F. De Mattia

A DNA barcoding approach to identify plant species in multiflower honey

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.

Analytical approaches for DNA barcoding data – how to find a way for plants?

Abstract 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.

The use of AFLP and morphological markers to study Iranian grapevine germplasm to avoid genetic erosion

Summary Molecular and morphological analyses have been used to define and clarify the relationships among the 54 most representative grapevine cultivars in Iran. A total of 459 scorable AFLP fragments, across all the accessions analysed, were detected. Among these, 185 (40.3%) were polymorphic. Values of genetic diversity, calculated by the Nei and Li index, ranged from 0 – 0.3. Our results highlight cases of different cultivars displaying the same AFLP profile and similar morphological characteristics, proving the occurrence of synonyms in Iranian germplasm. Combining AFLP results and morphological descriptions, two cases of wrong attribution were also observed: ‘Saghal Solian-2’ was renamed ‘Mosli’; and ‘Khalili Qermez’ was now called ‘Khalili Sefid’. Morphological characterisation, conducted using the IPGRI descriptors, showed large variability among the accessions analysed, mainly in young shoot and leaf colour, and in bunch size. Although synonyms were detected, and genomic similarities were verified, genetic differences and the richness observed in Iranian grapevine germplasm were not high, in contrast with their morphological characteristics. We hypothesise that many Iranian morphotypes have been derived from mutation events in a few ancient genotypes; however, the system of selection may result in the biodiversity of Iranian grapevine being endangered. For this reason, our molecular and morphological characterisation will help in the development of conservation plans and in the re-organisation of local germplasm collections.

Evaluating the efficacy of restoration plantings through DNA barcoding of frugivorous bird diets.

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.

A rapid diagnostic approach to identify poisonous plants using DNA barcoding data

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.

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Neis gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (HE ) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.

Genetic relationships between Sardinian and Spanish viticulture: the case of 'Cannonau' and 'Garnacha'

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.

Analysis of SSR and AFLP Markers to Detect Genetic Diversity Among Selected Clones of Grapevine (Vitis vinifera L.) cv. Keshmeshi

To assess the genetic differences between clones of grapevine (Vitis vinifera L.) cv. Keshmeshi, ten selected clones from a clonal selection programme were analysed by 23 Simple Sequence Repeats (SSR) and seven AFLP primer combinations. No intra-varietal differences between the clones could be detected by SSRs, whereas eight out of the 499 AFLP fragments generated by the seven primer combinations were polymorphic. The number of markers ranged from 44 (E34-M34) to 97 (E31-M32), with an average of 71.3 fragments per primer combination. Cluster analysis based on the AFLP data separated all the clones of Keshmeshi in two groups. The first group included nine white berry skin clones without any genetic differences, and the second group with only a red berry skin clone. AFLP could only distinguish the red berry clone of Keshmeshi from other white berry clones.

Genetic introgression of hybrid Rhododendron x intermedium Tausch is habitat mediated: Evidences from south-eastern Alps (Italy)

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.