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Nowadays, food authentication is more and more required given its relevance in terms of quality and safety. The seafood market is heavily affected by mislabelling and fraudulent substitutions/adulterations, especially for processed food products such as canned food items, due to the loss of morphological features. This study aims to develop new assays based on DNA to identify fresh mackerel (Scomber spp.) and commercial products. A new primer pair was de novo designed on the 5S rRNA gene and non-transcribed spacer (NTS), identifying a DNA mini-barcoding region suitable for species identification of processed commercial products. Moreover, to offer a fast and low-cost analysis, a new assay based on recombinase polymerase amplification (RPA) was developed for the identification of fresh ‘Sgombro’ (Scomber scombrus) and ‘Lanzardo o Occhione’ (Scomber japonicus and Scomber colias), coupled with the lateral flow visualisation for the most expensive species (Scomber scombrus) identification. This innovative portable assay has great potential for supply chain traceability in the seafood market.

Diversi marcatori plastidiali sono stati testati su varie specie della flora vascolare spontanea al fine di valutarne il potere discriminante come DNA barcoding, da integrare nell'identificazione tassonomica con i dati morfologici, cariologici, fenologici e biogeografici.

The Polygonaceae (40-50 genera and approximately 1,100 species) are characterized by the presence of an ocrea, an hyaline or membranous tubular sheath, derived from the fusion of the stipules. Their classifi cation is based principally on macromorphological characters. The broad Poly- gonum s.l. is a much debated genus which has been variously interpreted in the literature: its relation to associated genera is not clear and moreover, it has been subdivided into numerous more natural taxa considered at the rank of genus, subgenus or section. In the present study, using analysis of the rbcL sequence the monophyly of the various taxa that Polygonum s.l. has been subdivided are investigated by focusing on the species present in Europe in order to reach a classifi cation more in line with the molecular data. The phylogenetic tree obtained with the maximum parsimony method confi rms that Polygona- ceae is monophyletic, however, the subfamily Polygonoideae seems to be paraphyletic as it includes the Eriogonoideae. On the basis of the principal clades identifi ed the restriction of the Polygonoideae to only those taxa with an herbaceous habit is confi rmed and a new classifi cation articulated in 4 tribes is proposed: 1) Polygoneae, subdivided into Polygoninae (Atraphaxis, Polygonum -incl. Polygonella- and, probably, Calligonum, Oxygonum, Parapteropyrum and Pteropyrum) and Reynoutriinae (Fal- lopia, Homalocladium, Muehlenbeckia, ×Reyllopia, Reynoutria) as well as an incertae sedis genus (Knorringia); 2) Persicarieae, subdivided into Persicariinae subtrib. nov. (Persicaria) and Koenigii- nae (Aconogonum, Bistorta, Koenigia, Rubrivena); 3) Fagopyreae (Fagopyrum, Harpagocarpus and, probably, Eskemukerjea and Pteroxygonum); 4) Rumiceae (Emex, Oxyria, Rheum, Rumex). Lastly new combinations and/or new names are proposed in the genus ×Reyllopia, Persicaria and Rubrivena

To assess the haplotype diversity and genetic relationship between them, A set of 69 Iranian cultivated accessions, six European cultivars and an accession of Vitis labrusca along with 63 wild grapevine individuals were studied using chloroplast microsatellite markers. Results showed that among analyzed cpssr loci only ccmp 3 and ccmp10 were polymorphic within cultivars and only ccmp3 was polymorphic in wild grape individuals. The size variants of both loci combine in a total of 4 different haplotypes. All the 4 haplotype are displayed in the cultivars while only 2 are presented in wild grapes. Sultani or keshmeshi Bidane cultivar has the haplotype III that there is not this haplotype among the wild grapes of studied regions. Concerning to existence of both haplotypes I and II in the number of Iranian cultivated and wild grapes, it is possible to consider that the wild grapes are ancestor of some of our native cultivars.

In this contribution, new data concerning the distribution of vascular flora alien to Italy are presented. It includes new records, confirmations, exclusions, and status changes for Italy or for Italian administrative regions of taxa in the genera Bunias, Calocedrus, Calycanthus, Celosia, Clerodendrum, Convolvulus, Crassula, Cyclamen, Datura, Dicliptera, Eragrostis, Erigeron, Gamochaeta, Gazania, Impatiens, Kolkwitzia, Leucaena, Ludwigia, Medicago, Muscari, Nigella, Oenothera, Opuntia, Paulownia, Petroselinum, Phyllostachys, Physalis, Pseudosasa, Quercus, Reynoutria, Roldana, Saccharum, Sedum, Semiarundinaria, Senecio, Sisyrinchium, Solanum, Sporobolus, Tulipa, Vachellia, Verbena, and Youngia. Nomenclatural and distribution updates published elsewhere are provided as Suppl. material 1.

Rhododendron ferrugineum L. (Ericaceae) is a subalpine shrub that can be found throughout the Pyrenees and the Alps. The Northern Apennines are one of the southernmost limits of its geographical range. Local populations are very few, small and scattered over three mountain groups of the chain. In this study, the genetic diversity of such three Apennine populations (AP1, AP2 and AP3) was analysed and compared to the one of three populations from the near Maritime Alps and of six populations from the Central-Eastern Alps. Genetic variations across microsatellite markers revealed that the Apennine populations show some clonal individuals and the lowest genetic diversity values (AP1 and AP3 A[18] values are 2.46 and 2.31, respectively), as well as heterozygosity deficiency with respect to the Alpine populations. Genetic relationships among populations (Nei’s genetic distance) showed that, according to their geographical distribution, populations from the Central-Eastern Alps and from the Maritime Alps clustered in two separate groups, while the three Apennine populations were associated to either of the two main clusters. PCA analysis showed a clear separation of the three Apennine populations according to the high Fst values detected (AP1-AP2 = 0.427; AP1-AP3 = 0.446; AP2-AP3 = 0.325). Mantel test revealed a significant correlation between genetic and geographical distance matrices (r = 0.314, P = 0.001, 999 permutations). Considering the geographical and ecological peripheral condition of these populations and the high impact of the climatic changes on their habitat, we suggest an ex situ conservation strategy by a germplasm bank to preserve the most of their genetic variability.

Background: Identification keys are decision trees which require the observation of one or more morphological characters of an organism at each step of the process. While modern digital keys can overcome several constraints of classical paper-printed keys, their performance is not error-free. Moreover, identification cannot be always achieved when a specimen lacks some morphological features (i.e. because of season, incomplete development or miss-collecting). DNA barcoding was proven to have great potential in plant identification, while it can be ineffective with some closely related taxa, in which the relatively brief evolutionary distance did not produce differences in the core-barcode sequences. Methodology/Principal Findings: In this paper, we investigated how the DNA barcoding can support the modern digital approaches to the identification of organisms, using as a case study a local flora, that of Mt. Valerio, a small hill near the centre of Trieste (NE Italy). The core barcode markers (plastidial rbcL and matK), plus the additional trnH-psbA region, were used to identify vascular plants specimens. The usefulness of DNA barcoding data in enhancing the performance of a digital identification key was tested on three independent simulated scenarios. Conclusions/Significance: Our results show that the core barcode markers univocally identify most species of our local flora (96%). The trnH-psbA data improve the discriminating power of DNA barcoding among closely related plant taxa. In the multiparametric digital key, DNA barcoding data improves the identification success rate; in our simulation, DNA data overcame the absence of some morphological features, reaching a correct identification for 100% of the species. FRIDA, the software used to generate the digital key, has the potential to combine different data sources: we propose to use this feature to include molecular data as well, creating an integrated identification system for plant biodiversity surveys.

Medicinal plants have been widely used in traditional medicine due to their therapeutic properties. Although they are mostly used as herbal infusion and tincture, employment as ingredients of food supplements is increasing. However, fraud and adulteration are widespread issues. In our study, we aimed at evaluating DNA metabarcoding as a tool to identify product composition. In order to accomplish this, we analyzed fifteen commercial products with DNA metabarcoding, using two barcode regions: psbA-trnH and ITS2. Results showed that on average, 70% (44–100) of the declared ingredients have been identified. The ITS2 marker appears to identify more species (n = 60) than psbA-trnH (n = 35), with an ingredients’ identification rate of 52% versus 45%, respectively. Some species are identified only by one marker rather than the other. Additionally, in order to evaluate the quantitative ability of high-throughput sequencing (HTS) to compare the plant component to the corresponding assigned sequences, in the laboratory, we created six mock mixtures of plants starting both from biomass and gDNA. Our analysis also supports the application of DNA metabarcoding for a relative quantitative analysis. These results move towards the application of HTS analysis for studying the composition of herbal teas for medicinal plants’ traceability and quality control.

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91phox expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91phox expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis., Graphical Abstract, In a GLP study, Jofra Hernández and colleagues demonstrate that lentiviral vector-mediated HSPC gene therapy effectively corrects long-term X-linked chronic granulomatous disease in a mouse model of the disease. A small proportion of mice develops hematopoietic tumors originating from rare dominant clones harboring vector insertions near oncogenes.

The spread of food allergens is a topic of global importance due to its impact on public health. National and International regulations ask food producers and manufacturers to declare product compositions on the label, especially in case of processed raw materials. Wheat flour (Triticum aestivum) can be contaminated by a wide range of species belonging to the Brassicaceae in the field or during grain harvests, storage, and processing. Among them, mustards (Brassica nigra, Brassica juncea and Sinapis alba) are well known allergenic species. Often, food quality laboratories adopt an ELISA approach to detect the presence of mustard species. However, this approach shows cross-reactivity with other non-allergenic species such as Brassica napus (rapeseed). In the last few years, DNA barcoding was proposed as a valid identification method, and it is now commonly used in the authentication of food products. This study aims to set up an easy and rapid DNA-based tool to detect mustard allergenic species. DNA barcoding (matK and ITS2) and chromosome markers (A6, B, C1 genome regions) were selected, and specific primers were validated on incurred reference food matrices. The developed test was proven to be able to distinguish mustard from rapeseed and wheat, overcoming cross-reactivity with Brassica napus.

Tuna “Bottarga” is an artisanal food speciality prepared from the roe of Bluefin Tuna (Thunnus thynnus). Due to the recent restriction of Tuna catches fixed by EU, cheaper substitutes from yellowfin tuna (Thunnus albacares) are currently available in the mass distribution. Since the product is sold sliced or grated, morphological distinctive features may be lost, and fraudulent substitution is possible. In this study we tested DNA mini-barcoding and infrared spectroscopy as tools to trace its authenticity. A certified chemical standard, fresh (n = 4) and processed (n = 11) ovarian identified by the morphological observation of the tuna catches, commercial bottargasamples (n = 5) and artificial aged aliquots of all these samples (n = 50) were used in the experiments. The results showed that infrared spectroscopy failed in the taxonomic distinction, whereas enabled PLS-DA classification of aged samples (and thus the identification of fresh prepared artisanal products) with model non-error rate in fitting equal to 0.96, and 0.89 in cross-validation. Conversely, DNA mini-barcoding with mtDNA control region, success in species identification (100% maximum identity by the standard comparison BLAST approach against GenBank), regardless the processing state of the product.

Traceability, quality and safety of edible insects are important both for the producers and the consumers. Today, alongside the burst of edible insects in western countries, we are facing a gap of knowledge of insect microbiota associated with the microbial ecosystems of insect-based products. Recent studies suggest that the insect microbiota can vary between insect species and that can be shaped by additional factors, such as rearing conditions. Also, the production processes of raw materials (i.e. insect flour) into final food products can affect the insect microbiota too. This has consequences for the evaluation of food safety and food traceability. In this context, High-Throughput DNA Sequencing (HTS) techniques can give insight into the carryover of insect microbiota into final food products. In this study, we investigated the microbiota composition of insect-based commercial food products, applying HTS techniques coupled with bioinformatic analysis. The aim of this work was to analyse the microbiota variability of different categories of insect-based products made of A. domesticus (house cricket), T. molitor (mealworm beetle), and A. diaperinus (lesser mealworm or litter beetle), including commercial raw materials and processed food items, purchased via e-commerce from different companies. Our data revealed that samples cluster per insect species based on microbiota profile and preliminary results suggested that a small number of prevalent bacteria formed a “core microbiota” characterizing the products depending on the insect, suggesting that a resident microbiota is conserved. This microbial signature can be recognized despite the different food processing levels, rearing conditions selling companies. We showed that differences exist when comparing raw vs processed food made of the same insect, or similar products produced by different companies as well, laying the groundwork for further analyses. These results support the application of HTS analysis for studying the composition of processed insect food in a wider perspective, for food traceability and food quality control.

Food safety and quality depend on raw material characteristics and on the chemical, physical and biotechnological processes adopted during food transformation. Since a huge number of microorganisms are involved in food production, foodstuffs should be considered as complex matrices where any microbial component has a precise role and evolves in response to physical and chemical composition of food. Moreover, knowing the dynamics of microbial community involved in a food supply chain it is useful to reduce food spoilage, enhance the industrial processes and extend product shelf-life. In a more comprehensive vision, a precise understanding of the metabolic activity of microorganisms can be used to drive biotransformation steps towards the improvement of quality and nutritional value of food. High Throughput Sequencing (HTS) technologies are nowadays an emerging and widely adopted tool for microbial characterization of food matrices. Differently from traditional culture-dependent approaches, HTS allows the analysis of genomic regions of the whole biotic panel inhabiting and constituting food ecosystems. Our intent is to provide an up-to-date review of the principal fields of application of HTS in food studies. In particular, we devoted major attention to the analysis of food microbiota and to the applied implications deriving from its characterization in the principal food categories to improve biotransformation processes.

The purpose of this study was to test the ability of DNA barcoding to identify the plant origins of processed honey. Four multifloral honeys produced at different sites in a floristically rich area in the northern Italian Alps were examined by using the rbcL and trnH-psbA plastid regions as barcode markers. An extensive reference database of barcode sequences was generated for the local flora to determine the taxonomic composition of honey. Thirty-nine plant species were identified in the four honey samples, each of which originated from a mix of common plants belonging to Castanea, Quercus, Fagus and several herbaceous taxa. Interestingly, at least one endemic plant was found in all four honey samples, providing a clear signature for the geographic identity of these products. DNA of the toxic plant Atropa belladonna was detected in one sample, illustrating the usefulness of DNA barcoding for evaluating the safety of honey.

Among plants, several groups have toxicities that are dangerous for humans. Plant exposures are indeed one of the most frequent cases reported to poison control centers. A rapid and easy screening test is required for accurate medical treatment. However, it is usually based on the morphological analysis of ingested plant portions that is expensive in terms of time and resources requiring experience in systematic botany which could also be biased by the absence of clear diagnostic traits in stomach contents. In this study, we designed specific primer pairs for the amplification of a sequence-characterized amplified region within standard barcode regions (i.e., rbcL and trnH–psbA) to detect toxic plants belonging to the genera Atropa and Colchicum. Allied edible species were also included in our data-set to exclude the cross-amplification with the toxic ones when the detection system is applied. Using real-time polymerase chain reaction, we tested the specificity of the selected primer pairs in order to develop a fast and reliable tool to be used in poison centers.

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.

The objective of the present work is to evaluate the efficacy of a DNA barcoding approach as a tool for the recognition of commercial kitchen spices belonging to the Lamiaceae family that are usually sold as enhancers of food flavor. A total of 64 spices samples, encompassing six different genera (i.e. Mentha, Ocimum, Origanum, Salvia, Thymus and Rosmarinus) were processed with a classical DNA barcoding approach by amplifying and sequencing four candidate barcode regions (rpoB, rbcL, matK and trnH-psbA) with universal primers. Results suggest that the non-coding trnH-psbA intergenic spacer is the most suitable marker for molecular spices identification followed by matK, with interspecific genetic distance values ranging between about 0% to 7% and 0% to 5%, respectively. Both markers were almost invariably able to distinguish spices species from closest taxa with the exclusion of samples belonging to the genus Oregano. Moreover, in a context of food traceability the two markers are useful to identify commercial processed spice species (sold as dried plant material). We also evaluated the potential benefits of a multilocus barcode approach over a single-marker and although the most suitable combination was the matK. +. trhH-psbA, the observed genetic distances values were very similar to the discriminatory performance of the trnH-psbA. Finally, this preliminary work provide clear evidences that the efficacy of a DNA barcoding approach to the recognition of commercial spices is biased by the occurrence of taxonomic criticisms as well as traces of hybridization events within the family Lamiaceae. For this reason, to better define a more practical and standardized DNA barcoding tool for spices traceability, the building of a dedicated aromatic plants database in which all species and cultivars are described (both morphologically and molecularly) is strongly required. © 2010 Elsevier Ltd.

Pharmaceutically-active compounds are regularly and widely released into the aquatic environment in an unaltered form or as metabolites. So far, little is known about their potential detrimental effects on algae populations which can ultimately impact nutrient cycling and oxygen balance. For our analysis, the common microalga Pseudokirchneriella subcapitata (P. subcapitata) was exposed to a mixture of 13 drugs found in Italian wastewaters and rivers. Traces of pharmaceuticals investigated were detected in treated algal cells, except for cyclophosphamide and ranitidine, indicating that these algae are able to absorb pharmaceutical pollutants from the environment. The effects of the treatment were investigated by Amplified Fragment Length Polymorphism (AFLP) assessment of DNA damage and 2-DE proteomic analysis. While no genotoxic effect was detected, proteomic analysis showed that algae are sensitive to the presence of drugs and that, in particular, the chloroplast is affected. © 2010 Elsevier B.V.

SUMMARY To evaluate the relationship between Sardinian and Spanish viticulture, Simple Sequence Repeat (SSR) markers were applied to define the genetic profiles of 29 cultivated and 48 wild grapevine (Vitis vinifera L.) accessions. SSR data confirmed synonymy between ‘Cannonau’ and several Spanish accessions of ‘Garnacha Tinta’. SSR analysis also suggested that the ‘Garnacha’ group consisted of a heterogeneous pool of cultivars displaying different morphological and genetic traits (Link coefficient = approx. 0.5), probably caused by somatic mutation or accidental breeding events between closely-related grapevine accessions. In contrast, the ‘Vernaccia’ - ‘Granaccia’ Sardinian group was different from ‘Cannonau’ (Link coefficient = 0.8) and all Spanish ‘Garnacha Tinta’ and ‘Blanca’ accessions analysed. To understand the ‘Cannonau’ - ‘Garnacha’ relationship, we studied the origin of these accessions and their relationships with spontaneous wild grapevine. Both cultivars are ancient grapes that have been cultivated for many centuries in both Sardinia and Spain. Although the name ‘Garnacha’ may derive from the Italian word ‘Vernaccia’, molecular analysis excluded any direct genetic origin of the Spanish ‘Garnacha’, or Sardinian ‘Cannonau’ from the ‘Vernaccia’ ‘Granaccia’ Sardinian group. Structure analysis split the samples analysed into three clusters (K = 3). The first two clusters corresponded to the cultivated samples, while the wild accessions were in the third cluster. Based on this information, we can exclude any direct origin of the ‘Cannonau’ - ‘Garnacha’ group from the wild grapevines analysed and distributed on Sardinia.

Archaeobotanical-archaeological, cultural and historical data indicate that grapevine domestication can be dated back from 6000 to 7000 years ago and that it took place in the Caucasian and Middle East Regions. However, events leading to the domestication of this crop species are still an open issue. In this paper, 6 chloroplast microsatellites have been used to assess genetic similarities among, and within, domesticated and wild grapevine accessions representative of 7 distinct geographical regions from the Middle-East to Western Europe. Results show that 2 out of the 6 analyzed chloroplast loci are polymorphic within the 193 domesticated individuals and the 387 samples of 69 wild populations. Allele variants of the Cp-SSR loci combine in a total of 6 different haplotypes. The data show that the haplotype distribution is not homogeneous: the 6 haplotypes are present in the domesticated varieties, but only 5 (haplotype VI is absent) are observed in wild populations. The analysis of haplotype distribution allows discussion of the relationships between the two grape subspecies. The contribution of the wild grape germplasm to the domesticated gene pool still growing in different geographical regions can be, in cases, made evident, suggesting that beside domestication, gene introgression has also played a role in shaping the current varietal landscape of the European viticulture.

Vitis vinifera L. is largely diffused in Sardinia, where it occurs as both a spontaneous wild plant (V. vinifera subsp. silvestris) and a crop (V. vinifera subsp. vinifera). The huge number of grapevine cultivars is the result of the complex history of Sardinia. The island was part of the Spanish Empire from 1479 to 1714 when commercial and cultural exchanges between the two regions caused modifications of its agricultural products. Noticeably, in the case of viticulture, Spanish and Sardinian grapevine varieties often have similar-sounding names; for example, ‘Bobal’ and ‘Bovale’, ‘Cariñena’ and ‘Carignano’, and ‘Garnacha’, ‘Granaccia’ and ‘Granazza’. We may assume that, due to politic and economic relationships, exchanges of grape varieties could have been occurred thus improving the genetic composition of the local germplasm. At the same time, certain Sardinian cultivars, some of which derived from the domestication and cultivation of wild local grapevines, could have been introduced in Spain. The first aim of our work was to evaluate the genetic structure of grapevine germplasm of Sardinia and its relationships with Spanish cultivars. Simple Sequence Repeat (SSR) markers were analyzed to characterize each accession and to study the relationships between Sardinian and Spanish cultivars. Results suggest the occurrence of synonymies such as ‘Cannonau’ and several Spanish accessions of ‘Garnacha Tinta’. If genetic origin of cultivars is an element useful for assessing wine traceability, wine quality relies on different factors and it is largely influenced by the environmental conditions of the production area. The second goal of this study was to develop an multidisciplinary approach for the characterization of 'Cannonau' wine produced in different Sardinian localities. We conducted a high-throughput sequencing analysis of different samples to understand the relationship between wine identity and the microbiome, specifically concerning bacteria and fungi. Since microbiome depends on different conditions, we planned a sampling able give us a lot of information: different grapes of the same cultivar, cultivated with traditional and biodynamic agriculture, in different regions of the island characterized by different type of soil; musts pre-fermentation and post-fermentation in different wineries, with or without starter (S. cerevisiae). Research on wine microbiome focuses primarily on those microorganisms influencing wine quality and determining its organoleptic properties. Such properties are mainly due to secondary metabolites that are produced while grapes ripen and then due to fermentation. For this reason, each sample collected was also analyzed for metabolomic characteristics, by Nuclear Magnetic Resonance. Analyses of microbial distribution and of the interactions occurring within and between bacteria and fungi, can represent the key of identification of Sardinian wines identity. In addition, the integration of metabolomic data can permit the identification of specific characteristics of each Sardinian wine. This type of study belongs to Foodomics, a new comprehensive approach to food and nutrition. The described integrative approach could represent a valuable tool for a better characterization of other foods and beverages, even including their transformation, storage, safety and their impact on human health.

Hybridization between Rhododendron ferrugineum L. and R. hirsutum L. in south-eastern Alps was examined in order to (i) evaluate the breeding direction and the extent of backcrossing between hybrids and the parental species, (ii) define which processes facilitate speciation and maintain species identities and (iii) clarify the role of rock geochemistry in hybridization events. Individuals of three hybrid populations were analysed by morphological and molecular markers. The internal transcribed spacer and trnH–psbA distinguished the parental species and F1 hybrids while only the simple sequence repeat markers recognized genotype classes: F, H, F1, F2, BxF (backcross to R. ferrugineum) and BxH (backcross to R. hirsutum). Combining morphological and molecular data, we found that the tested populations had complex genetic structure: the F1 individuals produce F2 hybrids and backcross to parental species. Due to R. hirsutum phenology, most backcrossing events were with this parental species (asymmetric hybridization). Geochemical analyses indicate that alkaline soil conditions linked to calcareous dolomitic rocks promoted the genetic assimilation of R. hirsutum. In addition, R. x intermedium shows a higher edaphic adaptation than R. hirsutum as it can be found on a wide range of calcareous-dolomitic rocks as well as on weakly acidic soils of natural or anthropogenic origin.

Aim: The objective of the present study is to investigate the molecular characteristics of Sardinian grapevine cultivars to evaluate cases of synonyms and false attributions to protect local agro-biodiversity.Methods and results: The SSR analysis (13 loci) has been used to define the DNA fingerprint and the relationships with Sardinian grapevine cultivars. Results highlighted a high genetic variability among the accessions, with the Dice coefficients performing from 0 to 0.8. Despite the genetic richness, thirteen groups of redundant genotypes were detected. Molecular analysis refers of cultivars harbouring the same SSR profile but different berry colours such as cultivars Licronaxu Bianco and Nero and Moscatello Bianco and Nero. It could by hypothesized that Licronaxu and Moscatello could derive from a specific retrotransposon-induced mutation event in genes regulating anthocyanin biosynthesis.Conclusion: Sardinian germplasm has a real problem of cultivar identification probably due to different factors such as the absence of an exhaustive ampelography, problems in the language to name varieties and the existence of cultivars sensitive to biotic and abiotic stresses producing evident morphological modifications leading to mistakes in recognising and identifying properly the affected plants. However, our molecular results suggest that high grape-biodiversity is still preserved in this region.Significance and impact of study: Results of this work clarified the relationships among grapevine cultivars and provided a solid basis to improve a regional grapevine collection.

Frugivores are critical components of restoration programs because they are seed dispersers. Thus, knowledge about bird-plant trophic relationships is essential in the evaluation of the efficacy of restoration processes. Traditionally, the diet of frugivores is characterized by microscopically identifying plant residues in droppings, which is time-consuming, requires botanical knowledge, and cannot be used for fragments lacking detectable morphological characteristics (e.g., fragmented seeds and skins). We examined whether DNA barcoding can be used as a universal tool to rapidly characterize the diet of a frugivorous bird, Eurasian blackcap (Sylvia atricapilla). We used the DNA barcoding results to assess restoration efforts and monitor the diversity of potentially dispersed plants in a protected area in northern Italy. We collected 642 Eurasian Blackcap droppings at the restored site during the autumn migration over 3 years. Intact seeds and fragmented plant material were analyzed at 2 plastidial barcode loci (rbcL and trnH-psbA), and the resulting plant identifications were validated by comparison with a reference molecular data set of local flora. At least 17 plant species, including 7 of the 11 newly transplanted taxa, were found. Our results demonstrate the potential for DNA barcoding to be used to monitor the effectiveness of restoration plantings and to obtain information about fruit consumption and dispersal of invasive or unexpected plant species. Such an approach provides valuable information that could be used to study local plant biodiversity and to survey its evolution over time.

In this study, Hypnum cupressiforme moss bags were used to examine the atmospheric deposition of trace elements in the oil refinery region of Sardinia (Italy) compared with surrounding natural zones. The concentrations of 13 elements [arsenic (As), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn)] were determined using inductively coupled plasma optical emission spectrometry. A significant accumulation of pollutants was detected using active biomonitoring with moss bags compared with a control site. The most relevant contaminants for all of the tested sites were Cr, Cu, Ni, and Zn. Moreover, the accumulation of Cr and Zn in the refinery industrial areas, IA1 and IA2, was more than five times greater than that detected at the control site. Levels of Cd, Mg, and Pb were also higher at all of the monitored sites compared with the control site. Both genomic and proteomic methods were used to study the response of H. cupressiforme to air pollution. No DNA damage or mutations were detected using the amplified fragment length polymorphisms (AFLP) method. At the protein level, 15 gel spots exhibited differential expression profiles between the moss samples collected at the IA1 site and the control site. Furthermore, among the 14 spots that showed a decrease in protein expression, nine were associated with ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and proteins of the light-harvesting complexes of photosystem (PS) II, three were associated with protein synthesis, and three were stress-related proteins. Thus, some of these proteins may represent good moss biosensors which could be used as pre-alert markers of environmental pollution.

In this study, the genetic constitution of wild grape populations distributed in the Zagros Mountains in the north-west part of Iran was investigated to assess their risk of extinction and to identify the most suitable conservation strategy. A total of 65 wild grapevine samples from five different populations were analysed using 23 SSR loci. Consistent allelic richness was detected in all the analysed populations (from 4.01 to 5.35). The observed heterozygosity and expected heterozygosity were high with mean values of 0.67 and 0.69, respectively. A modest heterozygosity deficiency was detected only in the Piranshahr and Sardasht-Shalmash populations. The neighbour-joining dendrogram clustered the accessions into three groups corresponding to the geographical distribution of the populations. Molecular data combined with field evaluation revealed that anex situconservation approach is the most suitable strategy for preserving the genetic richness of Iranian wild grape populations with consistent rare alleles (about 20%). We proposein situpreservation only in the case of the Sardasht-Ghasmarash population to ensure that evolutionary dynamic forces continue to influence plant adaptation and survival in response to environmental alterations.

Measuring levels of population genetic diversity is an important step for assessing theconservation status of rare or endangered plant species and implementing appropriateconservation strategies. Populations of Ribes multiflorum subsp. sandalioticum andR. sardoum, two endangered endemic species from Sardinia, representing the wholegenus on the island, were investigated using ISSR and SSR markers to determine levelsand structure of genetic variability in their natural populations. Results indicatedmedium to low genetic diversity at the population level: Nei’s gene diversity for ISSRmarkers ranged from 0.0840 to 0.1316; the expected heterozygosity (H E ) for SSR ran-ged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoumshowed a high level of inbreeding, in accordance with its very small size. Regardingthe structure of the six R. sandalioticum populations, both principal coordinatesanalysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low pop-ulation structure, although two populations appeared to be clearly distinct from theothers. The genetic pattern of the two taxa associated with their different ecologicalpositions indicated resilience of R. sandalioticum populations in fresh and humidhabitats and uncertain future resistance for the residual R. sardoum population inxeric calcareous stands. Hence, this study highlights the importance of an integratedconservation approach (genetic plus in situ and ex situ conservation studies/measures)for activating management programmes in these endemic and threatened taxa thatcan be considered as crop wild relatives of cultivated Ribes species.INTRODUCTIONEndangered species with small and isolated populations havebeen the subject of numerous conservation studies in the lastdecade. Their aims were to avoid biodiversity loss and under-stand survival/extinction mechanisms in an evolutionary con-text (Beissinger 2000; Freville et al. 2007; Abeli et al. 2009).The extinction process can be related to ecological factors suchas competition, predation, parasitism, disease and environ-mental stresses (Jones 2008). In recent years, habitat alterationdue to shifts in climatic characters, human activities and sto-chastic events are among the main current causes of biodiver-sity loss (Chapin et al. 2000). As a consequence, threatenedspecies may have low numbers of populations or small popula-tion size (e.g. Ellstrand & Elam 1993). In particular, island en-demics are thought to be even more susceptible to extinction(Frankham 1997, 1998) due to an increased probability of sto-chastic events on populations and phenomena of genetic driftand inbreeding depression, resulting in reduced genetic diver-sity (Izawa et al. 2007; Bruni et al. 2013). Given these threats,conservation of endangered species in their biogeographic con-text requires a thorough knowledge of the genetic structure oftheir populations (Rao & Hodgkin 2002). In this framework,maintaining these species’ genetic diversity is of pivotal impor-tance as it is associated with population viability and the evolu-tionary potential to adapt to environmental changes (Reedet al. 2002; Reed & Frankham 2003).Ribes L. (Grossulariaceae) is a genus of ca. 200 taxa distrib-uted in temperate regions of the Northern Hemisphere and inthe Andes (Mabberley 2008). Ribes sardoum Martelli andR. multiflorum Kit ex Roem et Schult. subsp. sandalioticumArrigoni (hereafter, R. sandalioticum) are the only two membersof this genus present in Sardinia, and both are endemic to theisland. Sardinia is the only Mediterranean island where thisgenus is represented exclusively by endemic taxa, while on thenearest island systems the genus is absent (i.e. Tuscan archipel-ago) or represented only by widely distributed species (see Fenuet al. 2012 and references therein). R. sardoum is a threatenedspecies with only one small population found in the Supramon-tes region (CE Sardinia; Bacchetta et al. 2008) and categorisedas critically endangered (CR) on the IUCN Red List. R. sandali-oticum has 13 populations established in three biogeographicsubsectors, mainly distributed in CE and NE Sardinia, and iscategorised as endangered (EN; Fenu et al. 2012).

Pharmaceutically active compounds (PACs) are continuously dispersed into the environment due to human and veterinary use, giving rise to their potential accumulation in edible plants. In this study, Eruca sativa L. and Zea mays L. were selected to determine the potential uptake and accumulation of eight different PACs (Salbutamol, Atenolol, Lincomycin, Cyclophosphamide, Carbamazepine, Bezafibrate, Ofloxacin and Ranitidine) designed for human use. To mimic environmental conditions, the plants were grown in pots and irrigated with water spiked with a mixture of PACs at concentrations found in Italian wastewaters and rivers. Moreover, 10× and 100× concentrations of these pharmaceuticals were also tested. The presence of the pharmaceuticals was tested in the edible parts of the plants, namely leaves for E. sativa and grains for Z. mays. Quantification was performed by liquid chromatography mass spectroscopy (LC/MS/MS). In the grains of 100× treated Z. mays, only atenolol, lincomycin and carbamazepine were above the limit of detection (LOD). At the same concentration in E. sativa plants the uptake of all PACs was >LOD. Lincomycin and oflaxacin were above the limit of quantitation in all conditions tested in E. sativa. The results suggest that uptake of some pharmaceuticals from the soil may indeed be a potential transport route to plants and that these environmental pollutants can reach different edible parts of the selected crops. Measurements of the concentrations of these pharmaceuticals in plant materials were used to model potential adult human exposure to these compounds. The results indicate that under the current experimental conditions, crops exposed to the selected pharmaceutical mixture would not have any negative effects on human health. Moreover, no significant differences in the growth of E. sativa or Z. mays plants irrigated with PAC-spiked vs. non-spiked water were observed.

This outlook paper addresses the problem of the traceability of minor crops. These kinds of cultivations consist in a large number of plants locally distributed with a modest production in terms of cultivated acreage and quantity of final product. Because of globalization, the diffusion of minor crops is increasing due to their benefit for human health or their use as food supplements. Such a phenomenon implies a major risk for species substitution or uncontrolled admixture of manufactured plant products with severe consequences for the health of consumers. The need for a reliable identification system is therefore essential to evaluate the quality and provenance of minor agricultural products. DNA-based techniques can help in achieving this mission. In particular, the DNA barcoding approach has gained a role of primary importance thanks to its universality and versatility. Here, we present the advantages in the use of DNA barcoding for the characterization and traceability of minor crops based on our previous or ongoing studies at the ZooPlantLab (Milan, Italy). We also discuss how DNA barcoding may potentially be transferred from the laboratory to the food supply chain, from field to table.

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands. Copyright

La presente invenzione si riferisce a un prodotto detergente multiuso a base di acido citrico, cellulosa e oli essenziali. L’acido citrico fornisce l’acidità sufficiente per sciogliere il calcare (anticalcare) e per acidificare i panni dopo il lavaggio con detersivi alcalini rendendo le fibre elastiche (ammorbidente). L’acidità del prodotto e la presenza di oli essenziali rendono la miscela antimicrobica e sulla base dei test eseguiti igienizzante. In particolare, l’invenzione concerne una composizione solida, ricostituibile in soluzione acquosa, comprendente: - acido citrico, - cellulosa, - oli essenziali o estratti vegetali

This study investigated the conservation status of Marsilea quadrifolia, an endangered fern found in paddy fields, irrigation ditches and ponds. An evaluation at the European level based on IUCN criteria showed that the extent of occurrence (EOO) of M. quadrifolia has decreased from 5,930,000 km2 to 5,774,000 km2 during the past decade, whereas its area of occupancy (AOO) has decreased from 620 to 400 km2 (approximately 35.5%). These findings allowed the species to be upgraded from the IUCN classification of Near Threatened to Vulnerable. The agricultural chemical treatments seem to be the main extinction cause of M. quadrifolia, therefore we performed toxicological with 7 most common rice herbicides. Young plantlets were incubated for 96 h with each herbicide at three different concentrations: TQ (Tale Quale, chosen in accordance with the suggested dose for rice fields described on the product label), 1:100 and 1:1.000. Results suggested that survival of the plantlets depended on the herbicide and concentration used, and ranged between 0 and 80%, and no survival at ambient concentrations for 4 (Aura, Aura + Dash, Clincher and Viper) out of the 8 chosen herbicides. We conclude that herbicides represent one of the principal threats to the survival of this species. Finally, a DNA analysis using the AFLP approach was employed to identify the most suitable genetic pool for plant reintroduction efforts. The data show that the analysed populations of M. quadrifolia suffered from low genetic variability (Nei's gene diversity varied from 0.025 to 0.036). However, the analysis of the distribution of genetic variability suggested that 4 populations were characterised by different genetic traits that are useful in defining a genetic pool for plant conservation. This study highlights a strategy for implementing a plan of action for species growing in agro-ecosystems based on an integrated approach that is able to clarify the species conservation status, the principal threat factor and the genetic pool to be used for species conservation and reintroduction.

We evaluated the utility of the core barcode regions (matK and rbcL) and the plastid intergenic spacer trnH-psbA to distinguish between Thymus spp. This is a taxonomically complex group that has been investigated so far mainly using morphological approaches. Thirty-six samples representing nine different morphospecies were collected and used for molecular analysis. The three markers showed clear amplification and sequencing. However, the genetic variation and the resulting haplotype networks showed that only Thymus capitatus forms a well-defined ‘barcoding gap’ compared with the other taxa. The identification problems observed in the other Thymus spp. may be related to reduced gene flow among populations, resulting in high intraspecific and low interspecific genetic variation. This situation does not permit the definition of species-specific barcodes. A second hypothesis suggests that morphological traits used for the delimitation of Thymus spp. do not reflect real biological and molecular species boundaries. If this is the case, the taxonomy of Thymus should be revised through extensive sampling and analyses with different tools (i.e. molecular variability, morphology, geographical distribution, etc.) to define the natural units at the species level

Food safety and quality are nowadays a major concern. Any case of food alteration, especially when reported by the media, has a great impact on public opinion. There is an increasing demand for the improvement of quality controls, hence addressing scientific research towards the development of reliable molecular tools for food analysis. DNA barcoding is a widely used molecular-based system, which can identify biological specimens, and is used for the identification of both raw materials and processed food. In this review the results of several researches are critically analyzed, in order to exploit the effectiveness of DNA barcoding in food traceability, and to delineate some best practices in the application of DNA barcoding throughout the industrial pipeline. The use of DNA barcoding for food safety and in the identification of commercial fraud is also discussed. (c) 2012 Elsevier Ltd. All rights reserved.

Identification of living beings is a core problem in biology. Identification can be achieved by several methods, but in recent decades, molecular techniques have become more and more common. DNA barcoding is an initiative that was launched less than 10 years ago, but which has already gained a reputation in biological studies. The method was originally applied to metazoans, but it was rapidly used to discriminate plants. However, in plants there were several problems, including the choice of the right markers, their universality and the discrimination power. On the whole, DNA barcoding in plants is slightly behind when compared to animals, but it is likely that the gap will be filled in a short period of time. In a DNA barcoding approach, molecular and bioinformatics are deeply linked to generate the identification system. In this paper, we summarize the recent advances in DNA barcoding bioinformatics, by reviewing the principal approaches and critically analyzing the procedures.

Vegetation surveys have a long tradition in ecological studies, but several limitations in the morphological identification of species have been recognized. The objective of this study was to evaluate the effectiveness of DNA barcoding in plant species identification to save field technicians time and resources. Vegetation surveys were performed in four plots of semi-dry grassland in the Italian subalpine region of Lombardy. Two identification approaches were employed: a conventional morphological identification and a molecular multi-marker DNA barcoding method. Results showed that morphological identification of 49 species collected from the study area (five field inspections) required a substantial amount of time to complete relative to the molecular method. The same 49 samples were analysed using the following DNA multi-marker barcodes: rbcL, matK and trnH-psbA. rbcL showed 100% amplification success with standard primers, but low interspecific genetic variability. matK demonstrated some amplification problems with standard primers; however, consistent genetic diversity was observed. Finally, the trnH-psbA spacer region exhibited reliable amplification success and the highest molecular variability. In a comparison with publicly available databases, trnH-psbA and matK returned the highest proportion of identified samples, whereas rbcL returned several misidentifications. The DNA barcoding approach is a powerful tool in vegetation surveys and may significantly reduce the time and cost spent for species identification. However, to effectively apply DNA barcoding in vegetation surveys, exhaustive local or regional molecular databases must be defined. (c) 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 518529.

Human activities have increased the levels of environmental palladium (Pd) worldwide. Due to the growing evidence of its toxicity, Pd pollution has become the focus of serious concern. Several studies have given an account of the increasing concentration of Pd in aquatic ecosystems. The aim of the current study is to analyze the physiological and molecular effects induced by Pd on freshwater unicellular green algae. To do this, Pseudokirchneriella subcapitata (P. subcapitata) was exposed in vitro to different concentrations (0.1, 0.25 and 0.5 mg l(-1)) of K(2)PdCl(4), a soluble salt of Pd, corresponding to 0.03, 0.075 and 0.15 mg l(-1) of Pd. The uptake and the effects on algal growth and morphology were determined. The main results are that Pd is able to induce damage in P. subcapitata at a concentration of 0.1 mg l(-1) of K(2)PdCl(4), with the damage becoming more evident at a concentration of 0.25 mg l(-1)of K(2)PdCl(4); at a concentration of 0.5 mg l(-1) of K(2)PdCl(4), cellular degeneration occurs. The main cellular target of Pd is the chloroplast, as shown by TEM and proteomic analysis. TEM analysis also showed accumulation of precipitates, probably of Pd, in the chloroplasts, although further experiments are necessary to confirm that these are Pd-precipitates. Amplified fragment length polymorphism analysis (AFLP) demonstrated that Pd, even at the lowest concentration tested, induced randomly distributed DNA changes either directly or indirectly in the algal genome and that oxidative processes were involved.

The plant exposures are one of the most frequent poisonings reported to poison control centres. The diagnosis of intoxicated patients is usually based on the morphological analysis of ingested plant portions; this procedure requires experience in systematic botany, because the plant identification is based on few evident traits. The objective of this research is to test DNA barcoding approach as a new universal tool to identify toxic plants univocally and rapidly. Five DNA barcode regions were evaluated: three cpDNA sequences (trnH-psbA, rpoB and matK) and two nuclear regions (At103 and sqd1). The performance of these markers was evaluated in three plant groups: (1) a large collection of angiosperms containing different toxic substances, (2) congeneric species showing different degrees of toxicity and (3) congeneric edible and poisonous plants. Based on assessments of PCR, sequence quality and resolution power in species discrimination, we recommend the combination of plastidial and nuclear markers to identify toxic plants. Concerning plastidial markers, matK and trnH-psbA showed consistent genetic variability. However, in agreement with CBOL Plant Working Group, we selected matK as the best marker, because trnH-psbA showed some problems in sequences sizes and alignments. As a final and relevant observation, we also propose the combination of matK with a nuclear marker such as At103 to distinguish toxic hybrids form parental species. In conclusion, our data support the claim that DNA barcoding is a powerful tool for poisonous plant identifications.

Vitis vinifera ssp. silvestris, the spontaneous subspecies of V. vinifera L., is believed to be the ancestor of present grapevine cultivars. In this work, polymorphism at 13 SSR loci was investigated to answer the following key question: are wild plants (i) true silvestris, (ii) hybrids between wild and cultivated plants or (iii) or 'escapes' from vineyards? In particular, the objective of the present study was to identify truly wild individuals and to search for possible hybridization events. The study was performed in Sardinia, the second largest island in the Mediterranean Sea, which is characterized by a large and well-described number of both grape cultivars and wild populations. This region was ideal for the study because of its spatial isolation and, consequently, limited contamination from outside material. The results of this study show that domesticated and wild grapevine germplasms are genetically divergent and thus are real silvestris. Pure lineages (both domesticated and wild) show very high average posterior probabilities of assignment to their own clusters, with a low level of introgression. © 2010 German Botanical Society and The Royal Botanical Society of the Netherlands.

DNA barcoding is a recent and widely used molecular-based identification system that aims to identify biological specimens, and to assign them to a given species. However, DNA barcoding is even more than this, and besides many practical uses, it can be considered the core of an integrated taxonomic system, where bioinformatics plays a key role. DNA barcoding data could be interpreted in different ways depending on the examined taxa but the technique relies on standardized approaches, methods and analyses. The existing reference towards a common way to treat DNA barcoding data, analyses and results is the Barcode of Life Data Systems. However, the scientific community has produced in the recent years a number of alternative methods to manage barcoding data. The present work starts from this point, because users should be aware of the consequences their choices produce on the results. Despite the fact that a strict standardization is the essence of DNA barcoding, we propose a tour of six questions to improve the users' awareness about the method, the correct use of concepts and alternative tools provided by scientific community. © The Author 2010. Published by Oxford University Press.