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Forin, Niccolò (2018) A next generation sequencing approach for the study of ancient fungal specimens belonging to the Pier Andrea Saccardo collection preserved at the University of Padua. [Ph.D. thesis]

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Abstract (italian or english)

The mycological collections represent a huge source of molecular information that may be exploited to obtain important DNA data. Indeed, it has been demonstrated that DNA barcoding projects of fungarium material have the potential to enlarge the coverage of species-level DNA sequence information deposited in public databases. However, these collections are an underused resource for building up voucher-based reference datasets, due to the difficulty to obtain DNA sequences from herbarium material. The over one century old Saccardo mycological collection preserved in the herbarium of the Botanical Garden of Padua contains about 70,000 specimens including more than 4,000 type specimens. The types in this collection have been borrowed by mycologists from all over the world for morphological revisions and consequent taxonomic reclassifications, but they have never been involved in sequencing projects so far. Accordingly, the aim of this research was to apply a DNA barcoding approach to obtain internal transcribed spacer (ITS) sequences, the consensus barcode for fungal species identification, from specimens preserved in this collection. This DNA region has been identified as a suitable marker for molecular studies that involve ancient mycological material, because only short DNA regions can be obtained from ancient and degraded DNA. In addition, in case of initial PCR failure of the ITS region, it is possible to increase the amplification/sequencing success by analyzing separately the two non-coding regions ITS1 and ITS2 that form the entire ITS. In this thesis work, an Illumina MiSeq sequencing method was applied to recover ITS1/ITS2 sequences overcoming the problems of the high level of DNA degradation of the Saccardo fungarium samples and the presence of contaminations by exogenous fungal DNA. The method required the setup of the steps involved in the samples preparation for the sequencing and in the bioinformatic data analysis, and then its efficacy was first tested to obtain ITS2 sequences from 36 non-type Peziza specimens. Despite the presence of both external fungal contamination and cross-contamination between fungarium specimens, this high-throughput sequencing method has permitted to recover ITS2 sequences from 23 out of the 36 specimens studied and also a taxonomic re-evaluation of some samples at the species level and others at genus or higher taxonomic level. Then, this next-generation sequencing (NGS) approach was used to retrieve ITS1/ITS2 sequences from type specimens belonging to the genus Nectria and from Nectria-like types classified in the collection as members of other genera. Several of these types were morphologically revised in the past by expert mycologists and placed in synonymy with other species or reclassified as members of new genera. The ITS1/ITS2 sequences were obtained for 25 different types (30 in total considering multiple specimens) out of 76 specimens involved in the study. The combined morphological and molecular data analysis suggests that there is a need to reclassify some Nectria/Nectria-like types previously reclassified only on a morphological basis and some types never considered for taxonomic revisions. In fact, for 11 types the original species name has been confirmed, for four and five types new nomenclature combinations and synonymies have been proposed respectively, while for other five types the taxonomic assignment has been possible only at genus level. Since type specimens constitute an integral part of fungal classification and nomenclature and given the outstanding and worldwide importance of the Saccardo collection, these findings provide material for a taxonomic revision of invaluable types. Moreover, the method proposed in this research not only has provided an additional scientific value to the Saccardo collection, but it can be applied to obtain important voucher-sequences from problematic herbarium material, thus expanding the databases with well-annotated ITS barcode sequences.

Abstract (a different language)

Le collezioni micologiche rappresentano un’enorme risorsa di informazioni molecolari che può essere sfruttata per ottenere importanti sequenze di DNA. Infatti, è stato dimostrato che i progetti di DNA barcoding che coinvolgono materiale fungino proveniente da collezioni micologiche possono aumentare la quantità di informazioni molecolari relative alle diverse specie fungine nei database pubblici. Tuttavia, queste raccolte sono una risorsa poco sfruttata per la creazione di datasets di riferimento basati su campioni accuratamente identificati, a causa della difficoltà nell’ottenere sequenze di DNA dal materiale conservato all’interno degli erbari. La raccolta micologica di Pier Andrea Saccardo, di oltre un secolo, conservata nell'erbario dell'Orto Botanico di Padova, contiene circa 70.000 campioni fungini di cui oltre 4.000 sono esemplari tipo. I tipi di questa collezione sono stati spesso richiesti da micologi di tutto il mondo per revisioni morfologiche e conseguenti riclassificazioni tassonomiche, ma non erano mai stati coinvolti in progetti di sequenziamento finora. Di conseguenza, lo scopo di questa ricerca è stata quella di applicare un approccio di DNA barcoding per ottenere sequenze della regione ITS (internal transcribed spacer), il barcode utilizzato per l'identificazione delle specie fungine, dai campioni conservati in questa collezione. Questa regione è considerata un marcatore adatto per studi molecolari che coinvolgono materiale micologico antico, poiché solo corte regioni di DNA possono essere ottenute da DNA antico e degradato. Inoltre, in caso di fallimento nell’amplificazione della regione ITS, è possibile aumentare il successo dell'amplificazione/sequenziamento analizzando separatamente le due regioni non codificanti ITS1 e ITS2 che formano l'intera regione ITS. In questo lavoro di tesi, è stato applicato un metodo di sequenziamento Illumina MiSeq per ottenere sequenze ITS1/ITS2 superando i problemi relativi all'alto livello di degradazione del DNA dei campioni della collezione micologica e la presenza di contaminazioni da DNA fungino esogeno. Il metodo ha richiesto l'ottimizzazione delle diverse fasi coinvolte nella preparazione dei campioni per il sequenziamento e nell'analisi bioinformatica dei dati, e la sua efficacia è stata prima testata per ottenere sequenze ITS2 da 36 esemplari fungini non-tipo appartenenti al genere Peziza. Nonostante la presenza sia di contaminazioni fungine esterne che di contaminazione incrociata tra i campioni della collezione, questo metodo di sequenziamento ha permesso di recuperare sequenze della regione ITS2 da 23 dei 36 campioni studiati e anche una rivalutazione tassonomica di alcuni campioni a livello di specie e altri a livello di genere o livello tassonomico superiore. Successivamente, questo approccio di sequenziamento di nuova generazione è stato utilizzato per ottenre sequenze ITS1/ITS2 da campioni tipo appartenenti al genere Nectria e da tipi Nectria-simili classificati nella collezione come membri di altri generi. Molti di questi tipi furono morfologicamente revisionati in passato da esperti micologi e posti in sinonimia con altre specie o riclassificati come membri di nuovi generi. Le sequenze ITS1/ITS2 sono state ottenute per 25 diversi tipi (30 in totale considerando campioni multipli) su 76 campioni coinvolti nello studio. L'analisi combinata dei dati morfologici e molecolari suggerisce la necessità di riclassificare alcuni tipi di Nectria/Nectria-simili precedentemente riclassificati solo su base morfologica e alcuni tipi mai considerati per una revisione tassonomica. Infatti, per 11 tipi è stato confermato il nome originale della specie, per quattro e cinque tipi sono state proposte rispettivamente nuove combinazioni di nomenclatura e sinonimie, mentre per altri cinque l'assegnazione tassonomica è stata possibile solo a livello di genere. Poiché i campioni tipo costituiscono una parte integrante della classificazione e della nomenclatura dei funghi e data l'importanza eccezionale e mondiale della collezione Saccardo, questi risultati forniscono materiale per una revisione tassonomica di inestimabili campioni tipo. Inoltre, il metodo proposto in questa ricerca non solo ha fornito un valore scientifico aggiuntivo alla collezione Saccardo, ma può essere applicato per ottenere importanti sequenze da materiale d’erbario, espandendo così i database con sequenze ITS ben annotate.

EPrint type:Ph.D. thesis
Tutor:Baldan, Barbara
Ph.D. course:Ciclo 31 > Corsi 31 > BIOSCIENZE
Data di deposito della tesi:30 November 2018
Anno di Pubblicazione:30 November 2018
Key Words:DNA barcoding / next-generation sequencing / fungi / herbarium collection /nuclear ribosomal internal transcribed spacer / ITS / Pier Andrea Saccardo
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/02 Botanica sistematica
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Centri > Centro di Ateneo Orto Botanico
Codice ID:11535
Depositato il:06 Nov 2019 10:16
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