Carmine Guarino

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross‐tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Genetic diversity in a collection of ancient cultivars of apple (Malus × domestica Borkh.) as revealed by SSR-based fingerprinting

Summary A collection of 48 accessions of Malus domestica Borkh. from the apple germplasm repository of the Campania Region in Italy, several of which were suspected to be “synonyms”, was screened together with eight ancient well-known cultivars, added for reference, using a set of nine microsatellite or Simple Sequence Repeat (SSR) primer pairs to determine genetic identities, to estimate genetic diversity and to identify genetic relationships between the accessions. All microsatellite (SSR) primers revealed polymorphism, with seven to 14 alleles per main locus and the expected heterozygosity (He) ranging from 0.713–0.884. Three SSRs (CH01h02, CH02c11 and CH04c06) amplified a second locus that was less polymorphic and clearly distinguished from the main locus. The frequency of each allele at the main loci was generally low, with many alleles detected in only one or a few cultivars. Of the 56 genotypes studied, as many as 27 were identified as “synonyms” and were excluded from the genetic analysis. Synonymy not only included accessions classified with the same name, but also accessions with different names. In a few cases, accessions with the same name resulted in absolutely different SSR marker profiles. The remaining 29 unique genotypes showed great genetic diversity, with the eight reference cultivars (‘Belle de Boskoop’, ‘Calville blanc’, ‘Delicious’, ‘Golden delicious’, ‘Limoncella’, ‘McIntosh’, ‘Permain dorée’, and ‘Stark splendor’) distributed throughout the dendrogram. Finally, this paper discusses the problems in dealing with incorrect allele-sizing and “binning”, which still do not allow comparison of SSR-based profiles produced by different laboratories.

Assessment of three approaches of bioremediation (Natural Attenuation, Landfarming and Bioagumentation - Assistited Landfarming) for a petroleum hydrocarbons contaminated soil.

Contamination with total petroleum hydrocarbons (TPH) subsequent to refining activities, is currently one of the major environmental problems. Among the biological remediation approaches, landfarming and in situ bioremediation strategies are of great interest. Purpose of this study was to verify the feasibility of a remediation process wholly based on biological degradation applied to contaminated soils from a decommissioned refinery. This study evaluated through a pot experiment three bioremediation strategies: a) Natural Attenuation (NA), b) Landfarming (L), c) Bioaugmentation-assisted Landfarming (LB) for the treatment of a contaminated soil with petroleum hydrocarbons (TPHs). After a 90-days trial, Bioagumentation - assistited Landfarming approach produced the best results and the greatest evident effect was shown with the most polluted samples reaching a reduction of about 86% of total petroleum hydrocarbons (TPH), followed by Landfarming (70%), and Natural Attenuation (57%). The results of this study demonstrated that the combined use of bioremediation strategies was the most advantageous option for the treatment of contaminated soil with petroleum hydrocarbons, as compared to natural attenuation, bioaugmentation or landfarming applied alone. Besides, our results indicate that incubation with an autochthonous bacterial consortium may be a promising method for bioremediation of TPH-contaminated soils.

Differentiation in DNA fingerprinting among species of the genus Acer L. in Campania (Italy)

Abstract Natural populations of species in the Acer genus occurring in Campania (southern Italy) were surveyed by screening seven microsatellite loci. Primer pairs for Acer pseudoplatanus L. microsatellite loci were analysed in six different species: Acer lobelii Ten., Acer campestre L., Acer pseudoplatanus L., Acer obtusatum W. et K., Acer neapolitanum Ten. and Acer monspessulanum L. The aim of the present study was to survey the genetic variability and genetic structure of natural populations of the Acer genus in Campania. The high degree of polymorphism observed in six different species of Acer makes these markers useful for investigating genetic variation at various spatial scales, and for the analysis of gene flow and of the mating system.

Proteomic analysis of eucalyptus leaves unveils putative mechanisms involved in the plant response to a real condition of soil contamination by multiple heavy metals in the presence or absence of mycorrhizal/rhizobacterial additives.

Here we report on the growth, accumulation performances of, and leaf proteomic changes in Eucalyptus camaldulensis plants harvested for different periods of time in an industrial, heavy metals (HMs)-contaminated site in the presence or absence of soil microorganism (AMs/PGPRs) additives. Data were compared to those of control counterparts grown in a neighboring nonpolluted district. Plants harvested in the contaminated areas grew well and accumulated HMs in their leaves. The addition of AMs/PGPRs to the polluted soil determined plant growth and metal accumulation performances that surpassed those observed in the control. Comparative proteomics suggested molecular mechanisms underlying plant adaptation to the HMs challenge. Similarly to what was observed in laboratory-scale investigations on other metal hyperaccumulators but not on HMs-sensitive plants, eucalyptus grown in the contaminated areas showed an over-representation of enzymes involved in photosynthesis and the Calvin cycle. AMs/PGPRs addition to the soil increased the activation of these energetic pathways, suggesting the existence of signaling mechanisms that address the energy/reductive power requirement associated with augmented growth performances. HMs-exposed plants presented an over-representation of antioxidant enzymes, chaperones, and proteins involved in glutathione metabolism. While some antioxidant enzymes/chaperones returned to almost normal expression values in the presence of AMs/PGPRs or in plants exposed to HMs for prolonged periods, proteins guaranteeing elevated glutathione levels were constantly over-represented. These data suggest that glutathione (and related phytochelatins) could act as key molecules for ensuring the effective formation of HMs-chelating complexes that are possibly responsible for the observed plant tolerance to metal stresses. Overall, these results suggest potential genetic traits for further selection of phytoremediating plants based on dedicated cloning or breeding programs.

Molecular characterisation of ancient Prunus avium L. germplasm using sweet cherry SSR markers.

Summary A collection of 60 accessions of Prunus avium L. from the sweet cherry germplasm repository of the Campania Region in Italy, together with seven reference accessions of P. avium, were screened to estimate genetic diversity and to identify genetic relationships among accessions using a set of 28 microsatellite (SSR) markers. Of the 28 primer pairs used, 27 generated polymorphic amplification products and one was found to be monomorphic. Between twoand 14 alleles were revealed for the polymorphic loci, and the expected heterozygosity (He) ranged from 0.031 to 0.822. The total probability of identity was 5.59 10–16. The population structure was investigated using a model-based inference framework. Among the 67 accessions studied, the presence of seven groups was inferred using genetic data, without using prior information on the origin of the accessions. The probabilities of assignment of individual accessions to cultivated gene pools with proportions of membership >0.90 indicated that two gene pools accounted for the genomic origin of 46% of all the P. avium accessions sampled. A dendrogram was generated using the unweighted pair group method with arithmetic averages (UPGMA) based on a ‘Nei72’ geneticdistance analysis. This dendrogram classified all 60 ancient sweet cherry genotypes into one well-defined group that revealed the autochthonous character of most of the varieties cultivated in the Campania Region of Italy.The information obtained from SSR fingerprinting will be useful for optimising the conservation of cherry genetic resources present in the Campania Region. Moreover, this approach can be applied to optimise the conservation of local genetic resources of other fruit tree species.

The effectiveness and efficiency of phytoremediation of a multicontaminated industrial site: Porto Marghera (Venice Lagoon, Italy)

The Venice Lagoon is worldwide considered as a typical example of the human impact on the surrounding ecosystem. The development of the industrial zone of Porto Marghera begun in 1917 as an extension of the Venice Port, in order to sustain activities related to oil and coal, as well as to exploit the railway system. Despite the recent decrease in the number of employees, Porto Marghera is still one of the most important chemical districts in Italy. This study reports early results from the ongoing in-situ phytoextraction of potentially toxic elements (Cd, Hg, Zn) within the industrial area of Porto Marghera. Two agronomic plant species with high annual biomass yield (Helianthus annuus L., Brassica juncea (L.) Czern.) were used. This paper also reports the microcosms and mesocosms tests to evaluate the efficacy of the treatments to be applied to the in-situ phytoextraction process of the polluted site. The combined use of EDTA and Ammonium Thiosulfate during phytoextraction increases the efficiency of Cd, Hg, Zn removal from contaminated soil.

The Proteomic Changes in Cynara Cardunculus L. var. altilis DC Following the Etiolation Phenomena Using De Novo Sequence Analysis

Etiolation of vegetables is a complex phenomenon implying qualitative and quantitative protein changes. The 2-DE protein profile of green and etiolated fleshy stalk samples has shown great differences in the 4–7 pH and 10 to 250 kDa mass range. Currently, a shortage of Cynara DNA or protein sequences has required the use of de novo sequencing and BLAST similarity searches for protein identification. Highlighted herein is an application of proteomics to the identification of proteins, which in a great part matched those from the Viridiplantae order. Quantitative evaluation, statistical analyses, and MALDI-TOF MS characterization of the resolved spots in green and etiolated samples of C. cardunculus enabled us to identify 46 out of 60 spots, containing 21 spots included conserved Viridiplantae domains. The etiolation process is discussed in light of the identification of some proteins involved in specific biochemical and metabolic pathways.

Identification of native-metal tolerant plant species in situ: Environmental implications and functional traits

A study was undertaken to identify suitable native plants for the phytoremediation of the second largest integrated steelworks in Italy (Bagnoli brownfield site). A phytoecological survey allowed us to identify 139 plant taxa belonging to 58 different families. The most represented families were in the decreasing order Poaceae > Fabaceae > Asteraceae > Apiaceae. The biological spectrum showed a predominance of Therophytes > Hemycriptophytes > Phanerophyte. Seventy-six sites were selected on the presence of colonists plants and vegetation assemblage patterns. At each site, roots and leaves of the dominant plant and rhizosphere soils were sampled. Total content of metal(loid)s in soils and plant parts were determined. Agronomic soil parameters were studied. Anthropogenic sourced metal(loid)s were discriminated from geochemical ones, and plant metal(loid) accumulation and translocation efficiency were evaluated. The role of many native plant species in terms of TEs phytomanagement strategy was recognizable inside the investigated area. According to this survey of structural plant diversity, several combinations between plants and microorganisms are being further investigated to identify relevant biological system for the phytomanagement of this contaminated area.

Genetic characterization, micropropagation, and potential use for arsenic phytoremediation of Dittrichia viscosa (L.) Greuter

In the last decade, many scientists have focused their attention on the search for new plant species that can offer improved capacities to reclaim polluted soils and waters via phytoremediation. In this study, seed batches from three natural populations of Dittrichia viscosa, harvested in rural, urban, and industrial areas of central and southern Italy, were used to: (i) evaluate the genetic and morphological diversity of the populations; (ii) develop an efficient protocol for in-vitro propagation from seedling microcuttings; (iii) achieve optimal acclimatization of micropropagated plants to greenhouse conditions; (iv) test the response to arsenic (As) soil contamination of micropropagated plants. The genetic biodiversity study, based on Random Amplification of Polymorphic DNA (RAPD), as well as the morphometric analysis of 20 seedlings from each population revealed some degree of differentiation among populations. Based on these data, the most biodiverse plants from the three populations (10 lines each) were clonally multiplied by micropropagation using microcuttings of in-vitro grown seedlings. Three culture media were tested and Mureshige and Skoog medium was chosen for both seedling growth and micropropagation. The micropropagated plants responded well to greenhouse conditions and over 95% survived the acclimatization phase. Four clones were tested for their capacity to grow on soil spiked with NaAsO2 and to absorb and accumulate the metalloid. All clones tolerated up to 1.0mg As. At the end of the trial (five weeks), As was detectable only in leaves of As-treated plants and concentration varied significantly among clones. The amount of As present in plants (leaves) corresponded to ca. 0.10-1.7% of the amount supplied. However, As was no longer detectable in soil suggesting that the metalloid was taken up, translocated and probably phytovolatilized.