Chickpea (Cicer arietinum L.) is a grain legume widely cultivated in the warm temperate and semi-arid regions for its high nutritional value, given the 15-25% high quality protein seed content. In Italy, about 3,500 hectares are cultivated with chickpea, mainly in southern and island regions. We predisposed a chickpea germplasm collection consisting in 50 different south Italian landraces and other 100 different feral forms and landraces originated from different world areas. In order to assess the genetic variability on the whole collection, we used a set of microsatellites markers (SSR) specific for chickpea. A quite large polymorphism of SSR in the different genotypes was observed and this result, will be further exploited to study the genetic distance among genotypes. At the same time, to evaluate the response to drought stress of the whole collection, an assay on seedlings was set up in growth chamber, treating the plants with different concentrations of NaCl and PEG solutions. These experiments allowed to identify two accession (red-coloured seeds) showing a high degree of tolerance to the experimental conditions used. Moreover, a trial was also conducted for resistance to the seed-beetle Callosobruchus maculatus (Fabr.) that causes considerable economic losses worldwide. A preliminary laboratory bioassay was conducted on eighteen genotypes, that were evaluated by measuring the percentage damage to seeds, founding one black seed genotype (CA-100) that exhibited a complete resistance. This promising genotype will be studied at molecular level to highlight the bases of the resistance in this interaction plant-insect, and it will be incorporated in future breeding programme as bruchid resistance in chickpea lines.

Small interfering RNAs (siRNAs), play a vital role in epigenetics of plant virus-host plant interactions. It has been extensively studied at both the transcriptional and post-transcriptional levels. In plants, siRNAs initiate and manage gene silencing by directing DNA methylation and/or histone methylation. In Arabidopsis, the ~24 nt siRNAs directs DNA methylation (RNA-directed DNA methylation, RdDM) and chromatin remodeling at their target loci. Recent advances in highthroughput sequencing techniques has enabled thorough exploration of small RNAs populations and allow rapid analysis of massive datasets to assemble complete full-length genome sequence for different plant species. This large database of sequence information also allows identification of genome regions specifically matched by siRNAs that likely differ among tolerant, resistant or susceptible hosts and advance epigenetic studies on diseased plants. Resistance to Citrus tristeza virus (CTV), the most severe virus affecting Citrus spp., associated with a single dominant gene locus Ctv occurring in Poncirus trifoliata while all Citrus spp. are considered susceptible. This locus contains 22 putative genes, but their regulation and mechanism for resistance remains unknown. In our study, CTV was graft-inoculated on Carrizo citrange (Poncirus trifoliata x C. sinensis (I think) ) and C. aurantium (sour orange) seedlings, and the population of siRNA characterized by high-throughput sequencing using an ILLUMINA platform. The Ctv-derived siRNA (~2% of the total short reads) were dominated in both hosts by the 24-nt. However, CTV infection caused an increase in accumulation of 24-nt siRNA sequences homologous to the Ctv gene in Carrizo but it decreased in sour orange. Distribution of the 24nt along the Ctv gene locus (282Kb) had a clearly different distribution between the two host. The predominant hot spot of siRNA in Carrizo mapped in the putative gene Ctv-20, whereas in sour orange it associated to the intergenic region between the putative genes Ctv-11 and Ctv-12, where a Copia-like retrotransposon C is located. This distribution profile was conserved for each species between CTV-infected and uninfected plants but, as previously mentioned, the frequency of the 24nt siRNAs was altered by the presence of the virus. We supposed that the different profile of 24nt between the two host in the locus ctv is due to RdDM mechanisms. To demonstrate the methylation status of the resistance locus we performed a bisulfite treatment of DNA. in which unmethylated cytosine was converted to uracile, while methylated cytosine did not react. A methylcytosines mapping was carried out on Ctv-11 and Ctv-12 sequences. By specific software were found 5 different CpG islands in the Copia-likeretrotransposon sequence and 42 primer pair were designed. The PCR analyses have been carried out using MSP and BSP primers followed by combined bisulfite restriction analysis (COBRA).

Abscisic acid (ABA) is associated with regulating plant adaptive responses to various environmental stresses. In particular, drought stress signals are transmitted through at least two pathways: one is abscisic acid (ABA)-dependent, and the other is ABA-independent. In the first case, drought stress increases the cellular ABA levels, which induces the expression of drought stress-responsive genes, such as 9-cisepoxycarotenoid dioxygenase (NCED) and zeaxanthin epoxidase (ZEP). These genes belong to the carotenoid biosynthesis scenario. To date, most research of grapevines has focused on the physiological mechanisms of ABA during fruit ripening. Our interest is on studying the role of NCED and ZEP genes as candidate genes exhibiting up-regulation upon drought-stressed conditions. At the same time, several plant physiological parameters, such as leaf water status (ψl), net assimilation rate (A), stomatal conductance (gs), transpiration rate (E), and soil water potential (ψs), were monitored. To explain the complex molecular pattern undergoing these physiological changes, we investigated the levels of expression of one candidate gene encoding for VvNCED1. The results provided evidence of a different transcriptional pattern of the gene between the control and stressed plants, leading to a major accumulation of NCED1 transcripts in the stressed plants.

Mitochondria are considered to be the powerhouse of the cell. In mitochondria the degradation of the carbohydrates is coupled with the synthesis of high-energy molecules such as ATP, which powers up the vast majority of chemical reactions of the cell. The biogenesis and function of mitochondria is the result of coordination between the nuclear genome, where there is more than 95% of mitochondrial genes, and the mitochondrial genome. This coordination is particularly rigorous also because the protein complexes both of respiratory chain (OXPHOS) and mitochondrial ribosomes are assembled according to wellestablished stoichiometric relationships. In higher eukaryotes has been reported that this type of coordination is achieved mainly at the transcriptional level. Various experimental observations suggest the presence of a fine-tuned communication between mitochondrial and nuclear genomes that results in the interdependent expression of OXPHOS genes and ribosomal genes encoded by mtDNA and nuclear genome. Having previously identified, by using a series of bioinformatics analysis, a group of five DNA motifs that, for the distribution and frequency in putative regulatory regions of OXPHOS, TCA, and ribosomal genes, could be regarded as excellent regulatory sequences, we decided to validate this analysis by means of the One-Hybrid Assay. The One-hybrid assay was performed using two different DNA motifs as bait, the Site II motif (ref) and the Ac/tTGT motif. This analysis showed that some proteins are able to interact "in vivo" with those motifs. We have identified a transcription factor belonging of the family AP2/ERF/B3 (AtERF#115) that binds Site II motif. The transcription factor is part of the regulative cascade of ethylene, this finding could help explain the mechanisms by which ethylene is able to influence the respiration (e.g. climacteric). We have also identified two transcription factors belonging to the family of bZIP (AtbZIP18 and AtbZIP52), when we used the Ac/tTGT motif as a DNA-bait. The transcription factors are involved in the process of growth and development of the plant. The family of bZIP transcription factors is required in the process of growth and development of the plant and therefore it can be possible that AtbZIP18 and AtbZIP52 may be involved in response to those processes.

The widespread occurrence of epigenetic alterations as a consequence of polyploidization in plants indicates that DNA methylation systems may be perturbed by polyploidy changes. DNA methylation genes are involved in many vital developmental and physiological processes of plants. In this work, we seek novel information on epigenetic consequences of autopolyploidization in alfalfa (Medicago sativa). In particular, genes responsible for the DNA methylation status are studied in 2x, 3x and 4x genotypes progenies obtained by crossing two 2x plants that produce both n and 2n eggs and pollen, respectively. To identify DNA methyltransferase genes and their expression patterns we examined some of the elements of three major DNA methyltransferase families MET1, CMT and DRM. Methylation at CG nucleotides is maintained in plants by the the enzymes of the MET1 family typical of higher eukariotes. The second family, called Domain Rearranged Methyltransferase (DRM) has characteristic rearrangement of conserved motifs in the catalytic domain and probably catalyzes methylation of native DNA. The chromomethylases (CMT) are unique of higher plants. These enzymes maintain methylation of CHG trinucleotides. In silico searches have lead to the identification of M. sativa methyltransferase genes homologous to known plant methyltransferase genes. Oligonucleotides have been designed from these sequences in order to analize the expression pattern of these genes. Gene expression changes induced by polyploidization are being investigated using qRT-PCR and expression data will be validated and jointly analyzed to identify ploidy-affected genes.

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5' UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its e V ects on the activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to a V ect signi W cantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.

Most plant species are either polyploidy or have experience polyploidization during their evolution: this indicates a selective advantage of polyploidization in plants. Polyploidization is known to affect gene expression in several ways, including epigenetic mechanisms. The best described epigenetic mechanism is DNA hypermethylation, or the predominant marking of CpG, CpCpG, CpHpHp, and CpNpG motifs in DNA. The sole methyl donor for all eukaryotes, S-adenosylmethionine, provides the methyl group essential for such Arabidopsis thaliana enzymes as CHROMOMETHYLASE 3 (CMT3) and DOMAINS REARRANGED METYLASE 2 (DRM2) responsible for marking of CpNpG motifs, and METHYLTRANSFERASE 1 (MET1), the marker of CpG islands. This study is aimed at gathering new information on the general “methylation state” in Medicago sativa, an important autopolyploid forage species affected by chromosome doubling via sexual polyploidization. 2x and 4x progenies obtained by crossing 2x plants that produce both n and 2n eggs and pollen respectively, are used. The Medicago truncatula sequence database of CpG islands in MET, CMT, DRM and DEMET (DEMETHYLTRASFERASE) family was analyzed by two different bioinformatic approaches to assess the methylation status of virtually any group of CpG sites within CpG island; this is a step toward understanding the methylation-affected biological processes. Methylase and demethylase gene expression changes were investigated by RT-qPCR and the first results will be presented. These studies can be useful to understand the basis for the polyploidy advantage in agricultural crops.

Polyploidization is a widespread mechanism in eukaryotes and is predominant in flowering plants. Polyploids are very common among plants and are produced by multiplication of a genome derived from a single species (autopolyploids) or combination of two or more divergent genomes from different species (allopolyploids). Alteration in DNA methylation could regulate gene expression or other important epigenetic processes including dosage compensation, genomic imprinting, nucleolar dominance, de-repression of dormant transposable elements and alterations in chromatin structure among others. Polyploidization is known to involve altered DNA methylation in plants and recent studies provided evidence for changes in newly formed allopolyploids. Several genes responsible for the DNA methylation status have been studied so far. We investigated some members of the three major DNA methyltransferase family genes (MET) and their expression pattern in tetraploid plant of alfalfa (Medicago sativa L. 2n=4x=32) obtained by crossing diploid parents producing 2n gametes (bilaterla sexual polyploidization). Gene expression changes induced by polyploidization have been investigated using qRT-PCR and expression data have been validated to identify genes whose expression is affected by ploidy changes. We also carried out an evolutionary analysis on a collection of methyltransferase homologous genes identified by database searching using as a query M. sativa genes. A comprehensive overview of the distribution of conserved domains was obtained as well as phylogenetic relationships between members of the MET family. Previous observations indicate that the genome methylation status is affected by polyploidization in alfalfa. This expression study may contribute to answer the question of how this occurs, and provide useful information for crop improvement

Le larve di Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) infestano le radici di drupacee causandone spesso la morte. Ciascuna femmina depone oltre un migliaio di uova nel suolo presso il colletto e nei primi millimetri di profondità. La temperatura influenza ampiamente la fisiologia dell’adulto, il conseguimento della maturità sessuale, la fertilità e altre attività e comportamenti delle larve e dell’adulto. In generale possiamo dire che queste sofisticate risposte metaboliche, si articolano principalmente attraverso variazioni della espressione genica e, non meno importanti, anche dall'attivazione di meccanismi epigenetici. La metilazione del DNA, in quanto uno dei meccanismi epigenetici, è parte integrante della risposta degli esseri viventi agli insulti ambientali. Anche per gli insetti vale il dogma che l'abilita' di alterare rapidamente e reversibilmente i pattern d'espressione di numerosi geni, è la chiave della flessibilità della risposta all'ambiente circostante. Non potendo disporre di alcuna informazione specifica sul DNA del buprestide e volendo saggiare la risposta epigenetica allo stress termico e nonché alla ovo-deposizione si è deciso di applicare la tecnica RAPD per identificare sonde genomiche in grado di osservare polimorfismi sul DNA di insetti sottoposti a blando stress termico. Adulti di C. tenebrionis sono stati prelevati da Mariotto (Ba) e Ripalta (FG) nel settembre 2010 e allevati in condizioni controllate (23 o 28±1°C, 16 ore di luce e 8 ore di buio, UR 60±10%) per una settimana. Gonadi di maschi e femmine, selezionati in base alla temperatura di allevamento e al sesso, sono state destinate all’estrazione del DNA genomico. Per la tecnica RAPD sono stati utilizzati 44 primers OPERON delle serie OPY, OPU, OPW e OPX. Inizialmente, è stato saggiato il DNA genomico dei maschi allevati a 28°C. Dai primi esiti è stato possibile selezionare 18 primers in base ai profili elettroforetici con il maggior numero di bande di restrizione. Nelle successive analisi, i primers selezionati sono stati saggiati sul DNA genomico tal quale e su DNA, digerito con le endonucleasi di restrizione HpaII e MspI (enzimi sensibili alla metilazione), di femmine allevate a 28 o 23°C. Sono stati scelti i profili RAPD che presentavano differenze tra DNA tal quale e DNA digerito, riducendo il numero dei primers a due (OPY8 e OPY12). I risultati del polimorfismo di banda sono stati confermati anche saggiando OPY8 e OPY12 sul DNA genomico dei maschi e delle femmine allevati sia a 28 che a 23°C. In questo modo le bande di DNA individuate sono state clonate e avviate al sequenziamento. Il DNA di tutti gli insetti precedentemente trattati è stato poi, digerito con due differenti coppie di enzimi di restrizione sensibili alla metilazione e ibridato con le sonde precedentemente isolate, tramite tecnica Southern, al fine di ottenere un overview degli eventuali siti genomici di C. tenebrionis differentemente metilati.

Citrus tristeza virus is one of the agent of devastating cultivated citrus trees especially if grafted on sour orange (Citrus aurantium), which appears to be the most susceptible species. The trifoliate orange (Poncirus trifoliata) appears to be resistant to the disease compared to the sour orange, the sweet orange and the grapefruit, while, the Citrange carrizo, derived from cross to C. sinensis L. x P. trifoliate, appears to be tolerant. In Citrange carrizo, the virus replicates and spreads throughout the tolerant plant without showing any symptoms. On the contrary in the resistant plants of Poncirus, the virus replication is not blocked, but it seems likely that it acts by preventing cell to cell and/or long distance moviment (Karasev et al., 2010). The resistance to the virus is due to the presence of a locus, called locus CTV, which is available an accurate genetic map (Yang et al., 2001; Rai, 2006). Within this region are present 22 genes seven of which (Ctv.4, Ctv.7, Ctv.8, Ctv.11, Ctv.17, Ctv.18 and Ctv.21), are denoted R(1-7) genes, since they show a high homology with Arabidopsis resistance genes encoding to CC-NBS-LRR proteins (coil-coil-nucleotide binding site-leucine rich repeat) (Deng et al., 2000; Yang et al., 2003). In addition, six other genes, with a high homology sequence to known function genes of other species are present. In particolar the Ctv.20 showed homology with a plant virus movement-like protein (Karasev and Hilf 2010). This membrane protein, through the formation of channels in plasmodesma, is involved in the transfer of proteins and viral RNA from cell to cell. Ctv.20 was predicted to contain three open reading frames (ORFs) by GenScan Web server (Burge and Karlin, 1997), but BLAST searches indicated that both the first and the third ORFs were highly homologous with Petunia vein-clearing virus (PVCV) ORF1 (Yang et al. 2003). Yang and colleagues (2003), performed the northern hybridization analyses using DNA fragments from the first and the third ORFs and both these fragment hybridized with the same band of about 9 kb, that presented a size compatible with the GenScan prediction (data not shown). Northern hybridization indicated that Ctv.20 and its orthologous are highly expressed in P. trifoliata and sweet orange leaves and in P. trifoliata bark tissues, but are relatively lowly expressed in the phloem of sweet orange (data not shown). The ortholog of Ctv.20 in sweet orange is about 8.5 kb, which is slightly smaller than Ctv.20 (9 kb) in P. trifoliata. CTV tends to accumulate in phloem tissue of infected plants, which suggests that Ctv.20 could also be considered as a candidate gene for Ctv resistance (Yang et al., 2003). The aim of the present research was to carry out different siRNA 21:24 nucleotides libraries by Illumina sequencing, from susceptible plants of sour orange and tolerant of Citrange carrizo ones, both healthy and infected with different strains of CTV virus. The presence of a homologous locus in susceptible species such as Citrus aurantium suggests that the mechanisms of resistance and therefore of regulating the expression of the genes present in the locus cannot be fully explained by DNA sequence alone. On this basis we moved to verify the role of siRNA through an epigenetic regulation of the methylation status of ctv locus with particular reference to Ctv.20 gene. The difference between methylated and unmethylated condition in tolerant and susceptible plants was performed by PCR analysis on DNA digested by sensitive and unsensitive enzymes to cytosine methylation.

Citrus tristeza virus (CTV) is a filamentous virion (genus Closterovirus, family Closteroviridae) that contains a single-stranded, positive-sense RNA genome of 19.3 kb consisting of 12 open reading frames (ORFs). CTV is the responsible of the current devastation of cultivated citrus trees especially of the widely used rootstock sour orange (Citrus aurantium), which appears to be one of the most susceptible species causing severe economic losses. Moreover, the relative species of trifoliate orange (Poncirus trifoliata) appears to be resistant to the disease while, the Citrange carrizo, derived from the cross between C. sinensis L. x P. trifoliate, appears to be tolerant. The resistance to the virus is due to the presence of a locus, called Ctv locus, which is available an accurate genetic map. Eight retrotransposons, 61 Simple Sequence Repeats (SSRs), more than 400 Miniature Inverted-repeats transposable Elements (mites) and several genes, with a high homology sequence to known function genes of other species are present into the Ctv locus. Among these the Ctv.20 gene showed homology with a plant virus movement-like protein. This membrane protein is involved, through the formation of channels in plasmodesma, in the protein transfer and viral RNA movement from cell to cell. The aim of the present work was to carry out different siRNA 21:24 nucleotides libraries by Illumina sequencing, from susceptible plants of sour orange and tolerant of Citrange carrizo ones, both healthy and infected with different strains of CTV virus. The presence of a homologous locus in susceptible species such as Citrus aurantium suggests that the mechanisms of resistance and therefore of regulating the expression of the genes present in the locus cannot be fully explained by DNA sequence alone. On this basis we moved to verify the role of siRNA through an epigenetic regulation of the methylation status of Ctv locus. The difference between methylated and unmethylated condition in tolerant and susceptible plants was performed by PCR analysis on DNA digested by sensitive and unsensitive enzymes to cytosine methylation

La pianta del cece (Cicer arietinum L.) è prevalentemente diffusa nelle regioni temperate calde o semi aride della terra e rappresenta tra le leguminose da granella, il legume più coltivato a livello mondiale dopo il fagiolo e il pisello. In Italia sono coltivati a cece circa 3.500 ettari, quasi tutti localizzati nelle regioni meridionali e insulari. Sotto il profilo chimico nutrizionale, il seme del cece contiene proteine (15-25% del peso secco) di qualità alimentare tra le migliori nell‟ambito delle leguminose da granella. Avendo a disposizione germoplasma di cece costituito da specie selvatiche e varietà coltivate, ottenuto da differenti Istituzioni pubbliche, abbiamo stabilito di valutarne la variabilità genetica. Questo prerequisito è importante per un futuro utilizzo del materiale in programmi di miglioramento genetico. La collezione costituita da 150 differenti individui, è stata analizzata tramite caratteri morfologici biochimici e molecolari. Sono state condotte analisi con marcatori molecolari del tipo SSR in quanto è noto in letteratura che sono capaci di distinguere bene i differenti genotipi.Nel particolare dopo l‟estrazione del DNA si sono avviate analisi PCR utilizzando differenti primer specifici per il cece (Niroj Kumar et al., 2006). In aggiunta, sono stati allestiti saggi per ottenere una stima nel tempo della tolleranza allo drought stress sulle plantule della collezione. In particolare le piantine sono state trattate a concentrazioni differenti di soluzioni di NaCl (5mM e 100 mM) e con differenti soluzioni di PEG. Infine, su una parte dell‟intera collezione, è stato condotto un test di resistenza a Callosobruchus maculatus (Fabr.). Al momento i risultati ottenuti hanno indicato la presenza di alcuni dei marcatori SSR polimorfici tra i differenti genotipi della collezione mentre i test di resistenza alla siccità hanno evidenziato due accessioni di cece rosso tolleranti. Anche il test di resistenza al bruchide ha evidenziato un cece nero completamente resistente. Il proseguo della ricerca si articolerà nell‟identificare ulteriori marcatori di classe SSR capaci di distinguere meglio i differenti genotipi e si cercherà di aumentare le informazioni su quelle accessioni risultate essere tolleranti e resistenti agli stress biotici ed abiotici attraverso analisi di RealTime PCR.