Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. To set up effective control strategy, the accurate detection and identification of the species responsible of the diseases is crucial. However, the characterization based on morphology and/or multilocus genetic approaches is time consuming, requires great expertise, and sometimes is not conclusive. Therefore, the setup of a rapid and efficient DNA-based assay might be of paramount importance. The High-Resolution Melting (HRM) analysis represents an interesting tool for the uncovering of nucleotide variations as small as one base difference, and as such, relevant to species characterization.

To characterize 31 different Aglianico accessions randomly collected in Southern-Italy, 30 ampelographic descriptors, 13 SSRs and 10 AFLP primer combinations were analysed. An appreciable variation of ampelographic descriptors was mainly revealed by mature leaf traits, while very few variations were recorded for shoot and berry traits. Similarly, all SSR loci revealed molecular monomorphism and AFLPs a very high genetic similarity (Dice Coefficient) among all the accessions considered. One of the aim of this study was to clarify the genetic assessment of Aglianico Nero and Aglianico del Vulture Nero; since they are registered as two different cultivars with distinct varietal codes at the Italian Register of Grape Varieties. Registered Aglianico Nero and Aglianico del Vulture Nero were included in the analyses, compared and used as reference material. Our plants showed that all the accessions tested, independent from the biotype, and the two registered cultivars, belong to the same genotype, suggesting that, as reported by the Vitis International Variety Catalogue, a case of synonymy occurred between Aglianico Nero and Aglianico del Vulture Nero. These cultivars could therefore be considered as a single cultivar. Moreover, the AFLP data revealed a partial match between morphological and molecular data, showing that the AFLP molecular method was able to discriminate different accessions belonging to the same cultivar.

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.

The need of accurate and reliable methods for DNA isolation and plant species identification in foodstuffs is of great importance, especially in the protection of high added value products. Fresh foods, which are not subjected to any modifications, are suitable for many kind of analysis; for processed products, such as musts, wines, olive oils, and pasta, the situation may be more complicated due to DNA fragmentation and, in the worst case, by its degradation. This work aimed to establish an exhaustive and reproducible analytical procedure for table grape DNA tracing in industrial musts. Three different DNA extraction methods were initially compared and DNA was tested in PCR for its suitability for the amplification reaction of microsatellite markers or simple sequence repeats (SSRs). An optimized DNA extraction method for microsatellite amplification was developed and adapted for industrial musts. Two SSR-based molecular methods, High Resolution Melting and capillary electrophoresis, were tested and the markers VrZAG62 and VrZAG79 were found to be the most informative. High Resolution Melting analysis, here applied for the first time on musts, proved to be the method of choice for a preliminary screening using four cultivars chosen as references and different DNA mixtures prepared in laboratory. Capillary electrophoresis, providing allele size, allowed a fine genotyping of musts in comparison with reference cultivars. The LOD6 of a single grape cultivar in mixture with other varieties was also determined at 2.5 ng. Merging the information of the two molecular analyses applied to real samples, we demonstrated that is possible to discover case of musts adulterated with table grapes, and we propose our procedure in controlling musts quality and origin certification.

Grapevines (Vitis vinifera L.) produce non-climacteric fruit that exhibit a double sigmoidal pattern of growth. Ripening occurs during the second growth phase when grapes change colour, start to soften, accumulate reducing sugars, metabolise organic acids and synthesise flavour compounds. All these biochemical and physiological changes affect the quality of the fruit and therefore of the wine. Although the physiological processes underlying the ripening have been described the mechanisms that control the ripening of grape berries are not well known. Abscisic acid, ethylene and brassinosteroids are considered as promoters of ripening, as treatments of immature berries with these hormones can advance ripening. In grape, auxin levels are high early in development, then decline towards the onset of ripening (veraison). Indole-3-acetic acid (IAA) is the most abundant auxin in grape berries. Auxins can delay ripening when applied at an appropriate time prior to veraison. One important mechanism for controlling the levels of free, biologically active IAA is its enzymatic conjugation to amino acids. GH3 enzymes, encoding IAA-amido synthetases, are responsible for this conjugation. Previous phylogenetic analyses of Arabidopsis thaliana GH3 proteins classified them into three groups based on sequence similarity. Group II enzymes have been shown to be active on IAA and a member of group I conjugates jasmonic acid to amino acids. In order to elucidate the involvement of GH3 genes in grape berry ripening, we studied seven GH3 genes, six of which are IAA-amido synthetases, the other is a jasmonic acid-amido synthetase. The primary objective was to determine the subcellular localization of these enzymes. GFP-protein fusion constructs for all seven enzymes were transiently expressed in capsicum by biolistic bombardment and the transformed cells were scanned by fluorescence microscopy. All of these proteins displayed a cytosolic localization, confirming the in silico prediction. In order to further understand the likely function of these genes their expression patterns were analysed in different tissues comparing the varieties Shiraz and Cabernet Sauvignon. All of the IAA-amido synthetase genes showed different patterns of expression suggesting that although they all conjugate IAA to amino acids there is a degree of specialisation at the organ level.

In oil-mills, olive-pomace recentrifugation is a common way to reduce pomace moisture and, at the same time, to recover the oil therein. According to current rules, the obtained oil is defined as “crude olive-pomace oil.” The aim of this work is to verify the effect of recentrifugation on specific chemical and molecular parameters of the crude olive-pomace oil, by comparing it with the corresponding virgin olive oil obtained from the same olive lots. In particular, the following were considered: (i) the polar compounds of the oils that include compounds originated from oxidative and hydrolytic degradation, analyzed by highperformance size exclusion chromatography (HPSEC), and (ii) the profile of DNA microsatellite molecular markers that was analyzed by using theHigh ResolutionMelting (HRM) technique.Theobtained results evidenced the significantly higher hydrolytic degradation of crude olive-pomace oil, compared with the corresponding virgin olive oil, but at an extent unlikely able to allow the detection of fraudulent admixtures with virgin olive oils. In addition, the findings demonstrated the feasibility of the application of the HRM analysis of DNA microsatellites to crude olive-pomace oil, able to reveal the alteration of the declared varietal profile of a virgin olive oil sample by simply checking the HRM curve profiles.

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).

Olive (Olea europaea L.) is a subtropical woody species distributed throughout the Mediterranean regions whose remarkable importance is due mainly to the oil production from its drupes. The ancient origin and the ancestral hybridisation between different species of genus Olea and between genetically distant populations has originated numerous varieties. Unlike other crops, olive germplasm has not suffered any genetic erosion because a turnover with new genotypes has not occurred and old plants are able to survive for a long time without cultivation. Therefore a large variability has been preserved until now, but it has not been depth studied jet. Knowledge about evolution and genetic relationships within available germplasm are helpful to allow a better choice of parental lines to be used in crosses and to enlarge the genetic basis in olive breeding programs. A new strategy for SNP detection and characterization in natural populations is represented by EcoTILLING, a variation of TILLING which has been successfully used to examine genetic variation and to genotype several species. In combination with sequencing, EcoTILLING is an high throughput and very cost-effective technology, that allows to simultaneously screen a big number of individuals and to detect natural polymorphisms in coding regions of target genes. Moreover, individuals can be grouped according to their aplotype and distinguished in homozygous and heterozygous; finally, the effect of each identified mutation on protein structure and function can be predicted. The screening of several web-databases and the ortholog sequences of the fad7 gene (responsible of the 18:2 to 18:3 fatty acid desaturation) led to the identification of two olive ESTs. The sequence encoding for the fad7chloroplastic isoform was chosen as candidate gene. The genomic sequence and the structure of the gene were obtained by sequencing several overlapping PCR products; moreover, bioinformatic analysis allowed to predict the most suitable region of the gene for EcoTILLING screening. The application of the EcoTILLING strategy to olive genome was optimized for a subset of accessions, including cultivars and clones of the same cultivar. The CelI-based mutation assay and the Li-COR electrophoresis were chosen as SNP detection system. The cultivar Leccino was used as reference for the constitution of the 2-fold pools. Few polymorphisms were identified between the cultivars, confirming the high level of conservation of this gene. For example, a SNP at 630bp of the analyzed fragment allowed to distinguish the Leccino, Nociara, Toscanina and Frantoio cultivars (aplotype 1) from Cima di Melfi and Ascolana Tenera cultivars (aplotype 2). In the case of Leccino, the analysis among the 5 clones has revealed no genetic variation at the screened locus, showing the uniformity of the Leccino samples. Additionally, to detect the heterozygosity level in the target gene, single DNA from each cultivar was analysed, supposing that an heterozygous mutation in a diploid genome could be identified by the heteroduplex formation between the wildtype allele and its mutated counterpart.

Altamura bread is an Italian baking product that obtained the European mark of protected designation of origin (PDO). The varietal requirements of the official production protocol of this bread require it to be prepared from the durum wheat cultivars Appulo, Duilio, Arcangelo and Simeto (single or in combination, accounting for minimum 80%), and eventually other cultivars diffused in the production area. The aim of this work was to set up a microsatellite-based method for verifying the presence of the four required durum wheat cultivars in PDO Altamura bread, also in the presence of other cultivars up to 20%. Ten microsatellites were tested and the combination of the amplification profiles of four of them, characterised by high polymorphism and simple electrophoretic patterns, enabled to distinguish and identify breads from all the possible combinations of the cultivars required for PDO mark. The obtained amplicons were all in the range of molecular weight between 115 and 272 bp, and were analysed by capillary electrophoresis. The contribution of the single cultivars was detectable in the amplification profiles, enabling to verify their presence. The analysis was also effective in the case of additional cultivars.

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.

In this paper we describe the morphological, histological and chemical characters of 20 native Helichrysum italicum (Roth) G. Don ssp. italicum genotypes collected from different locations in Italy and Corsica (France) and grown under similar edaphic and climatic conditions. An AFLP technique was used to examine the level of genetic variability among the genotypes with the aim to disclose a possible correlation between the genetic and chemical data. The chemical analysis recognizes at least three different chemotypes based on the major constituents in the essential oils. This was confirmed by the AFLP analysis resulting in a dendrogram divided into three main clades. Because of the polymorphic trait of Helichrysum species, an efficient micropropagation protocol was established for the 20 H. italicum (Roth) G. Don ssp. italicum genotypes to guarantee the availability of stable genetic material with defined chemical profiles for industrial applications. The genotypes were found to influence the in vitro performance and the chemical composition of the essential oils, but not the phenotypic traits of the plants.

Il germoplasma olivicolo è caratterizzato da ampia variabilità genetica. Di fatto, solo tra le varietà italiane, sono state descritte almeno 628 cultivar, comprendenti anche alcune varianti clonali, la cui esatta individuazione e descrizione vengono rese difficoltose dalla presenza di numerosi casi di sinonimia e omonimia. Alcuni autori ritengono che la caratterizzazione molecolare non sia esaustiva o completamente efficace e che pertanto, ai fini della discriminazione varietale, occorra associare la descrizione morfo bioagronomica. Essendo la maggior parte dei caratteri agronomici e di importanza economica in varia misura condizionati dall’ambiente e dalle tecniche di coltivazione, per poter valutarne la componente genotipica è necessario disporre di germoplasma allevato in condizioni pedoclimatiche e colturali uniformi. In questo studio sono state prese in considerazione 96 varietà di olivo presenti presso il campo collezione di Mirto Crosia (CS) del CRA- OLI per le quali è stata determinata la composizione acidica per i principali acidi grassi (Acido palmitico, Acido stearico, Acido Oleico, Acido linoleico, Acido α-linolenico) per un periodo di 3 anni. E’ stato calcolato il grado di correlazione tra gli acidi grassi e valutato l’effetto genotipico sulla composizione acidica utilizzando un’analisi della varianza multivariata (MANOVA 50-50). I risultati mostrano un livello di correlazione negativo statisticamente significativo tra acido palmitico e l’acido oleico e tra l’acido oleico e l’acido linoleico, mentre l’effetto genotipico sulla composizione acidica è risultato altamente significativo. I risultati ottenuti rappresentano uno strumento molto utile per integrare gli studi di genetica molecolare, quali studi di associazione, nonché, unitamente ad altri descrittori, per l’identificazione genetica delle varietà.

Blue mould, caused by Penicillium expansum, is one of the most economically damaging postharvest diseases of pome fruits, although it may affect a wider host range, including sweet cherries and table grapes. Several reports on the role of mycotoxins in plant pathogenesis have been published, but few focussed on the influence of mycotoxins on the variation in host preference amongst producing fungi. In the present study the influence of the host on P. expansum pathogenicity/virulence was investigated, focussing mainly on the relationship with patulin production. Three P. expansum strain groups, originating from apples, sweet cherries, and table grapes (7 strains per host) were grown on their hosts of isolation and on artificial media derived from them. Strains within each P. expansum group proved to be more aggressive and produced more patulin than the other two groups under evaluation when grown on the host from which they originated. Table grape strains were the most aggressive (81% disease incidence) and strongest patulin producers (up to 554 μg/g). The difference in aggressivenessamongst strainswas appreciable only in the presence of a living host, suggesting that the complex pathogen–host interaction significantly influenced the ability of P. expansum to cause the disease. Incidence/severity of the disease and patulin production proved to be positively correlated, supporting the role of patulin as virulence/pathogenicity factor. The existence of genetic variation amongst isolates was confirmed by the High Resolution Melting method that was set up herein, which permitted discrimination of P. expansum from other species (P. chrysogenum and P. crustosum) and, within the same species, amongst the host of origin. Host effect on toxin production appeared to be exerted at a transcriptional level.

The TILLING strategy has been successfully applied to the sunflower genome in our laboratory (“sunTILL platform”). The interest was first focused on some key enzymes of the fatty acid pathway, because of the interest in increasing the nutritional value of sunflower oil by the reduction of the ratio of saturated to unsaturated fatty acids. Moreover, P. halstedii is one of the most dangerous pathogens that affects sunflower cultivation in the Mediterranean area. Therefore the availability of a stable and effective system, as genetic resistance, for the pest-control results of prime importance. Thereby, a pilot assay on 1,152 sunflower M2 lines was carried out by the reverse genetic screening of four genes: the kasII and kasIII genes, respectively codifying the isoforms II and III of the β-keto-acyl-ACP-synthetase; the fad2-1 gene, encoding the enzyme responsible of the converting reaction of oleic acid to linoleic acid; the AY490791 gene, involved in P. halstedii resistance. Since few genomic sequences are publicly available for sunflower, the reverse genetic screening was preceded by an accurate reconstruction of candidate gene models, by the amplification and the subsequent sequencing of short overlapping fragments. For each candidate gene the most promising region for TILLING analysis was thereby identified. In this way, new primer pairs flanking this region were set on the intronic sequences, with the aim to improve the screening efficiency on the coding regions. In the pilot assay, nine mutant lines have been totally identified. The four mutations scored in the kasII gene were homozygous; three of them were localized in introns, while one caused a G/T transversion, resulting in a premature stop-codon (E139*). No mutant lines were identified for the kasIII gene. In the case of fad2-1 gene, three mutations were identified: one resulted in a missense change (F26L), a second caused a silent change (R46=) and a third was situated in the non-coding region. The AY490791 gene screening revealed two mutations, both localized in non-coding sequence. Each mutant line was then confirmed by sequencing and genotyped by microsatellite markers to exclude any individuals originating from cross-pollination events. The results of this first reverse genetic screening translated into an average mutation frequency of 1/475 kb.

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.

The present work was aimed at assessing the genetic diversity of 42 local cultivars and oleaster genotypes from the area of Bejaia in Algeria. Fifteen highly polymorphic Simple Sequence Repeat markers were evaluated and proved to be very informative, producing a total number of 160 alleles with an average value of 10.7 per locus; the SSRs DCA09 and DCA16 were the most informative, distinguishing 17 and 19 genotypes, respectively. Phylogenetic and population structure analysis split the accessions in two main groups corresponding to most of oleasters and most of traditional varieties, respectively. Interestingly, ten traditional varieties resulted strictly related to the oleasters, indicating hybridization between the two botanical varieties. Genetic parameters and private alleles of groups confirmed this observation and indicated a wide genetic variability in Algerian olive germplasm. The results suggest the need to preserve and characterize this germplasm in order to limit the risk of losing potential important genetic traits present in the crop wild relatives.

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

DNA markers technology, derived from research in molecular biology and genomics, offers great promise for plant breeding, allowing the "molecular breeding" via marker-assisted selection. Grapevine genomic resources allowed, in recent years, the characterization at molecular level of genes involved in interesting phenotypes such as stenospermocarpic seedlessness, a trait really appreciated by consumers. Recent studies in table grapes revealed that the VvAGL11 gene, member of the D-lineage MADS-box family, controls the ovule identity, and thus potentially playing an important role in stenospermocarpy. Intragenic markers of VvAGL11 have been found and tested for breeding purposes. In the present paper, we describe an in deep assay on a total of 475 genotypes derived by our own grape germplasm and seeded × seedless crosses F1 offspring, to evaluate and verify the "diagnostic" power of VvAGL11 in marker-assisted selection. We found only 8/475 that were seeded and carried the seedless-associated allele in the STS p3-VvAGL11. However, and most importantly, there were no seedless varieties without such allele. We validated the marker as a 100 % effective tool for early negative selection of stenospermocarpy in Vitis vinifera L. crosses.

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.