induced depression in HCV patients, whether separately or jointly considered and although notreciprocally associated

The main aim of this work was to isolate the entire chloroplast (cp) genome sequences of Cynara taxa, in order to retrieve the most useful resources for phylogenetic and population studies. Twenty-Two samples were analysed, of which 18 belonged to the three taxa of C. cardunculus L., and the remaining to four wild species (C. syriaca, C. cornigera, C. baetica, and C. humilis). First, we obtained reads by wholegenome and a BAC clone of the 'Brindisino' globe artichoke by means of highthroughput sequencing. This genome was assembled, its size was 152,529 bp and it consisted of two single-copy regions separated by a pair of inverted repeats of 25,155 bp. The large and the small single-copy regions spanned 83,578 and 18,641 bp, respectively. For the other genotypes, a long-range PCR approach was employed for producing partially overlapping amplicons. These fragments were high-Throughput sequenced and reads were assembled for each genotype using de novo and referenceguided methods. No structural differences regarding gene content and order were highlighted among all analysed genotypes. A conserved structure was also observed among other eight Asteraceae species analysed. Cynara cp genome consists of 114 unique genes, including 30 tRNA, 4 rRNA, and 80 predicted protein-coding genes. The comparative analysis among all newly sequenced genomes allowed the discovery of a large number of informative loci, in terms of variable microsatellites, INDELS, and SNPs. The Cynara complete cp sequence allowed the placing of this genus in the Asteraceae family tree and, thanks to the set of samples considered helped clarify the phylogenetic relationships within this genus.

The complete chloroplast genome of the wild thistle Cynara humilis L. (Asteraceae) is presentedin this study. The genome is 152,585 bp in length and has a quadripartite structure composedby a large single-copy (LSC) of 83,622 bp, a small single-copy (SSC) of 18,651 bp and twoinverted repeats (IRb/a) of 25,156 bp each. The GC content corresponds to 37.7%. The amountof unique genes is 114, in which 17 are duplicated in the IRs, for a total of 131 genes.A maximum parsimony phylogenetic analysis revealed that C. humilis chloroplast genome isclosely related to that of the globe artichoke within the Carduoideae subfamily

The allotetraploid durum wheat [Triticum turgidum subsp. durum (Desf.) Husn.] is a highly economically important species especially in the Mediterranean basin. However, its genomics, transcriptomics and in particular microRNAome are still largely unknown.Results: In the present work, two small RNA libraries from durum wheat Ciccio and Svevo cultivars were generated from different tissues at the late milk (Z77) developmental stage. A total of 167 conserved and 98 potential novel miRNAs were identified in the two libraries and interestingly, three novel miRNAs were found to be derived from ribosomal RNA. Putative target genes were predicted for conserved and novel miRNAs, the majority of which interact with nucleic acids, according to GO terms relative to molecular function. Quantitative qPCR analysis showed that several miRNAs identified were differentially expressed in the mature (Z77) developmental stage compared to young (Z14) tissues. Moreover, target gene expression analysis suggested that in roots, the putative genes encoding for the SQUAMOSA SPL2 and TGA1 proteins are regulated by ttu-miR156n, while MYB3 transcription factor by ttu-miR319f. Additionally, the Photosystem II P680 chlorophyll A apoprotein gene showed an expression level negatively correlated to that of ttu-novel-48 in leaves.Conclusion: Our results suggest that, in durum wheat, these genes may play important roles in root/leaf development and are subjected to miRNA regulation. The prediction of novel miRNAs putatively derived from ribosomal RNA opens new perspectives on the study of plant miRNAs.

Plant microRNAs (miRNAs) are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA) libraries can detect even poorly expressed miRNAs.No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots.

Nitrogen (N) is a critical macronutrient for plants and its utilization is fundamental to crop productivity. In the last decades, N fertilizers have been extensively used to increase both grain yield and grain protein content in wheat, helping support the human population growth while causing a serious pollution problem. Wheat is one of the most used cereal crops for the important amount of proteins it provides in human diet, and durum wheat (Triticum turgidum ssp. durum) is particularly important in the Mediterranean region for the production of pasta and other typical dishes. In order to use gene knowledge to improve N-use efficiency, it is crucial to increase the understanding of how this crop responds to growth under different N conditions. Here, high-throughput sequencing was used to provide a determination of transcript levels of N-starvation-induced genes. We constructed 24 cDNA libraries from roots, leaves/stems, flag leaf and spike of durum wheat cultivar Svevo plants grown under N-deficient or standard conditions during the grain filling stage. A RNA-Seq experiment on three biological replicates for each tissue provided a large amount of sequencing data, which were used for a differential expression analysis. A total of 4968 transcripts were found differentially expressed between treated and control samples. Among them, 3270 belonged to root tissues, while the other 1698 were genes found in leaves/stems, flag leaf and spike tissues. Results highlighted that 3057 genes were differentially expressed uniquely in root tissues, 681 in leaves/stems, 298 in flag leaf and 234 only in spike tissues. The Gene Ontology analysis revealed a number of categories involved in N-stress in all organ types, the most frequent down-regulated transcripts functioned for thiamin biosynthetic and metabolic processes, photosynthesis, protein processing, localization and maturation, photorespiration, respiratory chain. Other categories such as CTP biosynthetic and metabolic processes, DNA synthesis, and amino acid metabolism were also represented. Selected genes were analysed in quantitative PCR in order to confirm their expression levels. Our results provide valuable information about N-starvation-responsive genes involved in different genetic pathways and concerning several plant organs of durum wheat. This data will help shed light on signal transduction pathways related to N-utilization and will be useful for identifying candidate genes for crop breeding.

Nitrogen (N) is a key macronutrient representing a limiting factor for plant growth and developmentand affects productivity in wheat. In this study, durum wheat response to N chronic starvation duringgrain filling was investigated through a transcriptomic approach in roots, leaves/stems, flag leaf andspikes of cv. Svevo. Nitrogen stress negatively influenced plant height, tillering, flag leaf area, spike andseed traits, and total N content. RNA-seq data revealed 4,626 differentially expressed genes (DEGs).Most transcriptomic changes were observed in roots, with 3,270 DEGs, while 963 were found in leaves/stems, 470 in flag leaf, and 355 in spike tissues. A total of 799 gene ontology (GO) terms were identified,180 and 619 among the upregulated and downregulated genes, respectively. Among the mostaddressed GO categories, N compound metabolism, carbon metabolism, and photosynthesis weremostly represented. Interesting DEGs, such as N transporters, genes involved in N assimilation, alongwith transcription factors, protein kinases and other genes related to stress were highlighted. Theseresults provide valuable information about the transcriptomic response to chronic N stress in durumwheat, which could be useful for future improvement of N use efficiency

Cynara cardunculus is a complex representing a single biological species which includes the wild cardoon (C. cardunculus var. sylvestris), and two crops: the globe artichoke (C. cardunculus var. scolymus) and the cultivated leafy cardoon (C. cardunculus var. altilis). Wild cardoon taxon is distributed across the Mediterranean Region and mainly two possible genepools have been recognized, the eastern-and the western-Mediterranean types. Morphologically, wild cardoons from Spain and Portugal can be easily distinguished from the other wild cardoons and are quite similar to cultivated cardoon. During the domestication process, the artichoke was selected for the gigantism of flower heads and the loss of spinosity, while the leafy cardoon for bigger leaf stalks and less spines. Another major difference between the two crops is related to their reproductive system, the artichoke being traditionally clonally propagated, the cultivated cardoon being propagated by seeds. Population analyses obtained from a wide range of wild cardoon, artichoke and leafy cardoon samples, and based on SSRs distributed on all the Cynara cardunculus genome, together with data obtained from NGS chloroplast and nuclear genome sequencing suggest independent domestication events for artichoke and cardoon. In the most plausible scenario, the eastern wild cardoon would have given rise to both crops, and artichoke would have been domesticated in southern Italy/northern Africa, probably Sicily in earlier times compared to leafy cardoon. Among the three taxa, artichoke is the only one to exhibit a bottleneck, although not a severe one. Molecular data also suggest that western wild cardoon would represent a feral form of the cultivated leafy cardoon. Relevant aspects on the evolution of domestication under different selective pressures and distinct reproductive behaviors are discussed.

L"Italia possiede il più ampio pool genico di carciofo coltivato, includendo così una ricca biodiversità cinaricola. Le regionicentro-meridionali conservano una moltitudine di tipi varietali ed ecotipi che si differenziano sulla base di tratti morfologicie per composizione chimica, e di conseguenza presentano diverse proprietà nutrizionali e attività farmacologiche. Il carciofoè una rilevante fonte di polifenoli, composti bioattivi dall"elevata capacità antiossidante e dunque molto salutari per l"uomo.Acquisire informazioni sul contenuto e sulla biosintesi di polifenoli in differenti varietà di carciofo, risulta quindi difondamentale importanza per la promozione e l"utilizzo di risorse genetiche autoctone di questa specie. A tale scopo sonostate effettuate dettagliate analisi biochimiche per caratterizzare sei diverse varietà di carciofo (Mola, Tondo di Paestum,Sant"Erasmo, Bianco di Ostuni, Blanca de Tudela e Violetto di Provenza) diffuse non solo in Italia ma anche in altri Paesidel bacino del Mediterraneo. Analisi qualitative e quantitative (HPLC-DAD-ESI-MS/MS) del profilo polifenolico diciascuna varietà hanno evidenziato che i polifenoli presenti in quantità maggiore sono acido clorogenico, cinarina, luteolin7-O-rutinoside e luteolin 7-O-glucoside. Inoltre il contenuto varia a seconda del genotipo e del tessuto preso inconsiderazione (Negro et al. 2012). Uno studio approfondito è stato da tempo avviato su diversi geni coinvolti nellaproduzione di polifenoli (Sonnante et al. 2008, Sonnante et al. 2010) e, più di recente, sulla regolazione della biosintesi ditali composti nei diversi tessuti di carciofo. Da una libreria di cloni ricombinanti BAC sono stati isolati due geni codificantiper putativi fattori di trascrizione MYB coinvolti nella biosintesi di flavonoli e proantocianidine. Tali geni sono staticaratterizzati strutturalmente. La loro espressione è stata analizzata in diversi tessuti di carciofo tramite PCR quantitativaReal-Time. Le corrispondenti proteine ricombinanti sono state inoltre espresse in batteri, per stabilirne la funzione in vitrotramite lo studio delle interazioni proteine/DNA mediante saggi di mobilità elettroforetica. Bibliografia: Negro et al. (2012)Journal of Food Science 77: 244-252. Sonnante et al. (2008) Physiologia Plantarum 132: 33-43. Sonnante et al. (2010) PlantPhysiology 153: 1224-1238.

Le attività svolte dal CNR-IBBR nell'ambito del progetto BiodiverSO finanziato dalla regione Puglia (Misura214 azione 4 sub a, PSR 2007-2013) hanno riguardato un gran numero di specie e forme diverse di piante orticolereperite sul territorio pugliese durante le numerose missioni di raccolta di germoplasma.Particolare interesse ha suscitato il fagiolino dall'occhio [Vigna unguiculata (L.) Walp.], presente in Puglia condue tipologie: il fagiolino pinto e il fagiolino al metro. Si tratta di una specie di origine africana, le cui forme coltivatevengono suddivise in 5 gruppi; il fagiolino pinto appartiene al gruppo unguiculata, mentre il fagiolo al metro appartieneal gruppo sesquipedalis.Nonostante considerato raro e sempre più sostituito dal comune fagiolino di origine americana (Phaseolusvulgaris L.), il fagiolino pinto è ancora largamente apprezzato e spunta prezzi più alti nei piccoli e grandi mercatiortofrutticoli. In Puglia, il fagiolino dall'occhio viene consumato esclusivamente sotto forma di baccello fresco, alcontrario di quanto avviene nel centro-nord Italia, in Africa, in Asia ed in altri Paesi, dove viene impiegato soprattuttocome seme secco.Il monitoraggio del territorio regionale ha consentito la raccolta di oltre 22 campioni di varietà localiappartenenti alla specie V. unguiculata. Per quelle più note e/o tradizionali è stato definito l'areale di diffusione, lepratiche agricole, gli usi e le tradizioni legate al consumo di questo prodotto. La sua coltivazione, per la gran partelimitata a piccoli orti e destinata all'autoconsumo, è praticata in particolare nella provincia di Bari, in Salento e inmisura minore nelle altre province pugliesi. Il fagiolino a metro risulta molto meno diffuso ed è stato rinvenuto inpochissimi siti nelle province di Bari e Brindisi.La maggior parte delle varietà locali presenta semi generalmente di colore bianco opaco con una caratteristicamacchia scura intorno all'ilo ("occhio", da cui il nome). Altre mostrano seme nero, marrone chiaro o molto scurotendente al nero occasionalmente con variegature o piccole macchie circolari. In alcune forme l'ilo presenta unacolorazione differente.Dalle indagini molecolari basate su marcatori SSR (microsatelliti), questa coltura ha mostrato una discretavariabilità genetica tra le accessioni raccolte in diversi siti e più raramente anche all'interno della stessa accessione,dove possono essere presenti forme differenti volutamente conservate e riseminate di anno in anno dagli agricoltori.

Il carciofo e il cardo coltivato appartengono entrambi alla specie Cynara cardunculus e si differenziano siamorfologicamente che per il metodo di propagazione. Il carciofo gioca un ruolo importante nell"economia agricola delleregioni del bacino del Mediterraneo. L"utilizzo di marcatori molecolari ha permesso di conoscere in maniera piùapprofondita la storia evolutiva di queste due piante coltivate. I dati molecolari denotano uno scenario evolutivo nel quale ilcarciofo (Cynara cardunculus var. scolymus (L.) Fiori) e il cardo coltivato (Cynara cardunculus var. altilis DC) derivano dauna differente pressione selettiva operata dall"uomo: per il capolino di grande dimensione nel caso del carciofo e per lacosta delle foglie ingrandita per il cardo coltivato. Gli eventi di domesticazione sottolineano l"origine di entrambe dal cardoselvatico (C. cardunculus var. sylvestris Lam), distribuito in tutto il bacino del Mediterraneo, anche se l"area didomesticazione appare incerta. È stata valutata la variabilità genetica e la struttura di popolazione in C. cardunculusgenotipizzando 801 individui rappresentanti 60 popolazioni di cardo selvatico e varietà di carciofo e cardo coltivatoattraverso marcatori microsatelliti distribuiti su tutto il genoma. I dati genotipici sono stati analizzati per la costruzione di unalbero filogenetico UPGMA e per l"analisi delle coordinate principali (PCA) che hanno permesso di studiare le relazionigenetiche tra il progenitore selvatico e i due taxa coltivati. Infine è stata analizzata la struttura delle popolazioni tramiteSTRUCTURE. L"analisi dei marcatori molecolari ha evidenziato in carciofo un alto livello di eterozigosità osservata (Ho) e,in media, un numero piuttosto basso di alleli, probabilmente dovuto alla perdita di variabilità genetica durante il processo didomesticazione. Al contrario è stato osservato un più basso livello di eterozigosità nel cardo coltivato e nel cardo selvatico,indicando che è presente un certo livello di autogamia nei cardi. L"analisi filogenetica evidenzia una chiara suddivisione delmateriale analizzato. Si distinguono infatti tre raggruppamenti principali: il primo che include i carciofi, il secondo i cardicoltivati e cardi selvatici occidentali (Spagna e Portogallo), e il terzo comprendente i cardi selvatici orientali (Itali, Grecia,Malta e Tunisia). L"analisi STRUCTURE ha confermato la suddivisione del materiale analizzato negli stessi tre gruppiprincipali. I risultati ottenuti evidenziano un"alta similarità sia genetica che morfologica tra cardi selvatici occidentali e cardicoltivati. Questi dati hanno portato a supporre che il cardo selvatico occidentale possa derivare da una naturalizzazione dellaforma coltivata, diffusa in Spagna e in Portogallo. In conclusione si può ipotizzare che il cardo selvatico orientalerappresenti l"unica vera forma selvatica da cui hanno avuto origine sia il carciofo che il cardo coltivato.