Antonietta Di Maio

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.

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.

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).

Sanger and next generation sequencing in the characterisation of arbuscular mycorrhizal fungi (AMF) in Pancratium maritimum L. (Amaryllidaceae), a representative plant species of Mediterranean sand dunes

Main conclusionAn interesting AMF colonization microcosm has been detected in the roots of Pancratium maritimum (sea daffodil). Both sequencing techniques (Sanger and NGS) have been used for AMF characterisation, showing a balanced trade-off between pros and cons.By Sanger and next generation sequencing of rRNA nuclear molecular markers (SSU–ITS–LSU and ITS2, respectively), the presence of AMF communities in the roots of P. maritimum was evaluated. Our results shed light on the presence of AMF in sea daffodil and the diversity of assemblages of AMF detected after Sanger sequencing of the SSU–ITS–LSU marker is much higher than that determined following NGS sequencing of ITS2 alone.

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

[This corrects the article DOI: 10.1371/journal.pone.0178262.].