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With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.

Both inter- and intraspecific maps have been developed in eggplant. The former benefit from an enhanced frequency of marker polymorphism, but their relevance to marker-assisted crop breeding is limited. The first maps developed could be defined as ‘first generation,’ built up by means of pre-NGS (next-generation sequencing) molecular biology techniques (AFLP, RAPD, SSR, etc.). Unfortunately, the reduced polymorphism detected in intraspecific mapping populations in the ‘first-generation’ maps, along with the relatively low commercial importance in the ‘seed market’ of the species, hampered the construction of dense eggplant genetic maps. Recently, thanks to NGS-derived molecular markers, new marker-rich maps (‘second-generation maps’) were constructed. To assist selection in breeding programs, in particular to identify QTLs underlying key agronomic traits, biparental approaches as well as genome-wide association (GWA) mapping studies were conducted in this species, using the available linkage maps. Among the traits studied, great importance was given to the identification of QTLs linked to morphological and biological traits, including leaf, flower, plant, and fruit characteristics, as well as QTLs associated with parthenocarpy and to resistances to fungal (Fusarium oxysporum f. sp. melongenae and Verticillium dahliae) and bacterial (Ralstonia solanacearum) wilts. QTL studies to elucidate the genetic basis of biochemical composition, content in bioactive and antinutritional compounds, as well as other fruit quality traits were also carried out.

Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.

[EN] Single primer enrichment technology (SPET) is a new, robust, and customizable solution for targeted genotyping. Unlike genotyping by sequencing (GBS), and like DNA chips, SPET is a targeted genotyping technology, relying on the sequencing of a region flanking a primer. Its reliance on single primers, rather than on primer pairs, greatly simplifies panel design, and allows higher levels of multiplexing than PCR-based genotyping. Thanks to the sequencing of the regions surrounding the target SNP, SPET allows the discovery of thousands of closely linked, novel SNPs. In order to assess the potential of SPET for high-throughput genotyping in plants, a panel comprising 5k target SNPs, designed both on coding regions and introns/UTRs, was developed for tomato and eggplant. Genotyping of two panels composed of 400 tomato and 422 eggplant accessions, comprising both domesticated material and wild relatives, generated a total of 12,002 and 30,731 high confidence SNPs, respectively, which comprised both target and novel SNPs in an approximate ratio of 1:1.6, and 1:5.5 in tomato and eggplant, respectively. The vast majority of the markers was transferrable to related species that diverged up to 3.4 million years ago (Solanum pennellii for tomato and S. macrocarpon for eggplant). Maximum Likelihood phylogenetic trees and PCA outputs obtained from the whole dataset highlighted genetic relationships among accessions and species which were congruent with what was previously reported in literature. Better discrimination among domesticated accessions was achieved by using the target SNPs, while better discrimination among wild species was achieved using the whole SNP dataset. Our results reveal that SPET genotyping is a robust, high-throughput technology for genetic fingerprinting, with a high degree of cross-transferability between crops and their cultivated and wild relatives, and allows identification of duplicates and mislabeled accessions in genebanks., This work has been funded by the European Union's Horizon 2020 Research and Innovation Programme under the grant agreement number 677379 (G2P-SOL project: Linking genetic resources, genomes, and phenotypes of solanaceous crops).

The first high-quality genome assembly of the globe artichoke has been produced within the Compositae Genome Project and the resequencing analyses of four globe artichoke genotypes, representative of the core varietal types, as well as a genotype of the related taxa cultivated cardoon was, later on, carried out. The Web site “www.artichokegenome.unito.it” hosts all the available genomic sequences, together with their structural/functional annotations and project information are presented to users via the open-source tool JBrowse, allowing the analysis of collinearity and the discovery of genomic variants, thus representing a one-stop resource for Cynara cardunculus genomics. Pseudomolecules as well as unmapped scaffolds were used for the bulk mining of SSR markers and for the construction of the first globe artichoke microsatellite marker database. A database, called “Cynara cardunculus MicroSatellite DataBase” (CyMSatDB) was developed to provide a searchable interface to the SSR data. CyMSatDB facilitates the retrieval of SSR markers, as well as suggested forward and reverse primers, on the basis of genomic location, genomic versus genic context, perfect versus imperfect repeat, motif type, motif sequence, and repeat number.

The main use of Cynara cardunculus L. is food, but there are also alternative uses, of which this chapter gives an overview. All the possible alternative uses of C. cardunculus have emerged only in recent decades, with the exception of the florets as vegetable rennet, which has a very ancient tradition behind it. The need for renewable energy sources has led to studying C. cardunculus as an energy plant, from which biomass for direct combustion, biomethane, bio-ethanol and oil can be obtained. The latter is also suitable for human consumption. The aboveground biomass could also provide fiber for paper pulp production, as well as various livestock feed resources. The chapter, lastly, includes the above-mentioned use as vegetable rennet and the possible use as an ornamental plant.

Fusarium spp. and Verticillium spp. are widespread soil pathogens responsible for vascular wilts causing heavy yield losses in eggplant (Solanum melongena) as well as in many other crops. Here we report on the identification of QTLs affecting the resistance to Fusarium and Verticillium in an F2 intraspecific population of 156 individuals bred from the cross ‘305E40’ × ‘67/3’, we previously characterized for key agronomic and biochemical traits. The female parent (‘305E40’) is an androgenetic introgressed line carrying the resistance locus Rfo-Sa1 derived from Solanum aethiopicum. The line is fully resistant to Fusarium and also displays a previously uncharacterized partial resistance to Verticillium. The male parent (‘67/3’) is an F8 selection from the eggplant intra-specific cross cv. ‘Purpura’ x cv. ‘CIN2’ which, unexpectedly, revealed a not previously characterized partial resistance to Fusarium, but it is highly susceptible to Verticillium. The degree of resistance of the F2 population was assessed following artificial inoculation in greenhouse (Fusarium) or growth chamber (Verticillium) of F2:3 progenies obtained by selfing each F2 individual. Other than a major QTL for the resistance to Fusarium, which lies in the genomic region of the Rfo-Sa1 locus, major and minor QTL influencing the response to both Fusarium and Verticillium were spotted, and putative tomato orthologous genes were identified as well. The markers linked to the spotted QTL may find application in the context of marker-assisted breeding.

The growing area of European hazelnut (Corylus avellana L.) is increasing, as well as the number of producing countries, and there is a pressing need for new improved cultivars. Hazelnut conventional breeding process is slow, due to the length of juvenile phase and the high heterozygosity level. The development of genetic linkage maps and the identification of molecular markers tightly linked to QTL (quantitative trait loci) of agronomic interest are essential tools for speeding up the selection of seedlings carrying desired traits through marker-assisted selection. The objectives of this study were to enrich a previous linkage map and confirm QTL related to time of leaf budburst, using an F1 population obtained by crossing Tonda Gentile delle Langhe with Merveille de Bollwiller. Genotyping-by-Sequencing was used to identify a total of 9,999 single nucleotide polymorphism markers. Well saturated linkage maps were constructed for each parent using the double pseudo-testcross mapping strategy. A reciprocal translocation was detected in Tonda Gentile delle Langhe between two non-homologous chromosomes. Applying a bioinformatic approach, we were able to disentangle 'pseudo-linkage' between markers, removing markers around the translocation breakpoints and obtain a linear order of the markers for the two chromosomes arms, for each linkage group involved in the translocation. Twenty-nine QTL for time of leaf budburst were identified, including a stably expressed region on LG_02 of the Tonda Gentile delle Langhe map. The stability of these QTL and their coding sequence content indicates promise for the identification of specific chromosomal regions carrying key genes involved in leaf budburst.

We have characterized the simple sequence repeat (SSR) markers of the eggplant (Solanum melongena) using a recent high quality sequence of its whole genome. We found nearly 133,000 perfect SSRs, a density of 125.5 SSRs/Mbp, and also about 178,400 imperfect SSRs. Of the perfect SSRs, 15.6% were complex, with two stretches of repeats separated by an intervening block of

Cynara cardunculus, a member of the Asteraceae family, comprises the three taxa var. scolymus (globe artichoke), var. altilis (cultivated cardoon) and the ancestral var. sylvestris (wild cardoon). The substantial quantities of lignocellulosic biomass produced by these plants (up to 30.0 t/ha year−1 dry matter by the cultivated cardoon) can be used either as a source of bioenergy and/or as raw material for paper pulp production. Here, genotyping-by-sequencing to an F1 population derived from a cross between a globe artichoke (C3) and a cultivated cardoon (ALT) genotypes has been used to perform a genome-wide linkage analysis, leading to the elaboration of a pair of highly dense genetic maps, each derived from one of the two highly heterozygous parental genotypes. In both maps, the number of linkage groups (17) matched the species’ haploid chromosome number. The F1 population was phenotyped over two seasons with respect to plant height, stem number, capitulum number, leaf and stem fresh weight, and the dry weight of the whole plant, the leaves, the stems, the capitula and the achenes. The phenotypic data were combined with the linkage maps to identify 81 quantitative trait loci, of which 50 were placed on the C3 map and 31 on the ALT map. The loci were scattered over 13 linkage groups, and were clustered within 27 genomic regions, 22 of which harboured two or more QTL. Ten of these regions were specific to the C3 map and six to the ALT map, while the other 11 were represented on both maps. The 27 regions harboured in all 1960 genes, 83% of which could be functionally annotated. An enrichment for certain gene ontology terms was noted for the gene content of the genomic regions harbouring loci influencing seed yield and the number/weight of stems.

The bracts of the globe artichoke inflorescence vary in their pigmentation and the extent of fleshy thorn development. Here, a genetic analysis of these two traits is presented, based on a pre-existing and well-characterized mapping population derived from a cross between a globe artichoke variety and a cultivated cardoon. While both traits appeared to be simply inherited when the number of trait classes was limited to three (pigmentation) or two (thorniness), extending the classification to a larger number of states generated continuous distributions appropriate for a quantitative trait locus (QTL) analysis. The seven QTL identified by this means, mapping to five different linkage groups, were all expressed across two growing seasons. The largest effect QTL mapped onto homologues linkage group in each parental maps, and explained up to 69 and 73 % of the phenotypic variance respectively for bracts pigmentation and thorniness.

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

Globe artichoke (Cynara cardunculus var. scolymus), whose immature inflorescences (capitula) are consumed as a vegetable all over the world, contributes significantly to the agricultural economy of the Mediterranean basin. Here, we describe a QTL (quantitative trait loci) analysis aimed at elucidating the mode of inheritance of seven main and first-order capitulum traits. Mapping was carried out in an F1 population obtained by crossing a globe artichoke with a cultivated cardoon (C. cardunculus var. altilis). A total of 100 QTL associated with the seven capitulum traits were mapped to 23 chromosomal regions, scattered over 12 of the 17 linkage groups. Among these, 73 were expressed in both growing seasons, while the others were only detected in one season. Up to nine QTL per trait were identified, and major QTL, responsible for some 20 % of the phenotypic variation, were detected for capitulum length, diameter, shape index and fresh weight. The QTL for correlated traits frequently co-localized, most likely due to pleiotropy. This study represents the first report on yield traits QTL in globe artichoke. The QTL identified, along with linked markers, particularly those located in four hot-spot QTL regions are of practical interest for crop improvement based on marker-assisted breeding.

An F1 mapping population was bred by crossing an accession of wild cardoon with a single Argentinian globe artichoke plant of the variety Estrella del Sur FCA with a view to generating new Cynara cardunculus linkage maps. Genotyping was conducting using a set of 553 SRAP, SSR, AFLP and SNP markers. The 1,465.5 cM map based on the segregation of alleles present in the wild cardoon parent comprised 214 loci distributed across 16 linkage groups (LGs), while the 910.1 cM globe artichoke-based map featured 141 loci falling into 12 LGs covering the total length. Three of the morphological traits (head spininess, leaf spininess and head color) for which the parents contrasted were inherited monogenically, and the genes conditioning them were mapped. A set of 48 co-dominant loci was used to align the LGs with those derived from a reference SSR-based consensus map of the species.

Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds, and wild species within and beyond the Compositae, and will facilitate the identification of economically important genes from related species.

The European hazelnut (Corylus avellana L.) is the most economically important nut species in the Betulaceae family. Despite the need for new improved hazelnut cultivars, few breeding programs are carried out because of the large plant size, the long lifecycle of the plant, and the expense and time required. To date, there are no reports of maps with quantitative trait loci (QTL) in hazelnut. Our objective in the present study was to identify QTL associated with vegetative traits to allow marker-assisted selection (MAS). An F1 progeny (275 plants) of Tonda Gentile delle Langhe × Merveille de Bollwiller obtained in 2009 was used to develop a QTL linkage map for vigour, sucker habit, and time of bud burst, after three years of observations. A set of 163 plants were analysed with 152 microsatellite markers. A map of 11 linkage groups was obtained, covering 663.1 cM, and 15 QTLs were identified and mapped for the traits examined. Of them, 10 were 'major' QTL, including a stably expressed region on LG_02 for leaf bud burst. At least one major QTL for each year underlies the variation in each trait and a clustering of QTL for trunk circumference and suckers/trunk circumference ratio with high inter-trait correlations was observed on LG_05, suggesting a single pleiotropic locus. This research represents an initial step in the future identification of chromosomal regions carrying genes of interest, important for breeding programs and MAS.

The technology in which a gene(s) is moved between unrelated species has been termed ‘genetic transformation,’ ‘gene technology,’ or ‘genetic engineering.’ Its result is a ‘genetically modified (GM) organism.’ The principal GM food crops grown commercially to date are herbicide-tolerant and insecticide-producing soybean, maize, and canola (oilseed rape), but the technology has been, or is in the process of being, used in a variety of other crop species with a view to improving levels of resistance to diseases and tolerance of abiotic stress, as well as to enhance the nutritional content of the end product (sometimes termed ‘biofortification’). This article describes the key purpose of GM technology, the main types of GM food currently on the market, the geographic spread of GM crop cultivation, and the global volume of GM crop production. It also addresses some of the major concerns surrounding the safety of the technology and the possible environmental hazards associated with the production and consumption of GM foodstuffs.

Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds and wild species within and beyond the Compositae and will facilitate the identification of economically important genes from related species.

Cynara cardunculus (2n=2x=34) is a member of the Asteraceae family, which is the largest and one of the most ecologically successful botanical families, and possesses a medium-sized genome (flow cytometry estimation = 1.07 Gb). The species is native to the Mediterranean Basin and, in addition to globe artichoke (var. scolymus L), it includes the cultivated cardoon (var. altilis DC) and the wild cardoon [var. sylvestris (Lamk) Fiori]. The two cultivated and the wild forms are fully cross-compatible and their F1 segregating progenies have been developed for linkage studies on the basis of the two-way pseudo-testcross approach as well as for QTL analyses. Here, we report a survey on the state of the art of map development and the identification of genomic region influencing key agronomic traits in the species.

Eggplant berries are a source of health-promoting metabolites including antioxidant and nutraceutical compounds, mainly anthocyanins and chlorogenic acid; however, they also contain some anti-nutritional compounds such as steroidal glycoalkaloids (SGA) and saponins, which are responsible for the bitter taste of the flesh and with potential toxic effects on humans. Up to now, Quantitative Trait Loci (QTL) for the metabolic content are far from being characterized in eggplant, thus hampering the application of breeding programs aimed at improving its fruit quality. Here we report on the identification of some QTL for the fruit metabolic content in an F2 intraspecific mapping population of 156 individuals, obtained by crossing the eggplant breeding lines “305E40” × “67/3.” The same population was previously employed for the development of a RAD-tag based linkage map and the identification of QTL associated to morphological and physiological traits. The mapping population was biochemically characterized for both fruit basic qualitative data, like dry matter, °Brix, sugars, and organic acids, as well as for health-related compounds such chlorogenic acid, (the main flesh monomeric phenol), the two peel anthocyanins [i.e., delphinidin-3-rutinoside (D3R) and delphinidin-3-(p- coumaroylrutinoside)-5-glucoside (nasunin)] and the two main steroidal glycoalkaloids, solasonine, and solamargine. For most of the traits, one major QTL (PVE ≥10%) was spotted and putative orthologies with other Solanaceae crops are discussed. The present results supply valuable information to eggplant breeders on the inheritance of key fruit quality traits, thus providing potential tools to assist future breeding programs.

Cynara cardunculus (2n = 2× = 34) is a member of the Asteraceae family that contributes significantly to the agricultural economy of the Mediterranean basin. The species includes two cultivated varieties, globe artichoke and cardoon, which are grown mainly for food. Cynara cardunculus is an orphan crop species whose genome/transcriptome has been relatively unexplored, especially in comparison to other Asteraceae crops. Hence, there is a significant need to improve its genomic resources through the identification of novel genes and sequence-based markers, to design new breeding schemes aimed at increasing quality and crop productivity. We report the outcome of cDNA sequencing and assembly for eleven accessions of C. cardunculus. Sequencing of three mapping parental genotypes using Roche 454-Titanium technology generated 1.7 × 10⁶ reads, which were assembled into 38,726 reference transcripts covering 32 Mbp. Putative enzyme-encoding genes were annotated using the KEGG-database. Transcription factors and candidate resistance genes were surveyed as well. Paired-end sequencing was done for cDNA libraries of eight other representative C. cardunculus accessions on an Illumina Genome Analyzer IIx, generating 46 × 10⁶ reads. Alignment of the IGA and 454 reads to reference transcripts led to the identification of 195,400 SNPs with a Bayesian probability exceeding 95%; a validation rate of 90% was obtained by Sanger-sequencing of a subset of contigs. These results demonstrate that the integration of data from different NGS platforms enables large-scale transcriptome characterization, along with massive SNP discovery. This information will contribute to the dissection of key agricultural traits in C. cardunculus and facilitate the implementation of marker-assisted selection programs.

Globe artichoke (Cynara cardunculus var. scolymus L., Asteraceae) is a perennial crop traditionally consumed as a vegetable in the Mediterranean countries and rich in nutraceutically and pharmaceutically active compounds, including phenolic and terpenoid compounds. Its bitter taste is caused by its high content of sesquiterpene lactones (STLs), such as cynaropicrin. The biosynthetic pathway responsible for STL biosynthesis in globe artichoke is unknown, but likely proceeds through germacrene A, as has been shown for other Asteraceae species. Here, we investigated the accumulation of cynaropicrin in different tissues of globe artichoke, and compared it to accumulation of phenolic compounds. Cynaropicrin concentration was highest in old leaves. A putative germacrene A synthase (GAS) gene was identified in a set of ~19,000 globe artichoke unigenes. When heterologously expressed in Escherichia coli, the putative globe artichoke GAS converted farnesyl diphosphate (FPP) into (+)-germacrene A. Among various tissues assayed, the level of globe artichoke GAS expression was highest in mature (six week old) leaves. A sequence polymorphism within a mapping population parent allowed the corresponding GAS gene to be positioned on a genetic map. This study reports the isolation, expression and mapping of a key gene involved in STL biosynthesis in C. cardunculus. This is a good basis for further investigation of this pathway.

In Sicily, the increasing use of exotic globe artichoke germplasm is eroding the presence of autochthonous landraces, including the long established ‘Violetto di Sicilia’. Ten clones have emerged from a clonal selection programme in this landrace, and here we describe the variation that they capture both at the level of AFLP-based genotype and phenotypically with respect to key productivity traits, on the basis of two seasons of field evaluation. The clonal selections yielded, on average, 8.9 heads per plant (equivalent to a fresh weight yield of 1.28 kg). Two clones yielded particularly well in both growing seasons (10.6 heads, equivalent to 1.46 kg per plant), while another pair produced particularly large heads (on average 165 g) and a high receptacle incidence (on average 19.3 g 100 g−1 fresh weight). Both the number of days to first harvest and the quantity of head dry matter were subject to a significant degree of ‘clone × year’ interaction. Yield, the number of heads per plant and receptacle incidence were associated with a moderate (0.30–0.53) broad sense heritability, indicating that these traits could be successfully improved by phenotype-based clonal selection. AFLP fingerprinting was able to discriminate between all the clones, based on only three primer combinations. A principal component analysis based on the AFLP fingerprints was used to compare the selected clones with a set of individuals chosen on the basis of maximum genetic diversity. This comparison suggested that the new clone set was representative of the genetic variation present in ‘Violetto di Sicilia’, because the diversity captured by the two sets was largely overlapping, confirming the possibility of carrying out clonal selection in this globe artichoke landrace without compromising its preservation in situ.

The huge diversity of fungi may reflect both the heterogeneity of the niches they occupy and the diverse stresses they must cope with. In order to investigate the genetic and functional diversity in the ericoid mycorrhizal fungus Oidiodendron maius subjected to heavy metal stress, we isolated O. maius strains from a serpentine site naturally enriched by heavy metals. Despite the high Cr and Ni soil concentrations, a high level of diversity was found in the serpentine fungal community. The growth of these isolates in the presence of different metal contaminants identified some tolerant strains, suggesting a site-specific adaptation. To investigate within-species gene divergence in stressful environments, we then compared the sequence polymorphism of a neutral (internal transcribed spacer) and a functional (Cu,ZnSOD) gene in O. maius isolates derived from the serpentine site, from a site heavily polluted with industrial wastes and from unpolluted sites. For all isolates tested, the polymorphism was higher in the nucleotide sequence of the functional gene. However, when compared with isolates from the serpentine area, isolates from industrially polluted sites showed a significantly higher polymorphism in the Cu,ZnSOD promoter region, suggesting that environmental stress may influence the rate of mutations in specific regions of the Sod1 locus.

The maize pathogens Fusarium verticillioides (Fv) and Fusarium proliferatum (Fp) are morphologically very similar to one another, so Fp isolates have been often mistaken as Fusarium moniliforme (the former name of Fv). The only presently accepted morphological discriminator between these species is the presence/absence of polyphialides. Here, a collection of 100 Fusarium strains, isolated from infected maize kernels on plants grown in north-western Italy, were assigned as Fv or Fp on the basis of the presence/absence of polyphialides. This classification was tested on a subset of isolates by sexual crosses, ITS and calmodulin sequencing and AFLP profiling. An ITS-RFLP assay was extended to the full collection and to a number of Fv and Fp isolates of different geographical origin and hosts. The ITS region is proposed as taxonomically informative for distinguishing between Fp and Fv.

Metschnikowia pulcherrima occurs naturally on fruits, buds and floral parts of apple trees. Some strains are effective as biocontrol agents against postharvest decay of apples and other fruits. The usefulness of the amplified fragment length polymorphism (AFLP) technique was evaluated for the genetic analysis of 26 strains of M. pulcherrima, isolated from different sources in different geographical regions. With six AFLP primer pairs, 729 polymorphic bands were scored. The technique showed a high discriminatory power. Genetic relationships between strains were also estimated using AFLP. All the isolates from the carposphere of apple, previously tested as biocontrol agents, were grouped in a single cluster with a high bootstrap value (97), indicating robustness and reproducibility. AFLP patterns could clearly distinguish the different strains and research is in progress to use some putative specific bands for single tag sequence (STS) conversion to develop isolate-specific markers.

Various types of molecular marker have been used to conduct phylogenetic and mapping studies in pepper (Capsicum annuum), among which microsatellites (SSRs) have been shown to be particularly valuable because of their co-dominant and multi-allelic nature. Of the approximately 180 pepper SSRs available in public domain, only a few have been either inter-and/or intraspecifically mapped so far. This paper reports the development of a set of new EST-based SSR assays. This class of SSRs is more transferable between species than those based on genomic sequence, and so can provide anchor markers in cross-species comparative mapping. A search of >8000 Capsicum ESTs, corresponding to approximately 4.1 Mbp of DNA, yielded 1325 SSRs present in 899 sequences. A cluster analysis was employed to obtain non-redundant SSR-containing sequences, leading to the definition of 576 non-redundant sequences containing 783 SSRs. Primer pairs were designed to amplify 348 SSRs, and a subset of 204 was used to screen for polymorphism within a parental set of four C. annuum inbred lines and one C. frutescens accession, used as parents of three mapping populations. The resulting 49 polymorphic SSRs provided the basis for assessing genetic diversity within an extended Capsicum spp. genotype set.

The dietary value of many plant polyphenols lies in the protection given against degenerative pathologies. Their in planta role is associated with the host's defense response against biotic and abiotic stress. The polyphenol content of a given plant tissue is strongly influenced by the growing environment, but is also genetically determined. Plants belonging to the Cynara cardunculus species (globe artichoke and the cultivated and wild cardoon) accumulate substantial quantities of polyphenols mainly mono and di-caffeoylquinic acid (CQA) in their foliage. Transgressive segregation for CQA content in an F1 population bred from a cross between a globe artichoke and a cultivated cardoon led to the selection of eight segregants which accumulated more CQA in their leaves than did those of either of their parental genotypes. The selections were grown over two seasons to assess their polyphenol profile (CQAs, apigenin and luteolin derivatives and narirutin), and were also fingerprinted using a set of 217 microsatellite markers. The growing environment exerted a strong effect on polyphenol content, but two of the selections were able to accumulate up to an order of magnitude more CQA than either parent in both growing seasons. Since the species is readily vegetatively propagable, such genotypes can be straightforwardly exploited as a source of pharmaceutically valuable compounds, while their SSR-based fingerprinting will allow the genetic identity of clonally propagated material to be easily verified.

Artemisia umbelliformis Lam. (syn. A. mutellina Vill., A. laxa (L.) Fritsch) is an herbaceous alpine plant belonging to the Asteraceae family, and most commonly growing in the wild at altitudes between 2,000 and 3,700 m a.s.l. Its flowers are used for the production of an alcohol-based infusion called genepi in Italian and genepi in French. Uncontrolled picking of natural populations has endangered the species’ survival, despite bans in Switzerland and Italy and strict regulation in France. Limited quantities are produced under cultivation, but these are insufficient to meet market needs, so the selection of productive genotypes is a present priority. We report here an AFLP-based assessment of the genetic diversity represented in five ecotypes (Valle Gesso, Marmora, Gran Paradiso, Val Chisone and Elva), one selection (RAC12) and a wild population. The four ecotypes Valle Gesso, Gran Paradiso, Val Chisone and Elva together captured a broad coverage of the genetic variability present. A sample of ten plants was selected from each of these ecotypes for a more detailed analysis of genetic, phenological and morphological variation. The assays confirmed the distinctness of the four ecotypes, and were used to identify set of mother plants of clonal populations suitable for cultivation.

SUMMARYThe morphological (using International Union for the Protection of New Varieties of Plants (UPOV) descriptors) and field performance of five clones selected from the globe artichoke landrace Spinoso di Palermo were determined over two seasons, and their amplified fragment length polymorphism (AFLP) profiles detected using seven primer combinations (PCs). The number of heads produced averaged 13·8 per plant (equivalent to a fresh weight yield of 2·1 kg/plant), but two of the clones produced 15·6 heads per plant (2·4 kg/plant). Three clones produced noticeably larger second-order heads (mean of 156 g), and so were considered to be suitable for the production of desirable heads over a prolonged harvesting period. Head yield and the number of heads per plant were associated with a moderate level of broad sense heritability (0·29–0·46), implying that these traits could be viewed as primary selection criteria. From the list of 51 UPOV descriptors, 18 varied among the five clones, but variation at just six, simply scored, descriptors was sufficient to discriminate completely between the examined clones. Full discrimination was also achieved by applying only three of the seven selected AFLP PCs. According to the AFLP profiles, two of the clones were highly similar. The similarity matrices calculated from the UPOV descriptors and the AFLP profiles were highly correlated with one another. The data are optimistic and indicate that the performance of Spinoso di Palermo could be much improved via clonal selection.

SummaryAnemone coronaria is an out-crossing species grown widely as an ornamental. The genetic divergence between and within five widely commercialised cultivars (‘Cristrina’, ‘Monalisa’, ‘Tetraelite’, ‘Wicabri’ and ‘Mistral’) has been evaluated using AFLP markers. Within each cultivar three-to-eight sub-cultivars, which differ mainly in the colour of their flowers, were assayed. Four plants within each sub-cultivar were genotyped. Due to the large size of the A. coronaria genome, an AFLP protocol based on restriction with Sbf I (an 8 bp cutter) and Mse I (a 4 bp cutter) was developed. Clear and reproducible electrophoretic patterns displaying 56 – 79 bands (average 67) per primer combination were obtained by applying five (Sbf I+2 / Mse I+3) selective nucleotides. A total of 402 bands were observed, of which 152 were polymorphic. Genetic similarities between accessions were calculated according to the Simple Matching Coefficient and used to construct a dendrogram based on the unweighted pair group method...

Cynara cardunculus L. is a species native to the Mediterranean basin that comprises 2 crops, globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC), as well as wild cardoon (var. sylvestris (Lamk) Fiori). Globe artichoke represents an important component of the South European agricultural economy but is also cultivated in North Africa, the Near East, South America, the United States, and China. Breeding activities and molecular marker studies have been, to date, extremely limited. Better knowledge of the genome of the species might be gained by developing a range of molecular markers. Here, we report on the development of 14 microsatellites (simple sequence repeats (SSRs)) through a novel approach that we have defined as the microsatellite amplified library (MAL). The approach represents a combination of amplified fragment length polymorphism and a primer extension based enriched library, is rapid, and requires no hybridization enrichment steps. The technique provided a ~40-fold increase in the efficiency of SSR identification compared with conventional library procedures. The developed SSRs were applied for genotyping 36 accessions of C. cardunculus, including a core of 27 varietal types of globe artichoke, 3 accessions of cultivated cardoon, and 6 Sicilian accessions of wild cardoon. Principal coordinates analysis made it possible to differentiate both cultivated and wild forms from each other.Key words: globe artichoke, wild and cultivated cardoon, molecular markers, AFLP, MAL (microsatellite amplified library).

As part of the clonal selection carried out since 1978 by CRAS on "Spinoso sardo" artichoke, a number of clones having interesting characteristics for the fresh market have been observed. In order to characterise the most promising selections and make it possible to register them on the National Register of varieties, an agronomical comparison among nine clones was started. The experiment was carried out for three consecutive years at the CRAS farm in Oristano on a net area of 600 square metres, according to a randomised-block experimental scheme with four replications and simple 10-plant plots. The following measurements were carried out on the vegetation when the first head was harvested: plant height, height of insertion of first-order head and number of leaves per plant. Moreover the following main production parameters were observed: number of heads per plant subdivided per order and time of emission, polar and equatorial diameter and weight of head, total length and number of leaves of stalk, and total weight of head and stalk. Significant differences were pointed out (Duncan Test) among the clones for almost all measured parameters: clone 50 B was differentiated for its plant biometric characteristics, and clones 108 B /11, 39 B and 37 B for its yield and early maturity. The Meli clone has been pointed out as having the best head biometric characteristics. At the same time the Department of Agronomical Science and Agrarian Vegetable Genetics of the University of Turin carried out genetic characterisation. The 9 clones. were characterized by using 25 AFLP (amplified fragment length polymorfphysm) primer combination ad electrophoretic pattern scores were used to produce a dendrogram based on the Jaccard's Similarity Index (JSI) and UPGMA (Unweighted Pair Group Method using Arithmetic Averages) clustering.

Recognition by the European Community of elite landraces encourages farmers to grow these and earn more than is possible by growing modern varieties. However, farmers often exercise strong selection by collecting seed from a limited number of plants which best embody a few characters of prime interest. This, in the long run, may be responsible for considerable genetic erosion. In Southern Piedmont, North-West Italy, a local landrace of pepper (Capsicum annuum L.) known as ‘Cuneo’ is grown; it deserves particular attention because of its hardiness, late production and fruit quality. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess the effect, on genetic composition of a population, of seed sampling carried out using the selection criteria adopted by the farmer. After two reproductive cycles using selection, it was already possible to detect loss in genetic variation and a change in allele frequencies, while no significant effect was found after two cycles of random sampling. Over this period, farmer selection pressure led to disappearance of eight low-frequency alleles, while only three alleles were lost in randomly sampled populations. Our results stress the need to adopt appropriate techniques for seed sampling in order to avoid genetic erosion of local landrace ‘gene pools’.

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein.

Oleander is a Mediterranean evergreen shrub found along watercourses, gravelly places and damp slopes. It is grown widely 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). Nine primer combinations yielded a total of 603 bands of which 241 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 show that the AFLP technique, which can simultaneously and assay a large number of loci randomly distributed in the genome, is much more informative on the genetic relationship and origin of accessions than the limited number of morphological characters conventionally used for variety discrimination. 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 essentially derived varieties is discussed.

SummaryGlobe artichoke is a diploid (2n=2x=34), predominantly cross-pollinated, native plant of the Mediterranean basin, and Italy contains the richest gene pool of cultivated varieties. Commercial production is mainly based on perennial cultivation of vegetatively propagated clones, which are highly heterozygous and segregate widely when progeny-tested. Here we report on the application of inter-simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP) markers for fingerprinting and evaluating genetic distances among 24 selected clones belonging to varietal type ‘Spinoso sardo’. Twenty-two of the 26 ISSR primers tested gave good amplification, but only seven were efficient in generating polymorphic bands. Thirty-one of the 48 AFLP primer combinations applied gave clear and reproducible amplification patterns, and the number of polymorphic bands ranged from 4 to 19. Furthermore, AFLP led to the identification of two chimeras among the mother plants from which clones originated. AFLP...

In several regions of Italy as well as other parts of southern Europe, the heterogeneity of the land, the climate and the soil favour the survival in cultivation of a large number of landraces specifically adapted to local conditions. Knowledge on the level and distribution of their genetic variation can help to develop appropriate strategies, in order to suistainably manage in situ these germplasm resources at risk of genetic erosion. C. annuum is an herbaceous diploid species and is considered to be self-pollinating, although different rates of out-crossing have been recorded. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess genetic diversity within and between five populations of a landrace of Capsicum annuum L., grown in a limited area in north-west Italy and locally known as ‘Cuneo’ pepper. Partitioning the genetic variation with Shannon's diversity index revealed that 41.6% occurred between and 58.4% within populations. Analogous results were obtained when the analysis was based only on RAPD or AFLP markers. However, AFLP was more reliable, since a lower range of variation was observed among primer combinations in detecting the two components of genetic variation. Notwithstanding the rather high level of within genetic variation detected, the five populations were clearly differentiated and differed in the frequency of alleles exclusive and/or present at very low frequencies. Our results show the need for accurate estimation of allele frequencies, in order to identify populations to which priority should be given for dynamic conservation of landraces.

Globe artichoke is a vegetable native to the Mediterranean basin. Its commercial production is mainly based on perennial cultivation of vegetatively propagated clones, which are highly heterozygous and segregate widely when progeny-tested. The aim of this study was to measure genetic variation using random amplified polymorphic DNA (RAPD) in five populations of ‘Spinoso sardo’, the most widely grown and economically important cultivar in Sardinia (Italy). Analysis of molecular variance (amova) gave highly significant differences between populations (28.1% of the total genetic diversity); substantial within-population variation was detected (71.8% of total genetic diversity). The RAPD results show the need to apply clonal selection to narrow the broad genetic variability within the cultivar and to address important questions on the most suitable strategies for germplasm preservation.

Cynara cardunculus L., a member of the Asteraceae family, is a diploid (2n = 34) outcrossing perennial species native to the Mediterranean basin. It includes globe artichoke (var. scolymus L.), which today is grown as vegetable all over the world, cultivated cardoon (var. altilis DC), locally grown in Southern European countries, and their progenitor wild cardoon (var. sylvestris Lam). The species is also a valuable source of pharmaceutical compounds, and is exploitable for the production of lignocellulosic biomass as well as oil from seed, the latter being suitable for both edible and bio-fuel end-uses.