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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.

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.

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.

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.

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.

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.

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.

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

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.

The effect of chromium on Pseudokirchneriella subcapitata has been assessed using different approaches: growth rate, metabolic activity microscopy analyses, and amplified fragment length polymorphism (AFLP) assessment of DNA damage. Starting from 24 h of treatment all the tested concentrations resulted in consistent algal growth inhibition. The average daily growth rate after 72 h calculated for the control (0.53 0.01 was statistically higher than those estimated for cell treated with 1, 2.5, 5 and 7.5 mu g g(-1) potassium dichromate (0.46 0.02; 0.32 0.01; 0.25 0.01 and -0.02 0.04, respectively). A reduction of viable cell numbers, estimated with FDA approach, was also observed after 24 h treatment for all the tested chromium concentrations apart from the lowest (1 mu g g(-1) potassium dichromate). A recovery of esterase activity was detected after 48 and 72 h for all treatments with the except of the 7.5 mu g g(-1) potassium dichromate treated samples showing a very modest recovery. This data suggests that potassium dichromate is, even from the lowest tested concentration, highly toxic to P subcapitata, and that this algal strain is a sensitive organism suitable for monitoring chromium in water. Our data also suggest that although algal counting by microscope and the FDA test gave similar indications concerning chromium effect, the FDA test was not completely reliable. In fact, we observed a decrease in the FDA stained cell numbers in control samples after 48 and 72 h of treatment, when most cells were actively proliferating. This lack of fluorescence could be explained by an uncontrolled fluorescein efflux due to the interaction of many experimental factors. The genotoxic effects of the different chromium concentrations were investigated by analyses of the DNA from control and treated algal samples, 72 h after inoculation. AFLP analysis revealed a total of 258 bands, 109 of which were polymorphics. Analysis of the AFLP matrix suggests that potassium dichromate is a powerful genotoxic agent, inducing genetic mutations also at the lowest tested concentration (0.35 mu g g(-1)). Furthermore, we observed a correlation between the polymorphic bands with increasing chromium concentration. Finally, the absence of preferential mutation sites suggests that the chromium induced DNA changes are randomly distributed in the genome. (c) 2006 Elsevier B.V. All rights reserved.