京都大学 / 新制・課程博士 / 博士(農学) / 甲第23944号 / 農博第2493号 / 新制||農||1090(附属図書館) / 学位論文||R4||N5379(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 德地 直子, 教授 北山 兼弘, 教授 神﨑 護, 准教授 伊勢 武史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Thesis (PhD (Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Consumers have the right to accurate information on the fish products they purchase to enable them to make educated seafood selections that will not endanger their own wellbeing or the wellbeing of the environment. Unfortunately, marine resource scarcity, financial incentives and inadequate or poorly enforced regulations have all promoted the mislabelling of fish species on global markets, the results of which may hold economic, conservation and health consequences. The primary aims of this study were to determine the most commonly available fish species on the South African market, to establish and compare DNA-based methods for the unambiguous identification of these species and to utilise the most applicable methods to evaluate the extent of mislabelling on the local fisheries market. The results from surveys of n = 215 restaurants and n = 200 retail outlets in four South African provinces (Western Cape, Kwa-Zulu Natal, Eastern Cape and Gauteng) indicated that 34 and 70 nominal fish types were available in restaurants and retail outlets, respectively, the most common of which were kingklip, salmon and hake. Over 30% of the fish species being sold were of conservation concern, while several outlets marketed specially-protected, illegal-to-sell species in South Africa. Fish purveyors were poorly equipped to provide information on the identity, origin, production method (farmed/wild) and sustainability of the fish they were selling and the labelling of many packaged fish products was in contravention with South African regulations. Data were published for the first time comparing the efficiency of five methods (urea-SDS-proteinase K, phenol-chloroform, salt extraction, SureFood PREP kit and Wizard Genomic DNA Purification kit) for the extraction of DNA from the muscle tissue of fish species available in South Africa. The SureFood kit was identified as the most suitable method for DNA extraction from fish muscle, extracting significantly (P < 0.05) higher DNA yields than all other methods evaluated and being simple and safe to use. A comprehensive reference library of genetic information was compiled for the first time that contains sufficient DNA sequence data from different mitochondrial DNA loci (16S ribosomal RNA (rRNA), 12S rRNA and cytochrome c oxidase I (COI) genes, as well as the control region) to allow the explicit identification of 53 fish species in South Africa. Although 16S and 12S rRNA gene sequencing allowed the identification of most fish to the genus level, the discrimination of closely-related, congeneric species was problematic when based on these gene regions. Conversely, the vast majority (98%) of fish examined could be readily differentiated by their COI sequences, with only members of the genus Thunnus requiring supplementary control region sequencing for species confirmation. Lastly, sequencing of the COI region was used to show that 9% of fish samples collected from local seafood wholesalers and 31% of samples from retail outlets were mislabelled. This study has established that fish mislabelling is a reality on the South African market and that DNA-based methods should be applied by both industry and regulatory bodies to deter illegal activities and to promote transparency on the domestic fisheries market. / AFRIKAANSE OPSOMMING: Verbruikers het die reg tot akkurate informasie rakende die visprodukte wat hulle aankoop. Hierdie inligting sal hulle bemagtig om ingeligte seekos keuses te maak wat voordelig sal wees vir beide die verbruiker se eie, sowel as die omgewing, se voortbestaan. Ongelukkig het 'n gebrek aan seelewebronne, geldelike aansporings en onvanpaste of swak geïmplimenteerde regulasies gelei tot die verkeerde etikettering van visspesies op die wêreldmarkte. Dit mag ekonomiese-, bewarings- en gesondheidsgevolge inhou. Die primêre doelwitte van hierdie studie was om te bepaal watter visspesies die algemeenste beskikbaar is in die Suid-Afrikaanse mark, om DNS-gebaseerde metodes vir die duidelike identifisering van hierdie spesies te vind en te vergelyk, en om die mees gepaste metodes te gebruik om die omvang van verkeerde etikettering in die plaaslike vismarkte te evalueer. Die resultate van opnames van n = 215 restaurante en n = 200 winkels in vier Suid-Afrikaanse provinsies (Wes-Kaap, Kwa-Zulu Natal, Oos-Kaap en Gauteng) het gewys dat 34 en 70 nominale visspesies in onderskeidelik restaurante en kleinhandelaars beskikbaar was. Koningklip, salm en stokvis was die mees algemene spesies. Meer as 30% van die visspesies wat te koop was is van bewaringsbelang, terwyl verskeie winkels spesiaal-beskermde, onwettig-om-te-verkoop spesies in Suid-Afrika bemark het. Visverkopers was swak bemagtig om informasie oor die identiteit, oorsprong, produksiemetode (teel/wild) en volhoubaarheid van die vis wat hulle verkoop het te kon gee. Verder was die etikettering van baie verpakte visprodukte in stryd met Suid-Afrikaanse regulasies. Vir die eerste keer is data gepubliseer wat vyf metodes (ureum-SDS-proteïenase K, fenolchloroform, sout-ekstraksie, SureFood PREP stel en Wizard Genomic DNS suiwering stel) vergelyk in hul doeltreffendheid om DNS vanuit die spierweefsel van visspesies wat in Suid-Afrika beskikbaar is te ekstraheer. Die SureFood stel is as die mees geskikte metode vir DNS ekstraksie vanuit visweefsel geïdentifiseer aangesien die DNS opbrengs betekenisvol (P < 0.05) hoër was met hierdie metode, en dit ook 'n eenvoudige en veilige metode is om te gebruik. 'n Omvattende verwysingsbiblioteek van genetiese informasie wat voldoende DNS volgordebepalingsdata van verskillende mitokondriale DNS lokusse (16S ribosomale RNS (rRNS), 12S rRNS en sitochroom c oksidase I (COI) gene, sowel as die kontrolegebiede) bevat, is vir die eerste keer opgestel om die besliste identifisering van 53 visspesies in Suid-Afrika toe te laat. Alhoewel 16S en 12S rRNS geenvolgordebepaling die identifisering van meeste visse op genusvlak toegelaat het, was die diskriminasie van naby-verwante, gelyksoorting spesies problematies wanneer hierdie geengebiede gebruik is. Die oorgrote meerderheid (98%) vis wat ondersoek is geredelik onderskei op grond van hul COI volgordebepalings, met slegs lede van die genus Thunnus wat addisionele kontrolegebied volgordebepaling vir spesies bevestiging vereis het. Laastens, is volgordebepaling van die COI-gebied gebruik om te wys dat 9% van die vismonsters van plaaslike seekosgroothandelaars en 31% van die monsters van kleinhandelaars verkeerd geëtiketteer is. Hierdie studie het bevestig dat die verkeerde etikettering van vis in Suid-Afrika 'n realiteit is, en dat DNS-gebaseerde metodes gebruik moet word deur die industrie sowel as die regulerende liggame om onwettige aktiwiteite teen te werk en om deursigtigheid in plaaslike vismarkte te bevorder.

The genus Geosmithia contains 11 described and several tens of undescribed species of fungi living nearly exclusively in galleries of subcorticolous insects, especially bark beetles. In this work, a phylogenetic analysis of the genus was made using DNA sequences of four protein-coding genes, namely TEF-1, RPB2, Mcm7 and Tsr1. The analysis has confirmed that ecological strategies of these fungi (such as association with conifers or broad leaved trees or symbiosis with ambrosia beetles) have evolved several times in this genus. 51 species are recognized based on the obtained phylogenetic tree according to Genealogical Concordance Phylogenetic Species Recognition. I have also tested utility of the above mentioned genes to serve as "barcode" for identification of closely related Geosmithia species.

<p> Klauenerkrankungen sind die dritthäufigste Abgangsursache bei Milchkühen und 90 % aller Lahmheiten gehen auf verschiedene Erkrankungen der Klauen zurück. Im Rahmen des FUGATO-plus Forschungsprojektes GENE-FL wurden die genetischen Grundlagen der Fundamentstabilität beim Rind, Schwein, Pferd und Schaf untersucht. Für die Untersuchungen beim Rind wurde Probenmaterial von insgesamt 1.962 erstlaktierenden Kühen der Rasse Holstein Friesian gesammelt. Diese stammten von sieben großen Herden aus Mecklenburg-Vorpommern mit vergleichbarem Haltungssystem und TMR-Fütterung. Der Klauenstatus der Tiere wurde zum Zeitpunkt des Klauenschnitts erfasst. Als Grundlage der molekularbiologischen Untersuchungen wurden anhand von in silico Analysen positionell-funktionelle Kandidatengene mit den assoziierten biochemischen Reaktionswegen für Fundamentmerkmale beim Rind und anderen landwirtschaftlichen Nutztierspezies ausgewählt. Aus den mehr als 1.000 ermittelten Kandidatengenen wurde ein individuell gefertigter SNP-Chip (384 SNP; ein SNP/Gen) erstellt. Mit dem SNP-Chip wurden 1.183 der phänotypisierten Tiere untersucht. Die Schwellenwertmodell-Analyse ergab eine signifikante Assoziation des im IQGAP1 (BTA21) befindlichen intronischen SNP (rs29017173, A/G) mit dem Merkmal Sohlenhämorrhagien. Des Weiteren konnte dieser SNP mit Fundamentmerkmalen der klassischen linearen Exterieurbeurteilung an zusätzlichem Datenmaterial von 2.394 Besamungsbullen der Rasse Holstein Friesian assoziiert werden. </p> <p> Bei den robusten Fleischrindern White Galloway werden im Bezug auf die Fellfarbe der Tiere vier Phänotypen unterschieden. Neben den drei weißen Fellfarbschlägen, die unterteilt werden in sehr gut markiert (wsg), übermarkiert (wsü) und untermarkiert (wss), gibt es den vollkommen schwarzen Fellfarbtyp (wsch). Als bevorzugte Fellzeichnungen gelten wsg und wsü, während die unerwünschten Zeichnungen wss und wsch sind. Auch bei gezielten Anpaarungen der phänotypisch bevorzugten Tiere kommen die nicht preferierten Farbphänotypen vermehrt vor. Zur Aufklärung des genetischen Hintergrunds der verschiedenen Farbvarianten wurden zunächst vier für die Fellfarbe relevante Gene, mast/stem cell growth factor receptor (KIT, BTA6), KIT ligand (KITLG, BTA5), melanocortin 1 receptor (MC1R, BTA18) und Tyrosinase (TYR, BTA29) vergleichend sequenziert und auf kausale kodierende Sequenzvarianten untersucht. In den potentiellen Kandidatengenen konnten keine Polymorphismen detektiert werden, die die unterschiedlichen Fellzeichnungsvarianten erklären und somit wurde eine Beteiligung dieser Gene ausgeschlossen. Eine kürzlich beim Rind beschriebene KIT Gen Duplikation und Insertion auf BTA29 bzw. Re-Insertion auf BTA6 wurde deshalb mittels Fluoreszenz-in-situ-Hybridisierung (FISH), Whole genome sequencing und Polymerasekettenreaktion (PCR)-basierter Genotypisierung der Insertionsbruchpunkte an 178 White Galloway und 64 White Park Tieren untersucht. In allen Fällen konnten die Fellfarb-Phänotypen auf die Duplikation und Insertion des KIT Gens auf BTA29 zurückgeführt werden. </p> <p> Lebensmittelskandale werden in regelmäßigen Abständen aus mehreren Ländern berichtet. Im Jahr 2013 wurden Fertigprodukte mit undeklarierten Pferdefleischbeimischungen entdeckt. Eine genaue Quantifizierung der zugesetzten undeklarierten Menge war bisher nicht verlässlich möglich. Mit der droplet digitalen PCR (ddPCR) wurden im Rahmen dieser Arbeit Testsysteme zum Nachweis entwickelt. Als Zielgene wurden das mitochondriale CYTB und das chromosomale F2 verwendet. Für die Etablierung des Verfahrens wurden als Gewebe zunächst Muskel, Fett, Sehne und Leber verwendet. Während sich in diesen Geweben die Anzahl der mtDNA-Kopien pro Zelle etwa um den Faktor 5 unterschied, war der Gehalt nukleärer DNA nahezu konstant. Ausgehend von den Spezies Rind, Schwein und Pferd wurden verschiedene DNA- und Fleischmischungen hergestellt, wobei der prozentuale Anteil der beigemischten Spezies zwischen 50 % bis 0,001 % variierte. Mit dem F2-basierten Testsystem wurde eine zuverlässige Quantifizierung (LOQ) bzw. Detektion (LOD) von Beimengungen von nur 0,01 % bzw. 0,001 % erreicht. </p>

TITLE: Highschool students practical knowledge of nature subjects AUTHOR: Bc. Kamila Štěpánka Moravcová DEPARTMENT: Biology and Environmental Studies Department SUPERVISOR: PhDr. Petr Novotný, Ph.D. Abstract This text is aiming to species identification. Main target is how good or bad are students of secondary education (junior high school) in species identification. Theoretical part: Definition of basic terminilogy and detailed research and comparison (outcomes and methodics) of previous testings and questionaires of pre-school childern, primary, secondary and college students. Practical part: 1) Pre-research: Making lists of species, which would be used to test students. These lists were based on what contained students notes and what contained biology education itself. 2) Research: I was testing students and I compared outcomes with previous testings. Result is, that species identification is part of biology education, which still needs developing, because students aren't good at all in species identification. Key words: species knowledge, species identification, product of nature

The aim of this work is a literature review on the topic of the mitochondrial genome and DNA barcoding, building a dataset of mitochondrial sequences from GenBank database and creatione of a software function for extraction of individual genes that are present in the mitochondrial genome. This function was developed in Matlab. DNA barcoding is a method that uses short DNA sequence of mitochondrial genome for identification of species. There is no comprehensive work examining the appropriateness of different mitochondrial genes. This aim investigates the potential of other mitochondrial genes and evaluate their effectiveness for DNA barcoding and calculation of intra-and interspecific variability.

Many scholarly tasks involve working with contextualized fine-grain information, such as a music professor creating a multimedia lecture on a musical style, while bringing together several snippets of compositions of that style. We refer to such contextualized parts of a larger unit of information (or whole documents), as subdocuments. Current approaches to work with subdocuments involve a mix of paper-based and digital techniques. With the increase in the volume and in the heterogeneity of information sources, the management, organization, access, retrieval, as well as reuse of subdocuments becomes challenging, leading to inefficient and ineffective task execution. A digital library (DL) facilitates management, access, retrieval, and use of collections of data and metadata through services. However, most DLs do not provide infrastructure or services to support working with subdocuments. Superimposed information (SI) refers to new information that is created to reference subdocuments in existing information resources. We combine this idea of SI with traditional DL services, to define and develop a DL with SI (an SI-DL). Our research questions are centered around one main question: how can we extend the notion of a DL to include SI, in order to support scholarly tasks that involve working with subdocuments? We pursued this question from a theoretical as well as a practical/user perspective. From a theoretical perspective, we developed a formal metamodel that precisely defines the components of an SI-DL, building upon related work in DLs, SI, annotations, and hypertext. From the practical/user perspective, we developed prototype superimposed applications and conducted user studies to explore the use of SI in scholarly tasks. We developed SuperIDR, a prototype SI-DL, which enables users to mark up subimages, annotate them, and retrieve information in multiple ways, including browsing, and text- and content-based image retrieval. We explored the use of subimages and evaluated the use of SuperIDR in fish species identification, a scholarly task that involves working with subimages. Findings from the user studies and other work in our research lead to theory- and experiment-based enhancements that can guide design of digital libraries with superimposed information. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19360号 / 理博第4122号 / 新制||理||1593(附属図書館) / 32374 / 新制||理||1593 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中川 尚史, 教授 中務 真人, 教授 曽田 貞滋 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Snabb och korrekt artidentifiering är avgörande för effektiv behandling av svampinfektioner, särskilt bland immunsupprimerade patienter. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) används rutinmässigt på kliniska laboratorier för identifiering av karaktäristiska proteinmönster hos bakterier och jästsvampar genom tolkning av proteinspektra i en masspektradatabas för korrekt artidentifiering. Mögelsvamparnas hårda cellvägg och heterogena växtsätt med varierande proteinuttryck beroende på mognadsstadie, försvårar identifiering med MALDI-TOF MS. Metodens tänkbara fördelar mot traditionella metoden mikroskopering är förkortade svarstider, säkrare artidentifiering av fler arter och mindre beroende av subjektiv morfologisk bedömning. Studiens syfte var att undersöka om MALDI-TOF MS kunde anpassas och användas för identifieringen av mögelsvamp i klinisk rutindiagnostik. Fyra referensstammar (Aspergillus niger, A. fumigatus, A.terreus, A.flavus) och ett kliniskt isolat (A.terreus) undersöktes. Preparationsmetoderna (I) fullständig myrsyraextraktion, (II) direktapplicering och (III) suspension i destillerat vatten användes för analys av sporer och frontmycel hos yngre och äldre mögelkulturer. Två olika masspektradatabaser för artidentifiering jämfördes; rutindatabasen BDAL och den specialiserade mögeldatabasen Filamentous Fungi Library. Även plocktekniken av mögelmaterial inför analys med MALDI-TOF MS utvärderades. Vid vissa tillfällen förbättrades artidentifieringen efter extraktion av mögelkulturerna, medan i andra fall var direktapplicering fullt tillräcklig. Mögelmaterial med mycket sporer tenderade ge något fler artidentifieringar i BDAL oavsett kulturernas ålder.  Filamentous Fungi Library tenderade i vissa fall ge bättre resultat jämfört med BDAL för yngre kulturer. Fler studier krävs för att utvärdera och optimera MALDI-TOF MS som metod för artidentifiering av mögelsvamp. / Rapid and accurate species identification is crucial for successful treatment of fungal infections, especially among immunosuppressed patients. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used routinely at clinical laboratories to identify characteristic protein patterns of bacteria and yeast by the interpretation of protein spectra in a database for accurate species identification. The hard cell wall of the mold and the heterogeneous growth with varying protein expression due to maturation, complicates identification with MALDI-TOF MS. The potential benefits of this method compared to microscopy as traditional method are shortened turn-around times, safer species identification of more species that is independent on subjective morphological assessment. The purpose of the study was to investigate whether MALDI-TOF MS could be adapted and used for the identification of molds in clinical routine diagnostics. Four reference strains (Aspergillus niger, A.fumigatus, A.terreus, A.flavus) and a clinical isolate (A.terreus) were examined. The preparation methods (I) complete formic acid extraction, (II) direct application and (III) suspension in distilled water were used for analysis of spores and frontmycelium from younger and older mold cultures. Two different masspektradatabases for species identification were compared; routine database BDAL and the specialized mold database, Filamentous Fungi Library. Also the collecting technique of mold prior to analysis with MALDI-TOF MS was evaluated. Sometimes, the species identification improved after extraction of mold cultures, while in other cases direct application was sufficient. Cultures with a lot of spores tended to give slightly more species identifications in BDAL regardless of the age of cultures. Filamentous Fungi Library, in some cases, tended to improve the performance compared to BDAL for younger cultures. More studies are required to evaluate and optimize MALDI-TOF MS as a method of mold identification.

Species are one of the fundamental components of biology and the accurate delimitation of species is important in evolutionary, systematic and ecological studies, yet there is still confusion over how species can be recognised. Examining different characters allows multiple lines of evidence for successful and accurate species delimitation and identification. In this thesis, morphological, genetic and behavioural variation is investigated within an endemic species of ground weta, Hemiandrus pallitarsis, in the North Island, New Zealand. Twelve morphological characters were measured, and mitochondrial cytochrome oxidase I DNA sequences were analysed from populations across the distributional range of H. pallitarsis. Both methods provide no evidence of a species complex within H. pallitarsis. Instead, the morphometric results suggest females are significantly larger than males, and ground weta in Palmerston North are significantly smaller than weta further north. Additionally, genetic analyses found substantial population structuring, large genetic distances, and an historical south to north pattern of movement in the North Island. The pattern of vibratory drumming behaviour followed that predicted by morphology and geographic proximity – drumming signals were more similar between geographically close populations and did not match the patterns of genetic isolation. Overall, this thesis was able to show that H. pallitarsis is morphologically, genetically and behaviourally variable across the North Island.