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The effect of two priming treatments in PEG solutions on nuclear replication stages of pepper (Capsicum annuumL.) embryos were evaluated using flow cytometry. Priming at −1.1 MPa for 10 d induced almost 40% of nuclei in the embryo root tips to enter the synthetic phase of nuclear division, while no DNA synthesis occurred during 6 d at−1.5 MPa and in less than 10% of the nuclei at 10 d. Both priming treatments effectively increased seed performance. A decrease of 2.7 d in mean germination time (MGT) was observed after priming in PEG solution at −1.1 MPa, while the treatment at−1.5 MPa lowered the MGT by 2.1 d. Primed and untreated seeds were subjected to controlled deterioration treatments by exposure to 45°C after equilibration at 75% RH. Both osmotic treatments considerably lowered seed tolerance to adverse storage conditions as compared with untreated seeds. However, seeds in which DNA replication was induced by priming were more sensitive to controlled deterioration than seeds in which priming did not induce nuclei to enter the synthetic phase. This could be a consequence of a higher DNA content, which is a more vulnerable target for mutation inducing factors. Alternatively the increased sensitivity could be a consequence of their more advanced progress in germinative events, making seeds less resistant to deteriorative factors imposed during storage. On the whole, from the present results it appears that the nucleic acid DNA content of the embryo only plays an additive role in influencing seed storability.

The species Cynara cardunculus includes the globe artichoke (var. scolymus), the cultivated cardoon (var. altilis) and the wild cardoon (var. sylvestris). The three taxa are sexually compatible and originate fertile F1 progenies, which, given the high heterozygosity of the species, are highly segregating. We report the characterization of two F1 populations, one bred from a cross between globe artichoke and cultivated cardoon, and the other between globe artichoke and wild cardoon. Both populations featured a wide array of phenotypes in relation to several traits, and some of the newly developed genotypes are of interest for the ornamental market. The two populations were genotyped at 50 microsatellite (SSR) loci: in the globe artichoke × wild cardoon and globe artichoke × cultivated cardoon progenies 116 and 97 alleles were respectively detected. SSR pattern scores were used to produce an UPGMA dendrogram and a PCoA plot. A set of nine SSR loci, evenly dispersed across the genome, was shown to be sufficient to unambiguously identify each segregant. The molecular fingerprinting is useful for establishing the true to type correspondence of propagative materials in nurseries and ensures the effective correspondence between the real and the declared identity of a clone.

A number of horticultural crop landraces are still grown in Piedmont (NW Italy), despite the dominance of improved cultivars and hybrids. Conservation strategies, both in an ex situ and an in situ context are required to prevent their loss. Here we describe an AFLP-based assessment of the genetic structure of leek, garlic, celery, red beet, cultivated cardoon, sweet pepper and common bean autochthonous landraces. Each landrace was sampled by selecting 3–5 populations representative of the area of cultivation. The genotypic data showed that the crop's breeding system was less important for determining genetic structure than the selection criteria adopted by the producers, the extent of informal seed exchange among producers and natural selection imposed by the local environmental conditions. The genotypic data identified alleles which were common, some which were restricted to a particular locality and some which were either infrequent or rare. On this basis, the most representative population(s) for each landrace were recognised and targeted for conservation. The landraces in study differed markedly from one another with respect to their genetic structure, and so they may represent an appropriate reference model for the management of crop landraces grown in fragmented areas and at risk of genetic erosion or extinction.

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ∼144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.

Plants respond to UV stress by producing antioxidant molecules and by altering their metabolism through the regulation of specific gene family members. Globe artichoke (Cynara cardunculus var. scolymus L.-Compositae family) is an attractive model species for studying the protein networks involved in UV response, being characterised by high loads of antioxidants, which are boosted by UV-C rays. The effect of UV-C on leaf proteome was investigated 24 h after irradiation by applying a polyethylene glycol (PEG) fractionation method, which we previously developed to cope with the so-called high abundance proteins, followed by two-dimensional electrophoresis analysis. Thirty-eight UV-C modulated proteins were analysed by means of a nano-liquid chromatography-nanospray tandem mass spectrometry, and 27 were successfully identified using a customised Compositae protein database, including a set of 19,055 globe artichoke unigenes. Proteins were categorised according to Gene Ontology (GO) and visualised on their cellular pathway: Most of them were involved not only in primary metabolism (e.g. photosynthesis) and in abiotic stress responses (e.g. reactive oxygen species scavenging) but also in cell wall modification, secondary metabolism, and regulation of transcription. To date, this is the first report on globe artichoke proteomic expression analysis following abiotic stress. Our results confirm the effectiveness of the PEG fractionation method in excluding most of the ribulose bisphosphate carboxylase/oxygenase and highlighting some major occurring phenomena.

Using flow cytometry, we determined relative DNA amounts expressed as C-values, of large numbers of nuclei isolated from embryo root tips in three seed lots of pepper (Capsicum annuum) and tomato (Lycopersicon esculentum). The nuclei in embryos of fully matured dry pepper and tomato seeds predominantly revealed 2C signals, which indicates that the cells of maturing seeds have arrested at G1 phase of the cell cycle. Priming of pepper and tomato seeds in −1.1, −1.3 or −1.5 MPa polyethylene glycol (PEG) for 14 days reduced the mean time to germination. In all seed lots an induction of 4C signals was found after priming, indicating that during priming the cells of the embryonic root tip had replicated their DNA and arrested at the G2 phase of the cell cycle. For both tomato and pepper, the frequency of 4C signals was highest at the lowest PEG concentration (osmotic potential of −1.1 MPa). A significant inverse correlation was found between the frequency of root tip cells expressing 4C DNA signals and the mean time to germination for each individual pepper and tomato seed lot. However, the effect of a specific osmotic treatment differed between seed lots of the same cultivar, with regard to both the decrease in the mean time to germination and to the percentage of 4C cells, suggesting specific differences among seed lots in the response to priming. The present results indicate that flow cytometry can be used to follow the progress in nuclear replication activities during priming. The advancement in DNA synthesis can thus be used to measure the efficiency of a priming treatment with regard to enhancement of seed lot germination rate.

We report the development of nine microsatellite markers in globe artichoke (Cynara cardunculus L. var. scolymus). Four markers were obtained from sequences available in GenBank and five were isolated using a biotin/streptavidin capture technique for (CA)n and (CT)n motifs directly from artichoke genomic DNA. Polymorphism was explored in 15 artichoke accessions that represent the genetic variation within cultivated varietal types. Inter-specific amplification was tested using cultivated cardoon (C. cardunculus L. var. altilis DC.) and wild cardoon (C. cardunculus L. var. sylvestris Lam.). Primers and conditions for polymerase chain reaction (PCR) amplification of detected loci are described.

Background The eggplant (Solanum melongena L.) genome is relatively unexplored, especially compared to those of the other major Solanaceae crops tomato and potato. In particular, no SNP markers are publicly available; on the other hand, over 1,000 SSR markers were developed and publicly available. We have combined the recently developed Restriction-site Associated DNA (RAD) approach with Illumina DNA sequencing for rapid and mass discovery of both SNP and SSR markers for eggplant. Results RAD tags were generated from the genomic DNA of a pair of eggplant mapping parents, and sequenced to produce ~17.5 Mb of sequences arrangeable into ~78,000 contigs. The resulting non-redundant genomic sequence dataset consisted of ~45,000 sequences, of which ~29% were putative coding sequences and ~70% were in common between the mapping parents. The shared sequences allowed the discovery of ~10,000 SNPs and nearly 1,000 indels, equivalent to a SNP frequency of 0.8 per Kb and an indel frequency of 0.07 per Kb. Over 2,000 of the SNPs are likely to be mappable via the Illumina GoldenGate assay. A subset of 384 SNPs was used to successfully fingerprint a panel of eggplant germplasm, producing a set of informative diversity data. The RAD sequences also included nearly 2,000 putative SSRs, and primer pairs were designed to amplify 1,155 loci. Conclusion The high throughput sequencing of the RAD tags allowed the discovery of a large number of DNA markers, which will prove useful for extending our current knowledge of the genome organization of eggplant, for assisting in marker-aided selection and for carrying out comparative genomic analyses within the Solanaceae family.

Flow cytometric determination of nuclear DNA contents in embryos of dry, fully matured pepper seeds revealed only 2C signals. Therefore, pepper belongs to those species in which the quiescent embryo arrests nuclear division in the presynthetic G 1 phase. In whole seeds, two other peaks were observed which corresponded with the 3C and 6C DNA content and originated from the triploid endosperm. After 36 to 48 h of imbibition in water (48 to 60 h before visible germination), a rise in the 4C signal was observed in the root tip of the embryo. Osmotic preconditioning (priming) of pepper seeds for 7, 14 or 21 days in polyethylene glycol (PEG) or KNO 3 +K 3 PO 4 considerably reduced the time to 50% germination (t 50 ) and the mean germination time, and the effect was proportional to the duration of the priming treatments

Flow cytometric determination of DNA levels in embryos of fully matured seeds of various plant species revealed large amounts of 2C DNA signals, indicating that most cells had arrested the cell cycle at the presynthetic G1 phase of nuclear division. The accumulation of cells at G1 was found both in orthodox and in recalcitrant (i.e. Castanea sativa) seed species. As recalcitrant seeds are characterized by the absence of maturation drying, the arrest of the cell cycle in the presynthetic phase may not be linked to the seed water status. Apart from the 2C signal, 4C values were found in the embryo of some seed species (e.g. Raphanus sativus) indicating that cells were arrested in G2 Cells arrested in G2 were primarily located in the root-tip region of the embryo. In addition, combinations of higher C values (i.e. 8C, 12C, 16C and 64C) were observed in the endosperm of Solanum melongena and Lycopersicon esculentum, and in the root-tip cells of Phaseolus vulgaris and Spinacia oleracea. These mixtures of polyploid nuclei (also called 'polysomaty') may arise from a developmentally controlled cellular endoreduplication and indicates that in each cell type of the seed the amount of DNA is regulated both spatially and temporally.

Meiosis was studied in an F1 hybrid between domesticated Capsicum annuum and its close relative C. chinense. The parental accessions differed by two chromosomal interchanges. A hexavalent was present in 18% of pollen mother cells collected in spring and summer but no multivalents were found in pollen mother cells collected in autumn and chiasmata were also less frequent then. Interchange heterozygosity, often resulting in multiples of six or more chromosomes, is characteristic of interspecific hybrids in Capsicum.

An anther-derived doubled haploid (DH) population and an F2mapping population were developed from an intraspecific hybrid between the eggplant breeding lines 305E40 and 67/3. The former incorporates an introgressed segment from Solanum aethiopicum Gilo Group carrying the gene Rfo-sa1, which confers resistance to Fusarium oxysporum ; the latter is a selection from an intraspecific cross involving two conventional eggplant varieties and lacks Rfo-sa1. Initially, 28 AFLP primer combinations (PCs) were applied to a sample of 93 F2individuals and 93 DH individuals, from which 170 polymorphic AFLP fragments were identified. In the DH population, the segregation of 117 of these AFLPs as well as markers closely linked to Rfo-sa1 was substantially distorted, while in the F2population, segregation distortion was restricted to just 10 markers, and thus the latter was chosen for map development. A set of 141 F2individuals was genotyped with 73 AFLP PCs (generating 406 informative markers), 32 SSRs, 4 tomato RFLPs, and 3 CAPS markers linked to Rfo-sa1. This resulted in the assignment of 348 markers to 12 major linkage groups. The framework map covered 718.7 cM, comprising 238 markers (212 AFLPs, 22 SSRs, 1 RFLP, and the Rfo-sa1 CAPS). Marker order and inter-marker distances in this eggplant map were largely consistent with those reported in a recently published SSR-based map. From an eggplant breeding perspective, DH populations produced by anther culture appear to be subject to massive segregation distortion and thus may not be very efficient in capturing the full range of genetic variation present in the parental lines.

Most of the 24 viruses which infect globe artichoke are detrimental to the crop’s performance and hamper the development of a nursery activity in the respect of current EU legislation. We describe a procedure to sanitize globe artichoke “Brindisino” from Artichoke Italian latent virus (AILV) and Artichoke latent virus (ArLV), while preserving its valuable early flowering trait. ArLV was successfully eliminated by meristem-tip culture, while AILV was removed when two rounds of meristem-tip culture were spaced out with in vitro thermotherapy. In vivo thermotherapy, followed by meristem-tip culture, was also successful in producing virus-free material but was less efficient in terms of the number of plants recovered post treatment. Due to the multi-clonal composition of the populations at present in cultivation, the selected and sanitised clones were fingerprinted by applying microsatellite and AFLP (amplified fragment length polymorphism) markers. One AFLP primer combination produced 28 informative fragments used to evaluate genetic relatedness among the clones in study. Our results demonstrates that AFLP-based molecular fingerprinting enables to verify the true to clone correspondence in nurseries, ensure the effective correspondence between the real and the declared identity of a clone, so that to avoid commercial frauds, and might represents a valuable tool for assessing somaclonal variation occuring during ‘in vitro’ propagation.

The caffeoylquinic acid (CQA) biosynthesis pathway in plants is not fully understood, but the BAHD superfamily, a large class of acyl-CoA-dependent acyltransferases, may represent an interesting group for the isolation of enzymes playing a key role. Globe artichoke (Cynara cardunculus var. scolymus) represents a relevant model species for studying CQA biosynthesis, because the plant accumulates high amounts and a diverse spectrum of CQAs in its leaves. An in silico scan of globe artichoke ESTs was based on a search string targeting the conserved motifs of BAHD proteins and complete BAHD sequences present in a range of species. EST hits were assembled into 32 unigenes and their phylogeny characterized. Among which were three putative BAHD acyltransferases showing a high level of similarity to globe artichoke HCT and HQT enzymes. The full-length cDNAs of these three proteins were isolated; two of these acyltransferases were able to synthesize both chlorogenic acid and p-coumaroylquinate, assessing their involvement in CQA biosynthesis. Upon exposure to UV-C radiation, which is known to induce CQA biosynthesis, expression of two of the enzyme transcripts was up-regulated while that of the third was unaffected. By analysing the globe artichoke unigene consensus sequences, further two genes (i.e. C4H, 4CL), related not only to flavonoids but also to chlorogenic acid (a mono-CQA) biosynthesis, were also identified and were genetically mapped along with the three acyltransferase genes. The research has improved the knowledge base surrounding globe artichoke BAHD members. The mapping of the genes related to the chlorogenic acid biosynthesis pathway may contribute to a greater understanding of the genetic basis of the synthesis of these compounds of nutraceutical and pharmaceutical value.

Species within the genus Hibiscus (Malvaceae) are herbaceous or woody plants, annuals or perennials. Within the genus, most of the domesticated species are grown as ornamentals, and the most widely exploited is Hibiscus rosa-sinensis, which is particularly variable with respect to flower colour and shape. H. rosa-sinensis is thought to have originated following hybridization between two species complexes endemic to different geographic locations. Using AFLP fingerprinting, the genetic variation in a collection of 94 H. rosa-sinensis cultivars was assessed, and their phylogenetic relationships with other Hibiscus species was determined. All eight AFLP primer combinations (PCs) produced informative marker bands, and the use of four PCs was sufficient to uniquely differentiate each accession. Three major clusters were identified: group A included 87 of the 94 H. rosa-sinensis cultivars, including within it two sub-clusters related to flower shape and size; group B included seven old H. rosa-sinensis cultivars together with H. storckii, considered to be their progenitor; group C included H. denisonii, H. kokio. H. genevii and H. liliiflorus which, together with H. boryanus, H. arnottianus and H. schizopetalus, are thought to have contributed to the wide genetic variability characteristic of H. rosa-sinensis. The lowest level of genetic similarity with H. rosa-sinensis was shown by H. tiliaceus, H. syriacus, H. cannabinus and H. panduriformis, all of which are sexually incompatible with H. rosa-sinensis and thus are not likely to have been implicated in its origin. Our results demonstrated that AFLP fingerprinting provides a valuable means of assessing genetic variation within H. rosa-sinensis and elucidating its phylogenetic relationship with other Hibiscus spp.

The genome organization of globe artichoke (Cynara cardunculus var. scolymus), unlike other species belonging to Asteraceae (=Compositae) family (i.e. sunflower, lettuce and chicory), remains largely unexplored. The species is highly heterozygous and suffers marked inbreeding depression when forced to self-fertilize. Thus a two-way pseudo-testcross represents the optimal strategy for linkage analysis. Here, we report linkage maps based on the progeny of a cross between globe artichoke (C. cardunculus var. scolymus) and cultivated cardoon (C. cardunculus var. altilis). The population was genotyped using a variety of PCR-based marker platforms, resulting in the identification of 708 testcross markers suitable for map construction. The male map consisted of 177 loci arranged in 17 major linkage groups, spanning 1,015.5 cM, while female map was built with 326 loci arranged into 20 major linkage groups, spanning 1,486.8 cM. The presence of 84 loci shared between these maps and those previously developed from a cross within globe artichoke allowed for map alignment and the definition of 17 homologous linkage groups, corresponding to the haploid number of the species. This will provide a favourable property for QTL scanning; furthermore, as 25 mapped markers (8%) correspond to coding regions, it has an additional value as functional map and might represent an important genetic tool for candidate gene studies in globe artichoke.

Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3′-hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with p-coumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps.

Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana.

Globe artichoke (Cynara cardunculus var. scolymus) is native to the Mediterranean Basin, where it grows in close proximity with its ancestor wild cardoon (C. cardunculus var. sylvestris); its commercial production is mainly based on vegetatively propagated clones which guarantee high yields of marketable product (i.e. immature inflorescence or capitula). A collection of 24 landraces of globe artichoke was made from small-holdings in Sicily, which is assumed to be one of the possible centres of its domestication. These landraces have been cultivated for centuries by local farmers, mainly due to their culinary uniqueness. The collection was characterised for a combination of morphological traits and AFLP, gSSR and cpSSr markers. Molecular analyses included genotypes of wild cardoon collected from different sites in Sicily as well as accessions of the most widely grown Sicilian varietal types: the spiny ‘Spinoso di Palermo’ and the non-spiny ‘Violetto di Sicilia’. The landraces follow a gradient of ‘ennoblement’ towards either the domesticated spiny or the non-spiny types. ‘Cimiciusa di Mazzarino’ was an outlier, in that it resembled the cultivated forms with respect to its AFLP fingerprint, but was more closely related to the wild cardoon on the basis of SSR profile. This particular landrace presents an example of an intermediary form in the domestication process, although it could also have derived from introgression from sympatric wild cardoon, followed by farmer selection. The abundant genetic variation present demonstrates the key role of farmers’ practice in the maintenance of genetic diversity, which should be preserved because of its potential value for plant breeders.

Cynara cardunculus includes three taxa, the globe artichoke (subsp. scolymus L. Hegi), the cultivated cardoon (var. altilis) and their progenitor, the wild cardoon (var. sylvestris). Globe artichoke is an important component of the Mediterranean rural economy, but its improvement through breeding has been rather limited and its genome organization remains largely unexplored. Here, we report the isolation of 61 new microsatellite loci which amplified a total of 208 alleles in a panel of 22 C. cardunculus genotypes. Of these, 51 were informative for linkage analysis and 39 were used to increase marker density in the available globe artichoke genetic maps. Sequence analysis of the 22 loci associated with genes showed that 9 are located within coding sequence, with the repetitive domain probably being involved in DNA binding or in protein-protein interactions. The expression of the genes associated with 9 of the 22 microsatellite loci was demonstrated by RT-PCR.

The viability of Iceland poppy ( Papaver nudicaule L.) seeds kept at 16 combinations of temperature and moisture content was checked by germination tests over a period of 48 months. Under the majority of storage treatments, the periods of viability of individual seeds were normally distributed around the mean value. The relationship between storage environment and seed survival was evaluated by a viability equation, whose constants were determined by a means probit analysis. This allows us to predict the percentage viability of Iceland poppy seeds after any time when stored at various temperatures and moisture contents, as well as to estimate the time taken for viability to decrease below any given level.

Background The leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L.) have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ), and a range of flavonoid compounds. Results Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT) encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively) using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase). The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA) and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps. Conclusion A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP) biosynthesis in C. cardunculus.

Background Eggplant (Solanum melongena L.) is a member of the Solanaceae family. In spite of its widespread cultivation and nutritional and economic importance, its genome has not as yet been extensively investigated. Few analyses have been carried out to determine the genetic diversity of eggplant at the DNA level, and linkage relationships have not been well characterised. As for the other Solanaceae crop species (potato, tomato and pepper), the level of intra-specific polymorphism appears to be rather limited, and so it is important that an effort is made to develop more informative DNA markers to make progress in understanding the genetics of eggplant and to advance its breeding. The aim of the present work was to develop a set of functional microsatellite (SSR) markers, via an in silico analysis of publicly available DNA sequence. Results From >3,300 genic DNA sequences, 50 SSR-containing candidates suitable for primer design were recovered. Of these, 39 were functional, and were then applied to a panel of 44 accessions, of which 38 were cultivated eggplant varieties, and six were from related Solanum species. The usefulness of the SSR assays for diversity analysis and taxonomic discrimination was demonstrated by constructing a phylogeny based on SSR polymorphisms, and by the demonstration that most were also functional when tested with template from tomato, pepper and potato. As a results of BLASTN analyses, several eggplant SSRs were found to have homologous counterparts in the phylogenetically related species, which carry microsatellite motifs in the same position. Conclusion The set of eggplant EST-SSR markers was informative for phylogenetic analysis and genetic mapping. Since EST-SSRs lie within expressed sequence, they have the potential to serve as perfect markers for genes determining variation in phenotype. Their high level of transferability to other Solanaceae species can be used to provide anchoring points for the integration of genetic maps across species.

Recent studies indicate that Southern Italy and Sicily are thought to be the origin of globe artichoke domestication from its ancestor wild cardoon. Indeed, Italy has the richest globe artichoke primary cultivated gene pool and harbours many distinct clonal varietal groups classified on the basis of capitulum appearance or harvest time. At present Italian germplasm is at risk of genetic erosion due to the recent introduction of varieties selected abroad, as well as the concentration on a limited number of varietal types best fitting market demand. The aim of this study was to assess the level of genetic variation in autochthonous globe artichoke germplasm grown in family gardens in Sicily. Twenty-five accessions were collected and characterized for morphological characters as well as by applying AFLP (amplified fragment length polymorphism) markers. Some accessions of wild cardoon were also included as a reference for the molecular analysis. Our results show that the use of AFLP is suitable for precisely characterizing local artichoke selection, for synonymy identification as well as for phylogenetic studies. Furthermore molecular and morphological data supply important information for the implementation of 'on farm' germplasm preservation strategies.

Cynara cardunculus is a diploid (2n = 2x = 34) species, native to the Mediterranean basin, which belongs to the family of Asteraceae. It includes globe artichoke, cultivated cardoon, as well as their progenitor wild cardoon. The species is a source of biophenols and its leaf extracts have been widely used in herbal medicine as hepatoprotectors and choleretics since ancient times. Globe artichoke leaves have been found to be rich in compounds originating from the metabolism of phenylpropanoids however, to our knowledge, the leaf polyphenolic composition of the two other forms within the species, cultivated and wild cardoon, have not yet been properly investigated. Two main classes of polyphenols have been detected by HPLC/DAD and HPLC/MS analyses: caffeoyl esters and flavonoids. The compounds which are the result of esterification of caffeoylquinic acid moiety with succinic acid, previously detected in other members of the Asteraceae family, were detected in cardoon leaves for the first time.

An eggplant (Solanum melongena) association panel of 191 accessions, comprising a mixture of breeding lines, old varieties and landrace selections was SNP genotyped and phenotyped for key breeding fruit and plant traits at two locations over two seasons. A genome-wide association (GWA) analysis was performed using the mixed linear model, which takes into account both a kinship matrix and the sub-population membership of the accessions. Overall, 194 phenotype/genotype associations were uncovered, relating to 30 of the 33 measured traits. These associations involved 79 SNP loci mapping to 39 distinct chromosomal regions distributed over all 12 eggplant chromosomes. A comparison of the map positions of these SNPs with those of loci derived from conventional linkage mapping showed that GWA analysis both validated many of the known controlling loci and detected a large number of new marker/trait associations. Exploiting established syntenic relationships between eggplant chromosomes and those of tomato and pepper recognized orthologous regions in ten eggplant chromosomes harbouring genes influencing breeders' traits.

A high copy number of retrotransposon sequences are present and widely dispersed in plant genomes. Their activity generates a considerable degree of sequence polymorphism. Here, we report the cloning of CYRE-5, a long-terminal repeat carrying retrotransposon-like sequence in Cynara cardunculus L., and its exploitation to develop a DNA fingerprinting assay across 22 accessions, including both cultivated (globe artichoke and cultivated cardoon) and wild (wild cardoon) types. The effectiveness of the sequence-specific amplified polymorphism (S-SAP) platform is compared with that of amplified fragment length polymorphism (AFLP). A genetic linkage analysis, based on a hybrid population between 2 globe artichoke varietal types, resulted in the inclusion of 29 S-SAP loci in the core genetic map, confirming their dispersed distribution across the globe artichoke genome.

The heterogeneity of pedoclimatic conditions and the farmer selection for adaptation to local tastes and uses have favored the maintenance in cultivation in Italy of numerous pepper (Capsicum annuum L.) landraces. We have used amplified fragment length polymorphism (AFLP) fingerprinting and morphological variation in fruit type to assess the diversity of 19 pepper ecotypes representative of the autochthonous germplasm. Principal component analysis grouped together triangular and blocky fruited types, while elongate types were subdivided into long and half long, differing mainly with respect to the length/diameter ratio. The half-long types were divided into two further clusters on the basis of overall dimension, weight, and flesh thickness. Genetic similarities between the ecotypes were calculated from AFLP data and this allowed the separation of the accessions into two major and three minor clusters. One major cluster comprised five ecotypes with blocky or triangular fruits, while the other contained nine ecotypes with long or half-long fruits. AFLP markers were successful in both detecting genetic diversity and determining genetic relationships in Italian pepper germplasm. They also made it possible to distinguish most of the provenances within a given landrace, and to identify pairs of genetically similar ecotypes carrying different names. We believe that our analyses will help in the identification of rational strategies for the preservation of Italian pepper genetic resources.

We present the first genetic maps of globe artichoke (Cynara cardunculus var. scolymus L. 2n=2x=34), constructed with a two-way pseudo-testcross strategy. A F1 mapping population of 94 individuals was generated between a late-maturing, non-spiny type and an early-maturing spiny type. The 30 AFLP, 13 M-AFLP and 9 S-SAP primer combinations chosen identified, respectively, 352, 38 and 41 polymorphic markers. Of 32 microsatellite primer pairs tested, 12 identified heterozygous loci in one or other parent, and 7 were fully informative as they segregated in both parents. The female parent map comprised 204 loci, spread over 18 linkage groups and spanned 1330.5 cM with a mean marker density of 6.5 cM. The equivalent figures for the male parent map were 180 loci, 17 linkage groups, 1239.4 and 6.9 cM. About 3% of the AFLP and AFLP-derived markers displayed segregation distortion with a P value below 0.01, and were not used for map construction. All the SSR loci were included in the linkage analysis, although one locus did show some segregation distortion. The presence of 78 markers in common to both maps allowed the alignment of 16 linkage groups. The maps generated provide a firm basis for the mapping of agriculturally relevant traits, which will then open the way for the application of a marker-assisted selection breeding strategy in this species.

Wild cardoon [Cynara cardunculus L var. sylvestris (Lamk) Fiori] is a non-domesticated robust perennial plant characterized by a rosette of large spiny leaves and branched flowering stems. It is native to the Mediterranean basin where it grows in a variety of habitats, from mesic riparian sites to xeric rocky slopes. Wild cardoon is potentially exploitable for the production of lignocellulosic biomass, oil from seeds, and biopharmaceuticals, such as caffeoylquinic acids (cynarine) and luteolin from leaves, and inulin from roots. The genetic variation present at microsatellite (SSR) and amplified fragment length polymorphism (AFLP) loci has been assessed in seven Italian populations of wild cardoon, collected from Sicily and Sardinia. Thirty individuals, randomly sampled from each population were genotyped at five SSR loci and fingerprinted using seven AFLP primer combinations. Genetic distance estimates both within and between populations were consistent between the two marker systems. As a result of the geographical isolation, the Sardinian and Sicilian populations were clearly differentiated, forming two distinct gene-pools. Most of the genetic variation was partitioned within rather than between populations. The genetic distribution will allow the design of rational sampling strategies to capture the genotypic range present.

Globe artichoke is native to the Mediterranean basin, where it is presumed to have evolved from the wild cardoon. Italy is a leading producer and numerous varietal types, which are usually vegetatively propagated, are grown. At present, Italian germplasm is at risk of genetic erosion due to the recent introduction of varieties selected abroad, as well as the concentration on a limited number of varietal types best fitting market demand. The aim of this study was to assess the level of genetic variation, via AFLP fingerprinting, present in autochthonous globe artichoke germplasm in Sicily, thought to be one of the origins of its domestication. Seven populations were identified, of which three were of the spiny type ‘Spinoso di Palermo’ and four of the non-spiny type ‘Violetto di Sicilia’. A significant genetic differentiation between spiny and non-spiny types was found. In each type, the majority of the genetic variation was present within, rather than between populations, presumably as a consequence of the multiclonal composition of each varietal type. Most of the populations were genetically distinct, as a result of farm fragmentation and adaptation to local pedo-climatic conditions. The genetic distribution detected supplies important information for the implementation of ‘on farm’ germplasm preservation strategies, and stresses the need for clonal selection programmes in order to provide more uniform material to farmers.

DNA methylation in plants is related to a number of epigenetic (i.e. heritable, but potentially reversible) phenomena. Heavy methylation of cytosine residues plays an important role in gene expression, and significant differences in cytosine methylation levels have been observed among various tissue types, which can be explained as one of the regulatory mechanisms during development and differentiation. Here, we report on the analysis of cytosine methylation during pepper seed germination using an adaptation of the AFLP technique called methylation-sensitive amplified polymorphism (MSAP). Notable changes in MSAP profiles of genomic DNA obtained from embryo tissues of dry seeds and germinating seeds were detected. The changes were mainly: (i) fragments not detected in dry seeds were present after digestion with both EcoRI/HpaII and EcoRI/MspI at a certain stage during germination; (ii) fragments present after both digestions in dry seeds were no longer detected upon germination. Although changes in MSAP patterns during germination are not easily interpreted, our results can be mainly attributed to demethylation events, which appear to be necessary for transcriptional activation during germination. The potential and limits of MSAP in the analysis of DNA methylation status are discussed.

Globe artichoke ( Cynara cardunculus L. var. scolymus L.) is a diploid (2 n=2 x=34), predominantly cross-pollinated plant native to the Mediterranean basin, and Italy contains the richest primary cultivated 'gene pool'. Commercial production is mainly based on perennial cultivation of vegetatively propagated clones that are highly heterozygous and segregate widely when progeny-tested. Analysis of the artichoke genome by means of molecular markers has been limited to a few studies; here we report on the genetic relatedness among 118 artichoke accessions, including clones belonging to the same varietal type, two accessions of cultivated cardoon ( C. cardunculus L. var. altilis DC.) and four accessions of wild cardoon [ C. cardunculus L. var. sylvestris (Lamk) Fiori] as measured by amplified fragment length polymorphism (AFLP). Eight primer combinations yielded a total of 667 bands, of which 519 were polymorphic. Genetic similarities among accessions were calculated according to Jaccard's Similarity Index and used to construct a dendrogram based on the unweighted pair group method using arithmetic averages. Our results demonstrate that AFLP markers can be useful in evaluating Cynara cardunculus genetic diversity and in classifying accessions to phylogenetic groups based on their genetic similarity values. Genetic variation among artichoke clones belonging to the same varietal type was in some cases higher than that found among accessions differently named and coming from different areas. The lowest Jaccard's Similarity Index found within a varietal type can be considered as a threshold for the identification of accessions which share an analogous genetic background. This will enable the selection of representatives in order to develop and manage a germplasm 'core collection' as well as the identification of suitable material for future artichoke breeding efforts.

In spite of its widespread cultivation and nutritional and economic importance, the eggplant (Solanum melongena L.) genome has not been extensively explored. A lack of knowledge of the patterns of inheritance of key agronomic traits has hindered the exploitation of marker technologies to accelerate its genetic improvement. An already established F2 intraspecific population of eggplant bred from the cross '305E40' x '67/3' was phenotyped for 20 agronomically relevant traits at two sites. Up to seven quantitative trait loci (QTL) per trait were identified and the percentage of the phenotypic variance (PV) explained per QTL ranged from 4 to 93%. Not all the QTL were detectable at both sites, but for each trait at least one major QTL (PV explained ≥ 10%) was identified. Although no detectable QTL x environment interaction was found, some QTL identified were location-specific. Many of the fruit-related QTL clustered within specific chromosomal regions, reflecting either linkage and/or pleiotropy. Evidence for putative tomato orthologous QTL/genes was obtained for several of the eggplant QTL. Information regarding the inheritance of key agronomic traits was obtained. Some of the QTL, along with their respective linked markers, may be useful in the context of marker-assisted breeding.

Flow cytometric determination of DNA amounts in embryos of fully matured dry tomato (Lycopersicon esculentum Mill.) seeds revealed mainly 2C signals. Apparently, most embryonic cells had arrested the cell cycle activity during maturation at the presynthetic G1 phase of nuclear division. During imbibition of tomato seeds, both in water and in PEG solution, an augmentation in the 4C signal of tissues at the embryonic root tip region occurred. This increase was found to be related to cells entering the synthetic phase of nuclear division leading to a doubling of the chromosomal material. In the root tip cells, 4C 2C ratios rose 1 day after imbibition in water while radicle emergence started 2 days later. Apparently, DNA synthesis preceded germination. The induction of nuclear activities was compared with changes in protein expression patterns during seed imbibition. Using chemiluminescent immunodetection after Western blotting, we identified changes in β-tubulin expression during germination and priming of tomato seeds. Tubulin was found to be synthesized 8 h after the start of imbibition both in water and in PEG. The start of tubulin synthesis considerably preceded DNA replication, cell division and germination. The induction of cell cycle activities may be an early indicator of the initiation of cell division and the start of germination.

Both inter- and intra-specific maps have been developed in eggplant (Solanum melongena L.). The former benefit from an enhanced frequency of marker polymorphism, but their relevance to marker-assisted crop breeding is limited. Combining the restriction-site associated DNA strategy with high throughput sequencing has facilitated the discovery of a large number of functional single nucleotide polymorphism (SNP) markers discriminating between the two eggplant mapping population parental lines '305E40' and '67/3'. A set of 347 de novo SNPs, together with 84 anchoring markers, were applied to the F(2) mapping population bred from the cross '305E40' x '67/3' to construct a linkage map. In all, 415 of the 431 markers were assembled into twelve major and one minor linkage group, spanning 1,390 cM, and the inclusion of established markers allowed each linkage group to be assigned to one of the 12 eggplant chromosomes. The map was then used to discover the genetic basis of seven traits associated with anthocyanin content. Each of the traits proved to be controlled by between one and six quantitative trait loci (QTL), of which at least one was a major QTL. Exploitation of syntenic relationships between the eggplant and tomato genomes facilitated the identification of potential candidate genes for the eggplant QTLs related to anthocyanin accumulation. The intra-specific linkage map should have utility for elucidating the genetic basis of other phenotypic traits in eggplant.

Oleander (Nerium oleander L.) is a Mediterranean evergreen shrub widely grown as an ornamental for its abundant and long-lasting flowering as well as its moderate hardiness. Genetic relatedness among 71 accessions, including commercial varieties, different sources of the same varieties, and selections from the wild were investigated using amplified fragment length polymorphism (AFLP). Genetic similarities among accessions were calculated according to Jaccard’s Similarity Index and used to construct a dendrogram based on the unweighted pair group method, using arithmetic averages. Our results show that up to about 9 % molecular genetic differentiation was detected among morphologically indistinguishable provenances of the same variety. This can be partly attributed to scoring error but mainly to somatic variation occurring during vegetative propagation. On the other hand lower genetic distance values were sometimes found among varieties, which differ in morphological characters and are thus commercialised with different names. The possibility of considering the amount of genetic variation within a variety as the threshold value for discrimination of initial varieties and essential derivative varieties is discussed.

The correlation between physiological markers for seed maturity (acquisition of germinability, definitive morphological traits and desiccation tolerance) and timing of the shutdown of nuclear replication activity and β-tubulin synthesis has been investigated in developing pepper seeds. Nuclear replication activity was analysed by flow cytometry. Expression of β-tubulin, a cell-cycle related structural protein, was detected in western blots of total protein extracts. In pepper embryos, DNA synthesis stopped 42–49 days after anthesis (DAA), at which time seeds started to acquire desiccation tolerance. A clear β-tubulin signal was detected up to 63 DAA, only 1 week before the acquisition of complete desiccation tolerance. Both nuclear replication and β-tubulin synthesis can therefore be used as markers for the physiological stage reached by developing pepper seeds. Detection of β-tubulin signals in embryonic tissues can also be helpful in discriminating immature seeds within a commercial seed lot.

International audience; Plant secondary metabolites are highly evolved compounds performing different functions, and have been widely exploited from food to medicine. A constant supply of phenols, a class of secondary metabolites, provides preventive and defensive mechanisms to reduce the risk of chronic diseases in human beings; among them mono-and di-caffeoylquinic acids (monoCQAs, diCQAs) have attracted a growing academic and industrial interest in recent years. In Cynara cardunculus L. the biosynthetic pathway of chlorogenic acid (CGA, 5-O-caffeoylquinic acid) has been the subject of our several recent studies. Here, we report the state of the art on the isolation and in vitro functional characterization of the genes involved in the biosynthetic pathway of the CGA: HCT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl-transferase), HQT (hydroxycinnamoyl-CoA quinate hydroxylcinnamoyl-transferase), two HQT-like genes, we named Acyltransf_1 and Acyltransf_2, and C3'H (p-coumaroyl ester 3'-hydroxylase). Plant phenolics are known to be involved in the plant stress response and we found out that in globe artichoke the exposure to UV-C induces the production of diCQAs. In UV-C treated globe artichoke leaves, the expression level of C3'H, HCT, HQT, Acyltransf_1, Acyltransf_2 genes was strongly increased, thus confirming their involvement in the synthesis of chlorogenic acid. The development of DNA-based markers for the isolated genes made it possible to locate them within the previously developed genetic maps of the species.

Globe artichoke (Cynara cardunculus L. var. scolymus) provides a rich dietary source of bio-active compounds derived from phenylpropanoid metabolism, notably caffeoylquinic acids (CQAs) and flavonoids. Micropropagation techniques have been established for this species, but in vitro cultures have not yet been extended to generate an efficient system for the induction of callus tissue. In this study, we compared more than 100 combinations of media supplements (e.g., phytohormones, absorbers of polyphenols, and inhibitors of polyphenol oxidase), along with various light regimes, and three different genotypes of globe artichoke to define the optimal conditions for callus induction from leaf explants. This led to the elaboration of an in vitro culture protocol which resulted in a high frequency of callus induction after just 1 week in culture. The procedure used leaf explants from virus-free, meristem culture-derived plantlets. Quantitative HPLC analysis revealed that, as in globe artichoke leaves, the predominant phenolic esters present in callus were mono- and di-caffeoylquinic acids (diCQA). The concentration of diCQA was three- to five-fold higher in calli than in leaves. The exposure of calli to UV-C light further enhanced the levels of CQAs. In vitro callus culture combined with UV-C irradiation may thus represent a viable production system for diCQA that is suitable for the synthesis of pharmacologically-active compounds.