Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.

Das Afrikanische Schweinepest-Virus (ASPV) verursacht eine tödliche Viruserkrankung bei Schweinen. Dieses hat sich weltweit fortlaufend verbreitet und wurde im September 2020 erstmalig in Deutschland nachgewiesen. Der Ausbruch der Seuche kann schwere wirtschaftliche Verluste nach sich ziehen. Bis heute ist kein Impfstoff zugelassen, daher sind Überwachung der epidemiologischen Situation und der frühzeitige Erregernachweis unerlässlich für die Bekämpfung der Afrikanischen Schweinepest als Tierseuche. Die Polymerase-Kettenreaktion (PCR) gilt als Goldstandard für den Nachweis von ASPV und zeichnet sich durch eine hohe Sensitivität und Spezifität aus. Allerdings erfordert die PCR gut ausgestattete Testlabore und ist zeitintensiv. Point-of-Need-Tests können schnelle und zuverlässige direkt vor Ort liefern und stellen somit eine Alternative zum Goldstandard PCR dar. Ziel dieser Studie war es, einen Point-of-Need-Test zum Nachweis von ASPV zu entwickeln. Dieser beruht auf der Grundlage der Rekombinase-Polymerase-Amplifikation (RPA) und sollte vor Ort einsatzfähig sein. Es wurden drei Primersätze und eine Sonde auf der Grundlage des B646L-Gens, welches für das virale Kapsidprotein p72 vom ASP-Virus kodiert, entwickelt. Alle möglichen Kombinationen wurden getestet. Die analytische Sensitivität wurde mit acht Wiederholungen von Verdünnungsreihen des molekularen Standards (102-100 DNA-Kopien pro µl) ermittelt. Die Nachweisgrenze wurde anhand einer Probit-Analyse dieser Durchläufe berechnet. Die Spezifität wurde mit verschiedenen viralen Nukleinsäuren von anderen das Schwein infizierenden Erregern überprüft. Um den Test im Feld einsatzfähig zu gestalten, wurden mittels ASPV-RPA zwei verschiedene Extraktionsansätze mit allen 73 verfügbaren Schweineblutproben getestet: eine schnelle Hitze/Lysepuffer-Extraktionsmethode und ein standardisiertes Extraktionsverfahren auf Spin-Säule-Basis. Die diagnostische Sensitivität und Spezifität wurde für beide Testverfahren berechnet. Alle Ergebnisse wurden mit einer etablierten real-time PCR für ASPV verglichen. Eine kleine Pilotstudie zum Feldeinsatz des ASPV-RPA-Tests wurde in Uganda mit 20 Blutproben unter Verwendung des Kofferlabors durchgeführt. Die berechnete Nachweisgrenze von ASPV-RPA lag bei 3,5 DNA-Kopien pro µl. Alle untersuchten ASPV-Genotypen wurden detektiert, aber keine anderen viralen Nukleinsäuren. Bei Verwendung der standardisierten DNA-Extraktionsmethode mit anschließender Durchführung der ASPV-RPA lag die diagnostische Sensitivität und Spezifität bei 100%, wie auch mittels der real-time PCR. Auch das schnelle Hitze-/Lysepuffer Protokoll zeigte vielversprechende Ergebnisse und erreichte eine Positivrate von 97% mittels ASPV-RPA im Vergleich zu 38% bei der PCR. In Uganda wurden elf ASPV-RPA-Proben als positiv erkannt, darunter zwei fieberfreie asymptomatische Tiere. Der schnelle Erregernachweis stellt einen essenziellen Aspekt der ASP Seuchenbekämpfung dar. Die ASPV-RPA erwies sich als genauso empfindlich und spezifisch wie die Goldstandard-PCR zur Erregeridentifizierung. In Kombination mit dem Schritt der DNA-Extraktion durch Hitze/Lysepuffer benötigt der entwickelte Test etwa 25 Minuten von der Probenentnahme bis zum Ergebnis. Die Positivrate ist mit 97% vielversprechend, wobei die ASPV-RPA im Vergleich zur PCR eine höhere Toleranz gegenüber Inhibitoren aufwies. Wie die Pilot-Feldstudie in Uganda mit dem Kofferlabor zeigt, ist ASPV-RPA eine im Feld einsatzfähige Nachweismethode. Das Kofferlabor bedarf lediglich einer Grundausstattung und einer Solarbatterie. Somit stellt das Kofferlabor eine vielversprechende Diagnostikmethode dar, welche vor Ort in ressourcenarmen Umgebungen zum Nachweis des ASPV eingesetzt werden kann.:1. Introduction 2. Literature overview 2.1 African swine fever 2.1.1 Aetiology 2.1.1.1 Classification and taxonomy 2.1.1.2 Viral structure and genome 2.1.1.3 Genetic typing and antigenic variability 2.1.2 Epidemiology 2.1.2.1 Disease distribution 2.1.2.2 Host range and epidemiological cycles 2.1.2.2.1 Warthog-tick cycle 2.1.2.2.2 Domestic pig-tick cycle 2.1.2.2.3 Domestic pig cycle 2.1.2.2.4 Wild boar-environment cycle 2.1.2.3 Tenacity, transmission, and infectivity 2.1.3 Pathophysiology 2.1.3.1 Pathogenesis 2.1.3.2 Clinical signs and pathological findings 2.1.3.3 Differential diagnosis 2.2 Available diagnostic tools for ASFV 2.1.4 Diagnosis based on immune response 2.1.5 Diagnosis based on agent identification 2.3 Gaps in African swine fever diagnostics 3. Publication 3.1 Statement of contribution 3.1.1 Publication 4. Discussion 5. Summary 6. Zusammenfassung 7. References 8. Appendix 9. Acknowledgements / African swine fever virus (ASFV) causes a deadly viral disease in pigs. The virus has gradually spread throughout the world and was reported in Germany in September 2020. ASF outbreak can lead to huge economical loss. No vaccine is commercially available and thus, surveillance and early detection play a pivotal role to control an ASF outbreak. Polymerase Chain Reaction (PCR) is considered the gold standard for ASFV detection due to its superior sensitivity and specificity. However, it is time-consuming and requires well-equipped laboratories. Point-of-need tests can offer an alternative, delivering fast and reliable results directly in the field. The aim of this study was to establish a field-deployable point-of-need test based on Recombinase Polymerase Amplification (RPA) to detect ASFV. Material and Methods: Three sets of primers and one probe based on the B646L gene which encodes for the viral capsid protein p72 were designed. All possible combinations were screened. Analytical sensitivity was tested with eight replicates of serial dilutions of the molecular standard (102-10° DNA copies per µl). The limit of detection was calculated using probit analysis. ASFV-RPA’s specificity was tested using various viral nucleic acids of pathogens infecting pigs. To allow the deployment at point of need, two different extraction approaches were tested in ASFV-RPA with all 73 pig blood samples included in this study: a rapid heat/lysis buffer extraction method and a standardized spin-column based extraction kit. Diagnostic sensitivity and specificity were calculated for both test approaches. All results were compared to an established real-time PCR for ASFV. A small pilot study for ASFV-RPA assay deployment was done in Uganda with 20 blood samples of a suspected outbreak using the field-deployable suitcaselab. The calculated limit of detection of ASFV-RPA was 3.5 DNA copies per µl. All screened ASFV genotypes were detected while no other viral nucleic acids were identified. Using the standardized DNA extraction method in ASFV-RPA, and compared to real-time PCR, diagnostic sensitivity and specificity were 100%. The rapid heat/lysis buffer protocol showed very promising results, achieving 97% of positivity rate compared to a 38% of the real-time PCR. In Uganda, ASFV-RPA detected 11 samples as positive, including two known afebrile animals. Immediate agent detection is a key aspect of ASF outbreak control. ASFV-RPA is as sensitive and specific as a gold standard PCR for ASFV identification. Combined with the heat/lysis buffer DNA isolation step, the duration of the assay is around 25 minutes from sample collection to result readout, with a promising positivity rate of 97% which indicates tolerance against inhibitors. ASFV-RPA is a portable detection method, as revealed during the pilot field study in Uganda. Only requiring basic equipment and solar batteries, the suitcase lab is a promising tool for on-site diagnostics in resource limited settings to detect ASFV.:1. Introduction 2. Literature overview 2.1 African swine fever 2.1.1 Aetiology 2.1.1.1 Classification and taxonomy 2.1.1.2 Viral structure and genome 2.1.1.3 Genetic typing and antigenic variability 2.1.2 Epidemiology 2.1.2.1 Disease distribution 2.1.2.2 Host range and epidemiological cycles 2.1.2.2.1 Warthog-tick cycle 2.1.2.2.2 Domestic pig-tick cycle 2.1.2.2.3 Domestic pig cycle 2.1.2.2.4 Wild boar-environment cycle 2.1.2.3 Tenacity, transmission, and infectivity 2.1.3 Pathophysiology 2.1.3.1 Pathogenesis 2.1.3.2 Clinical signs and pathological findings 2.1.3.3 Differential diagnosis 2.2 Available diagnostic tools for ASFV 2.1.4 Diagnosis based on immune response 2.1.5 Diagnosis based on agent identification 2.3 Gaps in African swine fever diagnostics 3. Publication 3.1 Statement of contribution 3.1.1 Publication 4. Discussion 5. Summary 6. Zusammenfassung 7. References 8. Appendix 9. Acknowledgements

[ES] El impacto del cambio climático en los cultivos hortícolas es cada vez más evidente, lo que ha llevado a la pérdida y erosión de diversidad genética de manera drástica. Esto plantea importantes desafíos para la mejora de los cultivos, que requiere la exploración de los recursos fitogenéticos conservados en los bancos de germoplasma y el desarrollo de tecnologías que permitan evaluar el valor fenotípico y genotípico de estos materiales. Sin embargo, la situación actual de las colecciones de germoplasma es la existencia de duplicados no identificados entre colecciones, errores en la clasificación taxonómica, documentación insuficiente y no disponible para investigadores y mejoradores, añadido a la falta de financiación para la conservación y gestión adecuadas. Esto dificulta enormemente la utilización de estos recursos. En la presente Tesis se aborda este problema comenzando por la unificación de datos de pasaporte, fenotipado e imágenes de las principales colecciones de tomate, pimiento y berenjena en un mismo repositorio en el primer capítulo. El segundo capítulo se centra en el desarrollo y optimización de un método de extracción de ADN genómico de alta calidad, rápido y económico que combina las ventajas del método de extracción basado en el CTAB, añadido a la purificación de los ácidos nucleicos en una matriz de sílice. Es un método universal que puede utilizarse para diferentes especies y tejidos. Se ha evaluado la eficiencia del ADN genómico resultante en diferentes plataformas de secuenciación como SPET (Single Primer Enrichment Technology) y Oxford Nanopore, generando resultados muy prometedores. Esto facilita el paso previo al genotipado de las colecciones que es la extracción de ADN. En el tercer capítulo se aborda el genotipado de las colecciones. El elevado número de accesiones de cada cultivo, en particular el tomate, supone un problema de tipo económico, en ocasiones irresoluble. Por ello, el tercer capítulo está orientado a la evaluación del potencial de la tecnología de secuenciación SPET, más económica que otras conocidas, para el genotipado de alto rendimiento de colecciones de germoplasma de tomate y berenjena. Los resultados revelan que el genotipado SPET es una tecnología robusta y de alto rendimiento para estudios genéticos, incluyendo la posibilidad de identificación de duplicados y errores de clasificación taxonómica en las entradas conservadas en los bancos. Con la información generada en los primeros tres capítulos se establecieron las colecciones nucleares para cada cultivo, abarcando la máxima diversidad genética y fenotípica en un conjunto de 450 individuos. Finalmente, en el cuarto capítulo, se analiza y describe la colección nuclear de tomate a nivel genético y fenotípico, mediante un enfoque basado en el establecimiento de grupos genéticos basados en su proximidad genética. El análisis de la diversidad genética y fenotípica reveló patrones de variación distintos entre diferentes grupos genéticos, contradiciendo afirmaciones anteriores que proponían una disminución en la diversidad genética como consecuencia de la mejora genética y descubriendo correlaciones entre rasgos morfológicos únicas dentro de los diferentes grupos. En resumen, esta tesis aumenta el conocimiento y accesibilidad a las colecciones de Solanaceae en bancos de germoplasma y proporciona herramientas moleculares. Destaca la importancia de estos bancos como reservorios de diversidad genética, aunque enfrenten desafíos como datos limitados y duplicados. Estos avances sientan las bases para la conservación y programas de mejora futuros. / [CA] L'impacte del canvi climàtic en els cultius hortícoles és cada vegada més evident, la qual cosa ha portat a la dràstica pèrdua i erosió de la diversitat genètica. La reduïda diversitat genètica planteja importants reptes per a la millora dels cultius. Sent necessari l'exploració dels recursos genètics vegetals conservats en els bancs de germoplasma i el desenvolupament de tecnologies que permeten avaluar el valor fenotípic i genotípic d'aquests materials. Pel que fa a les col·leccions de germoplasma presenten duplicats no identificats entre col·leccions, errors en la classificació taxonòmica, falta de finançament per a la conservació i gestió adequades a banda de documentació insuficient i no disponible (investigadors i milloradors vegetals). En la present tesi doctoral en el primer capítol s'aborda aquest problema unificant les dades de passaport, fenotipat i imatges de les principals col·leccions de tomaca, pebre i albergínia en un mateix repositori. El segon capítol es focalitza en el desenvolupament i optimització d'un mètode d'extracció de ADN genòmic d'alta qualitat, ràpid i econòmic que combina els avantatges del mètode d'extracció basat en el CTAB amb l'ús de matrius de sílice. El mètode desenvolupat pot utilitzar-se de manera universal per a diferents espècies i teixits vegetals. S'ha avaluat l'eficiència del ADN genòmic resultant en diferents plataformes de seqüenciació com SPET (Single Primer Enrichment Technology) i Oxford Nanopore, generant resultats molt prometedors. Això facilita el pas previ al genotipat de les col·leccions que és l'extracció d'ADN. En el tercer capítol aborda l'optimització del procés de genotipat de les col·leccions generades. L'elevat nombre d'accessions de cada cultiu, en particular la tomaca, suposa un problema de tipus econòmic, a vegades irresoluble. Per això, aquest capítol està orientat a l'avaluació del potencial de la tecnologia de seqüenciació SPET per al genotipat d'alt rendiment de col·leccions de germoplasma de tomaca i albergínia a un preu econòmic. Els resultats revelen que el genotipat SPET és una tecnologia robusta i d'alt rendiment per a estudis genètics, incloent-hi la possibilitat d'identificació de duplicats i errors de classificació taxonòmica en les entrades conservades en els bancs de germoplasma. La informació generada va permetre establir col·leccions nuclears per a cada cultiu, abastant la màxima diversitat genètica i fenotípica en un conjunt de 450 individus. Finalment, en el quart capítol, s'analitza i descrigué la col·lecció nuclear de tomaca a nivell genètic i fenotípic, focalitzant-se en l'establiment de grups genètics basats en la seua proximitat genètica. L'anàlisi de la diversitat genètica i fenotípica va revelar patrons de variació diferents entre diferents grups genètics, contradient afirmacions anteriors que proposaven una disminució en la diversitat genètica a conseqüència de la millora genètica. També és descobriren noves correlacions entre trets morfològics únics dins dels diferents grups. L'estudi destaca la importància d'abordar les iniciatives de millora de la tomaca tenint en compte tant la diversitat genètica com la fenotípica, amb especial èmfasi en aspectes com la grandària, la forma, el color i la qualitat del fruit. En definitiva, els treballs realitzats en aquesta tesi doctoral augmenten, d'una banda, el coneixement i l'accessibilitat a les principals col·leccions de solanàcies conservades en els bancs de germoplasma. Per un altre, generen eines moleculars que permeten l' avaluació genotípica de les col·leccions analizades. En resum, aquests avanços suposen una base per al futur, proporcionant informació valuosa per a la pròpia conservació de les col·leccions i el seu ús en programes de millora. / [EN] The impact of climate change on horticultural crops is increasingly evident, leading to drastic loss and erosion of genetic diversity. This poses significant challenges for crop improvement, which requires the exploration of plant genetic resources conserved in germplasm banks and the development of technologies. However, the current situation of germplasm collections is characterized by the existence of unidentified duplicates among collections, taxonomic mislabelling, insufficient and unavailable documentation for researchers and breeders, and a lack of funding for proper conservation and management. This greatly hampers the utilization of these resources. This thesis addresses this problem by starting with the unification of passport, phenotyping, and image data from the main collections of tomato, pepper, and eggplant. Genotyping these collections enables the creation of core collections, enhancing knowledge of genotypic and phenotypic variability for researchers and breeders. In the first chapter, an inventory of available passport and phenotypic data of tomato, pepper, and eggplant accessions conserved in major European and non-European germplasm banks were conducted to improve the efficiency of plant genetic resource management. The second chapter focuses on the development and optimization of a high-quality, fast, and cost-effective genomic DNA extraction method that combines the advantages of the CTAB-based extraction method with nucleic acid purification on a silica matrix. The efficiency of the resulting genomic DNA was evaluated on different sequencing platforms, such as Single Primer Enrichment Technology (SPET) and Oxford Nanopore, yielding promising results. This facilitates the prerequisite step of DNA extraction before genotyping the collections. Chapter three addresses the genotyping of the collections. The high number of accessions for each crop, particularly tomato, poses an often insurmountable economic problem. Therefore, chapter three is focused on evaluating the potential of SPET sequencing technology, which is more cost-effective than other known methods, for high-throughput genotyping of tomato and eggplant germplasm collections. The results reveal that SPET genotyping is a robust and high-performance technology for genetic studies, including the identification of duplicates and taxonomic misclassifications in the accessions stored in the germplasm banks. Based on the information generated in the first three chapters, core collections were established for each crop, encompassing maximum genetic and phenotypic diversity in a set of 450 individuals. Finally, in the fourth chapter, the genetic and phenotypic analysis of the tomato core collection is examined and described using an approach based on establishing genetic groups based on their genetic proximity. Genetic and phenotypic diversity analysis revealed distinct patterns of variation among different genetic groups, contradicting previous claims of a decrease in genetic diversity due to genetic improvement and uncovering unique correlations between morphological traits within different groups. The study highlights the importance of considering both genetic and phenotypic diversity in tomato breeding initiatives, with a particular emphasis on aspects such as fruit size, shape, color, and quality. In conclusion, this thesis enhances knowledge and accessibility to major Solanaceae collections in germplasm banks, while providing molecular tools for genotypic evaluation. It underscores germplasm banks' role as genetic diversity reservoirs, despite challenges such as data limitations and inaccuracies, emphasizing the importance of data standardization and maintenance. These advancements lay a foundation for conservation and breeding programs in the future. / This work was supported by grants CIPROM/2021/020 from Conselleria d’Innovació, Universitats, Ciència i Societat Digital (Generalitat Valenciana, Spain), PID2021-128148OB-I00 funded by MCIN/AEI/10.13039/501100011033/ and by “ERDF A way of making Europe”, PDC2022-133513-I00 funded by MCIN/AEI/10.13039/501100011033/, and by “European Union NextGenerationEU/PRTR”, by Grant Agreement No. 677379 (G2P-SOL project: Linking genetic resources, genomes and phenotypes of Solanaceous crops) from European Union’s Horizon 2020 Research and Innovation Programme, by the Grant Agreement No. 101094738 (PRO-GRACE project: Promoting a Plant Genetic Resource Community for Europe) from the European Union’s Horizon Europe programme, as well as by the initiative "Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives", which is supported by the Government of Norway. This later project is managed by the Global Crop Diversity Trust with the Millennium Seed Bank of the Royal Botanic Gardens, Kew and implemented in partnership with national and international gene banks and plant breeding institutes around the world. For further information, see the project website: htp://www.cwrdiversity.org/. The overall work also partially fulfils some goals of the Agritech National Research Center and received funding from the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)–MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4—D.D. 1032 17/06/2022, CN00000022). David Alonso is grateful to Universitat Politècnica de València for a predoctoral (PAID-01-16) / Alonso Martín, D. (2023). Linking Genetic Resources, Genomes and Phenotypes of Solanaceus Crops [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/201550

Les archaea constituent l'un des quatre domaines connus du vivant. Contrairement à ce que leur nom laisse supposer, elles ont colonisé tous les écosystèmes et les microbiotes de certains hôtes dont l'Homme. Chez l'homme, certaines espèces d'archaea méthanogènes ont été associées aux muqueuses orale, intestinale et vaginale. Ces archaea méthanogènes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. Quatre archaea methanogènes seulement ont été isolées à partir de prélèvements humains y compris dans le microbiote digestif Methanobrevibacter smithii détectée dans 95,7% des individus, Methanosphaera stadtmanae retrouvée chez environ un tiers des individus et plus récemment dans notre laboratoire Methanomassilicoccus luminyensis détectée en moyenne chez 4% des individus avec une prévalence liée à l'âge ; et dans le microbiote orale Methanobrevibacter oralis isolée à partir de la plaque dentaire. / Archaea is one of four known domains of life. Unlike what their name suggests, they some species of methanogenic archaea have been associated with oral, vaginal and intestinal mucosa. These methanogenic archaea are obligate anaerobic prokaryotes and their culture conditions are fastidious and very poorly known. Only four methanogenic archaea have been isolated from human samples including the digestive microbiota; Methanobrevibacter smithii detected in 95.7% of individuals Methanosphaera stadtmanae found in approximately one third of individuals and more recently in our laboratory Methanomassilicoccus luminyensis detected on average in 4% of individuals with a prevalence of age-related, and in the oral microbiota Methanobrevibacter oralis isolated from dental plaque.

Les compartiments géologiques profonds suscitent un intérêt grandissant dans la communauté scientifique depuis les 50 dernières années. Néanmoins, ces écosystèmes demeurent largement méconnus du fait de leur difficulté d'accès. Le forage profond réalisé par l'ANDRA dans le Bassin parisien en 2008 a offert une opportunité unique de les étudier. Dans ce cadre, cette thèse avait deux objectifs majeurs ; i) caractériser, d'un point de vue microbiologique, quatre formations sédimentaires terrestres triasiques situées entre 1700 et 2000 m de profondeur et ii) étudier les effets combinés des paramètres de température, pression et salinité ainsi que de leur interaction sur l'activité métabolique de procaryotes anaérobies afin de mieux appréhender leur comportement au cours d'un enfouissement géologique.Malgré la recherche de microorganisme par la réalisation d'une gamme de milieux de culture diversifiée, ciblant préférentiellement les types trophiques fréquemment rencontrés en subsurface (méthanogènes, fermentaires, réducteurs de composés soufrés), aucun microorganisme viable et cultivable n'ait été isolé. En parallèle, une approche moléculaire complémentaire, composée (i) de l'étude comparative de l'efficacité de différentes méthodes d'extraction directe d'ADN et (ii) de l'analyse de la diversité bactérienne par la réalisation d'inventaires moléculaires, par DGGE (Denaturing Gel Gradient Electrophoresis) et clonage, a été réalisée sur le coeur des carottes de roches, conservées à pression atmosphérique ou sous pression, dans leurs états initiaux et post-incubation. L'exploration de ces formations sédimentaires profondes a indiqué la présence d'une très faible biomasse et d'une biodiversité microbienne pauvre principalement composée de membres aérobies et mésophiles appartenant au domaine Bacteria. Cette communauté bactérienne inattendue car a priori peu adaptée aux conditions régnant in-situ, également retrouvée dans divers écosystèmes de subsurface ainsi que dans des biotopes extrêmes, pourrait provenir en partie d'une paléo-recharge de l'aquifère du Trias par des eaux froides dérivées de la fonte des glaces formées lors de la dernière glaciation du Pléistocène.Le second objectif a été abordé à travers l'élaboration d'un plan factoriel complet dans le but d'identifier les effets des paramètres sur les activités microbiennes. Ainsi, les activités métaboliques de huit souches microbiennes halophiles et thermo-tolérantes ont été mesurées sous trente conditions distinctes de température (40, 55 et 70°C), pression (1, 90 et 180 bars) et salinité (13, 50, 110, 180 et 260 g.l-1). Toutes les souches originaires d'environnements profonds se sont révélées être au minimum piézo-tolérantes et capables de maintenir leur activité métabolique sous pressions hydrostatiques. Les métabolismes fermentaires (Thermovirga lienii et Halothermothrix orenii) et thiosulfato-réducteurs (Petrotoga mexicana et Thermosipho japonicus) se sont avérés particulièrement bien adaptées, d'un point de vue métabolique, aux hautes pressions, les plus hautes activités ayant été détectées sous pression. Certaines souches ont montré une résistance accrue aux hautes températures sous pression (Petrotoga mexicana). Toutefois une résistance variable à la salinité dans les différentes conditions de température et de pression a été observée pour chacune des souches, suggérant que certains mécanismes de résistance contre la pression osmotique seraient également efficaces pour lutter contre les températures et les pressions hydrostatiques élevées.Ce travail souligne que l'étude des écosystèmes terrestres profonds d'un point de vue microbiologique ne doit pas se restreindre à la recherche et à l'analyse de la diversité présente. L'étude des activités métaboliques de souches de subsurface en conditions profondes ouvre la voie à une meilleure compréhension des rôles joués par les communautés microbiennes en milieu extrême.

Humans evolved in a world of giant creatures. Current evidence suggests that most ice age megafauna went extinct around the transition to our current Holocene epoch. The ecological reverberations associated with the loss of over 65% of Earth’s largest terrestrial animals transformed ecosystems and human lifeways forever thereafter. However, there is still substantial debate as to the cause of this mass extinction. Evidence variously supports climate change and anthropogenic factors as primary drivers in the restructuring of the terrestrial biosphere. Much of the ongoing debate is driven by the insufficient resolution accessible via macro-remains. To help fill in the gaps in our understandings of the Pleistocene-Holocene transition, I utilized the growing power of sedimentary ancient DNA (sedaDNA) to reconstruct shifting signals of plants and animals in central Yukon. To date, sedaDNA has typically been analyzed by amplifying small, taxonomically informative regions. However, this approach is not ideally suited to the degraded characteristics of sedaDNA and ignores most of the potential data. Means of isolating sedaDNA have also suffered from the use of overly aggressive purification techniques resulting in substantial loss. To address these limitations, I first experimentally developed a novel means of releasing and isolating sedaDNA. Secondly, I developed a novel environmental bait-set designed to simultaneously capture DNA informative of macro-scale ecosystems. When combined, we identify a substantial improvement in the quantity and breadth of biomolecules recovered. These optimizations facilitated the unexpected discovery of horse and mammoth surviving thousands of years after their supposed extirpation. I followed up these results by extracting DNA from multiple permafrost cores where we confirm the late survival signal and identify a far more complex and high-resolution dataset beyond those identifiable by complementary methods. I was also able to reconstruct mitochondrial genomes from multiple megafauna simultaneously solely from sediment, demonstrating the information potential of sedaDNA. / Dissertation / Doctor of Philosophy (PhD) / A new addition to the rapidly growing field of palaeogenetics is environmental DNA (eDNA) with its immense wealth of biomolecules preserved over millennia outside of biological tissues. Organisms are constantly shedding cells, and while most of this DNA is metabolized or otherwise degraded, some small fraction is preserved through sedimentary mineral-binding. I experimentally developed new ancient eDNA methods for recovery, isolation, and analysis to maximize our access to these biomolecules and demonstrate that this novel approach outperforms alternative protocols. Thereafter, I used these methods to extract DNA from ice age permafrost samples dating between 30,000–6,000 years before present. These data demonstrate the power of ancient eDNA for reconstructing ecosystem change through time, as well as identifying evidence for the Holocene survival of caballine horse and woolly mammoth in continental North America. This late persistence of Pleistocene fauna has implications for understanding the human ecological and climatological factors involved in the Late Pleistocene mass extinction event. This effort is paralleled with megafaunal mitogenomic assembly and phylogenetics solely from sediment. This thesis demonstrates that environmental DNA can significantly augment macro-scale buried records in palaeoecology.

<p>Continuous monitoring of pathogens that may be present in water is one of the key preventive measures that can be used in rural areas of developed countries and developing countries to reduce chances of the water borne diseases outbreak. Off-site testing of microbiological contamination of water is conventionally done for monitoring water quality. However, such a process is time consuming and involves using a variety of hazardous reagents. To address these issues, a portable device for rapid detection of unsafe water is needed.</p> <p>One of the key components in this system is to extract DNA from the pathogens. The primary consideration for DNA extraction is to separate DNA from proteins and other cell debris in the lysate solution. The pure population of DNA molecules are then sent downstream for subsequent processing such as real-time PCR (Polymerase Chain Reaction) and BioFET sensors for further identification and analysis.</p> <p>The focus of the thesis will be on the fabrication of a microfluidic DNA extraction system that can achieve high DNA extraction efficiency and a good repeatability. It can also be easily automated, and integrated with other components of the DNA analysis system. The high surface-to-volume macro/mesoporous silica DNA binding column was synthesized using sol-gel silica technology and triblock copolymer F127 was added to form a crack-free mesoporous silica network. Furthermore, a monodispersed polystyrene microspheres soft-template was assembled using a simple but novel technique that employs controlled suction to enhance self-assembly into a periodically patterned structure in the extraction chamber/chambers. In combination of heat annealing treatment of this assembled polystyrene template, one can easily control the size of the macropores in the final macro/mesoporous silica structure to allow a lower pressure resistance for DNA sample flow at elution stage. The final macro/mesoporous silica structure synthesized using heat annealing temperature of 115^oC for 10 minutes was determined to have a porosity of 83.6%. Mesopores of this silica monolith was determined by BET test to be 3.65 nm and the macroporous ranging from 0.5μm to 0.86μm were observed. In addition, the fabrication of porous silica monolith can be easily integrated with the microfluidic system for achieving DNA extraction purposes</p> / Master of Applied Science (MASc)

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.

No description available.