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.

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.

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.

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.

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.

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 large wealth of genetic diversity is present in the diversity centers of durum wheat. However, modern breeding selection methods are mostly based on improvement of élite lines which have a narrow genetic base; this limits a lot the genetic pool which breeders can exploit for the production of new varieties ready to face the predicted climate changes or adaptation to new cultivation areas. The oncoming changing climatic conditions claim for the need of finding new adaptation traits to both abiotic and biotic stresses to be introduced in modern élite varieties. These traits are often found in landraces stored in germplasm collections. The Eurisco database reports a total of over 17,000 durum accessions, some 6,800 of which are traditional varieties and/or landraces: Besides their incontestable usefulness, their great number hampers the possibility to explore their genepool. A different approach to reduce the number of genotypes to screen for new useful traits to use in breeding programs while maintaining a good representation of the genetic background, is the constitution of a working durum wheat collection selected by mean of a single seed descent (SSD) approach so far called SSD genotypes. The working collection, accounts for over approximately 450 genotypes representative of 40 countries mainly concentrated in the Mediterranean area and Horn of Africa. The SSD genotypes were subjected to large scale SNP screening by ILLUMINA Infinium iSelect 90k wheat chip and the data were analysed to assess the heterozygosity and the genetic diversity present in the SSD population. Moreover the population was preliminary phenotyped following field growth. Both analyses give the picture of a good level of genetic variability represented in the SSD genotypes. This makes this collection a good candidate for a new "genetool" for durum wheat breeding and research.

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.

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.