Olga De Castro

Pcr amplification of Michele Tenore's historical specimens and facility to utilize an alternative approach to resolve taxonomic problems

A polymorphic non-coding region of chloroplast DNA (cpDNA) was amplified by the polymerase chain reaction (PCR) from herbarium specimens of Pinus brutia and P. halepensis. The samples belong to the historical collection of Michele Tenore. Several discordances and inconsistencies in taxonomic attribution are present for historical specimens of P. brutia. Moreover, there are inaccuracies as to their geographical distribution. In this work, we demonstrate the potential use of molecular methods of amplifying DNA from historical samples to resolve the identification of herbarium specimens.

SSR-Patchwork: An Optimized Protocol to Obtain a Rapid and Inexpensive SSR Library Using First-Generation Sequencing Technology

• Premise of the study: We have optimized a version of a microsatellite loci isolation protocol for first-generation sequencing (FGS) technologies. The protocol is optimized to reduce the cost and number of steps, and it combines some procedures from previous simple sequence repeat (SSR) protocols with several key improvements that significantly affect the final yield of the SSR library. This protocol may be accessible for laboratories with a moderate budget or for which next-generation sequencing (NGS) is not readily available. • Methods and Results: We drew from classic protocols for library enrichment by digestion, ligation, amplification, hybridization, cloning, and sequencing. Three different systems were chosen: two with very different genome sizes (Galdieria sulphuraria, 10 Mbp; Pancratium maritimum, 30 000 Mbp), and a third with an undetermined genome size (Kochia saxicola). Moreover, we also report the optimization of the sequencing reagents. A good frequency of the obtained microsatellite loci was achieved. • Conclusions: The method presented here is very detailed; comparative tests with other SSR protocols are also reported. This optimized protocol is a promising tool for low-cost genetic studies and the rapid, simple construction of homemade SSR libraries for small and large genomes.Premise of the study: We have optimized a version of a microsatellite loci isolation protocol for first-generation sequencing (FGS) technologies. The protocol is optimized to reduce the cost and number of steps, and it combines some procedures from previous simple sequence repeat (SSR) protocols with several key improvements that significantly affect the final yield of the SSR library. This protocol may be accessible for laboratories with a moderate budget or for which next-generation sequencing (NGS) is not readily available. Methods and Results: We drew from classic protocols for library enrichment by digestion, ligation, amplification, hybridization, cloning, and sequencing. Three different systems were chosen: two with very different genome sizes (Galdieria sulphuraria, 10 Mbp; Pancratium maritimum, 30000 Mbp), and a third with an undetermined genome size (Kochia saxicola). Moreover, we also report the optimization of the sequencing reagents. A good frequency of the obtained microsatellite loci was achieved. Conclusions: The method presented here is very detailed; comparative tests with other SSR protocols are also reported. This optimized protocol is a promising tool for low-cost genetic studies and the rapid, simple construction of homemade SSR libraries for small and large genomes.

Ethnobotanical investigation on medicinal plants in the Vesuvio National Park (Campania, Southern Italy).

ETHNOPHARMACOLOGICAL RELEVANCE This paper illustrates the results of an ethnobotanical study carried out in the Vesuvio National Park (VNP) (Campania, Southern Italy). It describes the medicinal uses of the plants in an ancient area rich in ethnobiodiversity investigated for the first time. AIM FOR THE STUDY The main aim of the study was to understand at what extent current knowledge on medicinal plant uses is still alive in VNP. MATERIALS AND METHODS The informations were collected using semi-structured and unstructured interviews performed on 136 persons living in the investigated area from March to November 2014 and from April to October 2015. The age of the informants ranged from 47 to 85 years old; more than half of the informants aged between 61 and 70. Local plant uses were listed and analyzed in a table and compared with uses in other localities in Italy and in other regions of the Mediterranean basin. RESULTS In VNP were recorded a total number of 132 plant species, belonging to 110 genera and 51 families mentioned for medicinal purposes. Among the recorded 132 plant species, 70 are spontaneous or subspontaneous and 62 are cultivated above all in the kitchen gardens or in the apartments, as food or as ornamental. Herbs represent the majority, followed by trees and shrubs or subshrubs. The investigated plants were used to cure 116 different human health diseases and 4 veterinary problems. The majority of plants are used in the treatment of gastrointestinal, skin and respiratory problems. CONCLUSION The number of medicinal plants reported in this paper reflects a well-preserved traditional popular knowledge (TPK) of the elderly people living in the rural areas and in the small villages of VNP. The conservation of TPK is owed to the persistence of an oral tradition that safeguard the use of plants as herbal medicine. We realized that while the use of some wild plants is decreasing, people continue to gather some cultivated and invasive plants for preparing remedies. Researches like this are necessary to protect ancient memories, to promote the transfer of information to the younger generations, to preserve ethno-biodiversity and to provide a starting point fur further biochemical investigations on medicinal entities.

Development and characterization of 21 microsatellite markers for Pancratium maritimum L. (Amaryllidaceae)

Pancratium maritimum L. (Amaryllidaceae) is a bulbous perennial plant growing on coastal sands of Mediterranean, Black and Caspian seas as well as on part of European coasts of Atlantic Ocean. The excess of flowers sampling, urbanization and tourism put serious threats to the species, resulting in a significant decrease of its populations. In order to investigate its conservation genetics, we developed and characterized microsatellite markers in P. maritimum. Twenty-one microsatellite loci were isolated using the SSR-patchwork protocol. The average number of alleles per locus was 4.4. Polymorphic information content value ranged from 0 to 0.75 with an average of 0.39. Eleven loci showed significant departures from Hardy–Weinberg equilibrium.

Disentangling Phylogenetic Relationships in a Hotspot of Diversity: The Butterworts (Pinguicula L., Lentibulariaceae) Endemic to Italy.

The genus Pinguicula (Lentibulariaceae) consists of about 100 carnivorous species, also known as butterworts. Eleven taxa are endemic to Italy, which represents a biodiversity hotspot for butterworts in Europe. The aim of our study was to provide a phylogenetic framework for the Italian endemics, in order to: a) investigate the relationships between species in this group; b) evaluate their actual taxonomic value. To achieve this, we analysed all the taxa endemic to Italy, along with several other species, by means of ITS nrDNA analysis. Our results clarify the relationships between Italian endemics and other Pinguicula taxa identifying a basal polytomy defined by five clades. All of the Italian endemics (with the exception of P. lavalvae) fall within a single large clade, which includes P. vulgaris and allied species. Among them, P. poldinii represents the most isolated lineage. Other taxa show strong molecular similarities and form a single subclade, although their taxonomic ranks can be retained. Pinguicula lattanziae sp. nov., seemingly endemic to Liguria (NW Italy), is also described.

Analysis of the genus Petagnaea Caruel (Apiaceae), using new molecular and literature data

In Southern Italy, an endemic monotypic genus belonging to family Apiaceae occurs: Petagnaea (P. gussonei), relict of Tertiary flora, belonging to subfamily Saniculoideae. At present, P. gussonei is an endangered species and is included in various lists of species deserving special protection. The genus belongs to scapose hemicryptophytes and shares a sciaphilous habitat (hygrophilous woodland). This study is aimed at doing a complete contribution about the evolutionary history of Petagnaea, using molecular markers as plastidial DNA (cpDNA), nuclear ribosomal DNA (rDNA) and data present in literature. We used nucleotide sequences from four regions of the chloroplast genome (rps16 intron, trnL(UAA) intron, atpB-rbcL intergenic spacer, and partial matK gene) to investigate possible haplotypes in Petagnaea populations. To have an idea of the molecular relationships of all populations of P. gussonei, the internal transcribed spacer (ITS) sequences, already employed in recent studies, were obtained for 18 populations. These sequences in combination with other Saniculoideae ITS sequences available from GenBank have been used for a further phylogenetic analysis. The results agree with the current classification of Saniculoideae in placing P. gussonei in tribe Saniculeae, since P. gussonei is in basal position to Sanicula. According to intraspecific chloroplast DNA diversity, no different haplotypes were detected. In addition to molecular data, morphology, cytology, phytochemistry and conservation status have been considered in the discussion.

Leaf and stem anatomy in eight Hypericum species (Clusiaceae)

Abstract - Foliar micromorphology, epicuticular wax morphology and anatomical features of leaves and stem, particularly secondary xylem, were examined with light microscopy, general and histochemical staining and scanning electron microscopy in eight Hypericum species. Outer tegument tissue and type of secondary xylem are determining characteristics. Secondary xylem is ring-porous in H. perforatum, H. perfoliatum, H. tetrapterum, H. triquetrifolium, H. androsaemum and H. hircinum. In H. aegypticum and H. pubescens xylem is diffuse-porous, which is considered to be a more primitive type. These characteristics may be considered an additional criterion for species identification.

Comparative transcriptional analysis reveals differential gene expression between Sand Daffodil tissues

Sand Daffodil (Pancratium maritimum) is a world-wide endangered Amayllidaceae species and represents an important anti-cancer medicinal resource due to alkaloids production. Despite its increasing pharmaceutical importance, there are not molecular resources that can be utilized toward improving genetic traits. In our research, the suppression subtractive hybridization (SSH) method conducted to generate large-scale expressed sequence tags (EST), was designed to identify gene candidates related to the morphological and physiological differences between the two tissues, leaves and bulbs, since lycorine, the main anti-cancer compound, is there synthesized. We focused on identification of transcripts in different tissues from Sand Daffodil using PCR-based suppression SSH to identify genes involved in global pathway control. Sequencing of 2,000 differentially screened clones from the SSH libraries resulted in 136 unigenes. Functional annotation and gene ontology analysis of up-regulated EST libraries showed several known biosynthetic genes and novel transcripts that may be involved in signaling, cellular transport, or metabolism. Real time RT-PCR analysis of a set of 8 candidate genes further confirmed the differential gene expression.

Plastid DNA sequencing and nuclear SNP genotyping help resolve the puzzle of central American Platanus.

BACKGROUND AND AIMS Recent research on the history of Platanus reveals that hybridization phenomena occurred in the central American species. This study has two goals: to help resolve the evolutive puzzle of central American Platanus, and to test the potential of real-time polymerase chain reaction (PCR) for detecting ancient hybridization. METHODS Sequencing of a uniparental plastid DNA marker [psbA-trnH((GUG)) intergenic spacer] and qualitative and quantitative single nucleotide polymorphism (SNP) genotyping of biparental nuclear ribosomal DNA (nrDNA) markers [LEAFY intron 2 (LFY-i2) and internal transcribed spacer 2 (ITS2)] were used. KEY RESULTS Based on the SNP genotyping results, several Platanus accessions show the presence of hybridization/introgression, including some accessions of P. rzedowskii and of P. mexicana var. interior and one of P. mexicana var. mexicana from Oaxaca (= P. oaxacana). Based on haplotype analyses of the psbA-trnH spacer, five haplotypes were detected. The most common of these is present in taxa belonging to P. orientalis, P. racemosa sensu lato, some accessions of P. occidentalis sensu stricto (s.s.) from Texas, P. occidentalis var. palmeri, P. mexicana s.s. and P. rzedowskii. This is highly relevant to genetic relationships with the haplotypes present in P. occidentalis s.s. and P. mexicana var. interior. CONCLUSIONS Hybridization and introgression events between lineages ancestral to modern central and eastern North American Platanus species occurred. Plastid haplotypes and qualitative and quantitative SNP genotyping provide information critical for understanding the complex history of Mexican Platanus. Compared with the usual molecular techniques of sub-cloning, sequencing and genotyping, real-time PCR assay is a quick and sensitive technique for analysing complex evolutionary patterns.

What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas

In this study, we used several molecular techniques to develop a fast and reliable protocol (DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a (healthy) beverage, its high selling value, the importation of barely recognizable raw product (i.e., crushed), and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta).