Дисертації з теми "Faecal DNA"

Johne’s disease, caused by Mycobacteria avium subsp. paratuberculosis (MAP), plagues cattle and dairy farmers worldwide. Infected animals suffer from chronic granulomatous enteritis, reduced fertility, decline in milk production and emaciation. It is spread through colostrum, milk and faeces. Johne’s disease has also become strongly associated with human Crohn’s disease due to the similarity in symptoms and the presence of MAP in samples taken from Crohn’s disease patients. Currently there is not cure for Johne’s disease, therefore, routine testing and isolation or culling of diseased animals are measures used to prevent infection. At present, the gold standard test for MAP is by faecal culture, which can take up to 4 months to reach a diagnosis. The low-cost ELISA test has a shorter diagnosis duration, however, it uses blood or milk samples for testing which do not correlate with the bacterial shedding in faecal samples. As a result of this mismatch in bacterial shedding between blood, milk and faeces, ELISA testing lacks in both sensitivity and specificity to MAP when compared to faecal culture and PCR tests. PCR can be sensitive and specific for MAP testing, although it is currently the most expensive test for MAP detection within the UK. Reducing the cost of PCR testing was one of the motivating factors during the development of this assay and device. In this thesis, surface acoustic waves (SAW) have been used to create, develop and test a diagnostic device and the assay for Johne’s disease. SAW is becoming an increasingly popular tool in the field of diagnostic devices due to its multi-functionality which allows for many pieces of laboratory equipment to be consolidated into one device. In using SAW there was no longer a need for laboratory equipment usually used to perform a DNA extraction because SAW could be used to mix, heat and move the sample droplet. Traditional, laboratory-based faecal DNA extraction was adapted for use with SAW. To date, faecal DNA extraction using SAW has not been published. The SAW-driven, droplet-based assay developed during this project used 90% less DNA extraction reagents than the traditional tube-style method. Due to the thermal resistant nature of MAP it is particularly difficult to lyse during DNA extraction. The newly created SAW faecal DNA extraction was used to retrieve and clean DNA sufficiently for successful PCR to follow. In the context of faecal samples, this was particularly challenging due to the PCR inhibitors found in these complex samples. To investigate the effectiveness of the SAW DNA extraction, K-10 strain of genomic MAP DNA, MAP cell cultures and pre-tested bovine faecal samples were tested to prepare the MAP DNA for amplification and detection. DNA extraction was followed by SAW PCR. The sensitivity and specificity of the SAW PCR was ensured by using both IS900 and F57 target sequences. IS900 is repeated 17 times in the MAP genome thereby providing sensitivity down to 102 CFU/g. F57 is only repeated once therefore providing specificity. Specificity of the assay was further improved by using TaqMan probes to quantify the PCR. In order to keep this PCR-based assay stable in the absence of cold-chain storage, the disaccharide sugar trehalose was added to the PCR reagents and freeze dried to determine its ability to maintain performance. These experiments enhanced the future portability of this assay. It was found that reagents maintained activity for at least 41 days after freeze drying and this duration is expected to be extended. During this thesis, a newly developed acoustically driven DNA extraction and qPCR was integrated onto one device which had the capability of detecting as few as 5 MAP genomes. This novel proof-of-concept research, lays the foundation for an acoustically driven portable device for use on faecal samples and in this case, for the detection of Johne’s disease.

Current studies of koalas in the wild mainly rely on information gathered by traditional field methods, such as community sightings, spotlighting, radiotracking, animal trappings, ear tagging and faecal pellet incidence. Collection of faeces is potentially the most reliable source of non-invasively obtaining DNA samples, which can be used to identify specific individuals. This thesis demonstrated a simple, rapid and reproducible method of extracting DNA from Koala faecal pellets using a commercially available DNA extraction kit, shows the maximum age of pellets from which DNA can be reliably extracted and defines the conditions required for the long term storage of pellets before DNA extraction is carried out. Mitochondrial DNA PCR analysis provided a simple and rapid indication of the success of both the faecal DNA extraction and pellet collection process. The faecal DNA was successfully used for microsatellite analysis and the subsequent genetic profiling of individuals from within the Campbelltown Koala population. The study paves the way for the analysis of microsatellite loci in koala faecal pellet DAN to study populations, which are too sparsely distributed to allow the capture of individual koalas
Master of Science (M. Sc.) (Hons.)

Abstract

Gaining accurate population information is crucial for the conservation and management of species. The National Monitoring Program for Large Carnivores monitors the Swedish lynx population (species Lynx lynx) by surveying family groups, non-invasive sampling and genetic analysis. Ten microsatellite regions were used as genetic markers to retrieve unique individual genotypes, through polymerase chain reactions (PCR) with specific primer-pairs and capillary-electrophoresis. Complete genotypes were matched using an internal database. The aim of this degree project was to show how monitoring of lynx through genetic analysis is carried out at the Department of Evolutionary Biology at Uppsala University, and to evaluate how effective these methods are and how they might be improved.

Even though most of the methods used were fairly robust and reproducible, non-invasive sampling and microsatellite analysis posed some problems regarding DNA quality and quantity, and increased the risks of certain genotyping errors. These risks might be worth taking though, as genetic analysis, in combination with field observations, gives a more comprehensive picture of the Swedish lynx population.

O gênero Mazama é composto por cinco espécies no Brasil. São animais de visualização dificultada por causa de comportamentos evasivos, o que torna as capturas e os estudos comportamentais quase impossíveis. Assim, o uso de metodologias não invasivas para estudos ecológicos e genéticos destas espécies se torna necessário. A análise do DNA fecal está dentro das técnicas mais promissoras para esse fim. Este estudo objetivou genotipar amostras fecais, de uma população de veado-mateiro (Mazama americana) para a obtenção de informações genéticas e ecológicas. Para tanto, foram coletadas, com auxílio de um cão farejador, georreferenciadas e estocadas em etanol absoluto, 52 amostras fecais de cervídeos. A coleta realizou-se em um fragmento (21o20S 47o17W) de 600 ha de floresta estacional semidecidual. Dessas amostras coletadas, 31% (n=16) foram classificadas como frescas e 69% (n=36) como não frescas. O DNA foi extraído em torno de 30 dias após a coleta, usando o kit comercial QIAamp® DNA Stool Mini Kit, seguindo o protocolo do fabricante. Das 52 amostras, 45 foram identificadas por PCR/RFLP como pertencentes a M. americana e as demais apresentaram problemas de amplificação e digestão, permanecendo sem identificação. Amplificaram-se por PCR cinco locos microssatélites, e o sucesso de amplificação, visualizado em gel de agarose, variou com o tamanho dos locos e com a classe das amostras. O sucesso de amplificação foi de 65% das amostras da categoria fresca e 35% das amostras da categoria não fresca. Encontrou-se uma correlação negativa (R= -0,82) entre o tamanho dos fragmentos dos locos de microssatélites e o sucesso de amplificação. Foi possível identificar o sexo do animal em 43,7% das amostras fecais, pela amplificação do gene da amelogenina. Os locos microssatélites amplificados foram analisados em sequenciador automático. Os eletroferogramas gerados pelo seqüenciador impossibilitaram a genotipagem da maioria dos locos e amostras, tornando inviável qualquer análise genética e ecológica com confiabilidade. Fica evidente a dificuldade de se trabalhar com a metodologia do DNA fecal para a identificação individual e sexagem de amostras obtidas de Cervídeos florestais em vida livre. Algumas melhorias metodológicas (coleta de amostras fecais frescas, seleção de iniciadores para locos menores e quantificação do DNA extraído por PCR em tempo real) são sugeridas para o aumento nos índices de sucesso na genotipgem em estudos futuros.
Mazama genus is composed by five species in Brazil. All of them are difficult to observe due to their evasive behaviors, what makes the captures and behavioral studies almost impossible. Thus, the use of non invasive methodologies is necessary to study the ecology and genetics of these species. The fecal DNA analysis is one of the most promising techniques for this purpose. This study aimed to genotype a Mazama americana population faecal samples for obtaining genetics and ecological information. For this, 52 deer faecal samples were collected in a 600ha seasonal semideciduos forest fragment (21o20S 47o17W), with the help of a detection dog, stored in ethanol and georeferenced. Of these samples 31% (n=16) was classified as fresh and 69% (n=36) as not fresh. About thirty days after the collection the DNA was extracted using the QIAamp® DNA Stool Mini Kit following the manufacturers instructions. From the 52 samples collected and extracted, 45 were identified by PCR/RFLP as M. americana and the others showed amplification and digestion problems, remaining without identification. Five microsatellite loci were amplified by PCR and the amplification success, visualized in agarose gel, varied with the loco size and age class. The amplifications success occurred in 65% of the fresh samples and in 35% of the non-fresh samples and a negative correlation (R= -0.82) was found between amplification success and loci sizes. It was possible to identify the animal sex in 43% of the samples by the amelogenin gene. The microsatellite loci amplifications were analyzed in an automatic sequencer. The majority of the samples and loci were impossible to genotype because of the quality of the elestroferograms, what made impossible any reliable genetic and ecological analysis. It is evident the difficulty to work with the faecal DNA methodology using field collected forests deer samples for individual and sexual identifications. Some methodological improvements (collect fresh samples, select primers for shorter loci and quantify the extracted DNA by real time PCR) are suggested to increase the genotyping success indexes in future studies

O veado-mão-curta (Mazama nana) é uma espécie de cervídeo que ocupa a região Sul do Brasil, norte da Argentina e leste do Paraguai, tendo sido severamente afetada pela redução drástica das áreas florestadas. Trata-se, também, da espécie de cervídeo neotropical menos estudada pela ciência. Frente a essa situação, o presente projeto propôs-se a entender como essa espécie se distribui em sua área de ocorrência, propôs áreas prioritárias para sua conservação e estimou a densidade de duas populações. Dada a raridade e a alta elusividade da espécie, propôs-se o uso de metodologias indiretas para se atingir o objetivo proposto. Assim, foram usadas metodologias baseadas na coleta de amostras fecais, extração do DNA e posterior análises molecular e genética. Foram coletadas amostras fecais com o auxílio de um cão farejador em unidades de conservação distribuídas ao longo do Sul do Brasil. Após a identificação da espécie por meio da amplificação de um fragmento do citocromo B e corte com enzimas de restrição (PCR/RFLP) e com os dados de localização das amostras, foram feitas modelagens de distribuição com o software Maxent. Foram escolhidas duas unidades de conservação, onde foram realizadas coletas de amostras fecais, baseadas em faixas, para possibilitar a estimativa da densidade de animais nestas populações. Estabeleceu-se a distribuição geográfica potencial de M. nana no Brasil para os Estados do Paraná, Santa Catarina, norte e centro do Rio Grande do Sul, extremo sul de São Paulo e Mato Grosso do Sul. Porção leste do Paraguai e, na Argentina para a província de Missiones. A densidade da espécie para a região norte do Parque Nacional do Iguaçu foi de 1,9 ind/km2 e para o Parque Estadual Vila Rica do Espírito Santo foi de 5,5 ind/km2. A população potencial da espécie foi de 152.991 indivíduos, sendo 15.524 indivíduos a população dentro das áreas protegidas. Sugere-se a manutenção do estado de conservação da espécie como Vulnerável, tanto na lista Brasileira como na lista Internacional de fauna ameaçada de extinção.
The Brazilian dwarf brocket (Mazama nana) is a deer species that occupies the forests of southern Brazil, north of Argentina and east of Paraguay. It has been greatly affected by the drastic reduction of forested areas. It is also the less studied neotropical deer. Considering this situation, this project aimed to shed light on the species distribution along its range, to indicate conservation priority areas and estimate the density of two populations. Given the rarity and high elusiveness of the species, it is proposed the use of indirect methods to achieve this goal. Fecal samples collection based methodologies were used, followed by DNA extraction and subsequent molecular and genetic analysis. Fecal samples were tracked and collected in protected areas spread over south Brazil, with the help of a scat detection dog. After species identification by PCR/RFLP and samples spatialization, species distribution modeling was carried out using Maxent software suit. Two protected areas were chosen for a faecal sampling based on transects, in order to estimate the population density. Potential geographical distribution of M. nana in Brazil was stablished at states of Paraná, Santa Catarina, northern and center Rio Grande do Sul, extreme South of São Paulo and Mato Grosso do Sul. Also at Eastern Paraguay and Missiones province in Argentina. Species density at the northern area of Iguaçu National Park was 1.9 ind/km2 and at the State Park of Vila Rica do Espírito Santo was 5.5 ind/km2. The species potential population was 152,991 individuals, including 15,524 individuals inside protected areas. It is suggested to maintain species conservation status as vulnerable on the Brazilian and on the International red list of threatened species.

The field of conservation genetics, in combination with non-invasive sampling, provides a powerful set of tools for investigating the conservation status and natural history of rare species that are otherwise difficult to study. A systematic literature review demonstrated that this is certainly the case for many forest associated antelope species, which are poorly studied and yet constitute some of the most heavily hunted wildlife in Africa. The aim of the present study was to use non-invasive sampling to investigate genetic patterns in forest antelope populations in the high-biodiversity Udzungwa Mountains, Tanzania, within the context of the conservation of these species and the wider ecosystem. Genetic information was derived from faecal samples collected across the Udzungwa landscape and assigned to five antelope species (N = 618, collected 2006-09). Faecal pellet length was measured for a subset of samples but statistical assignment to species by this method proved unreliable. Phylogenetic analysis using mitochondrial control region sequences unexpectedly revealed that Harvey’s duiker within the Udzungwas are paraphyletic with respect to sequences from a putative sister species from southern Africa. However, there was no corresponding pattern in the microsatellite dataset suggesting that these mitochondrial lineages do not represent contemporary genetic isolation. Instead, Harvey’s duiker nuclear variation is shaped both by isolation by distance, due to positive spatial autocorrelation at short distances, and clustering of distinct genotypes from western outlying forests. These forests also harbour the endangered Abbott’s duiker and therefore require effective conservation management. Despite being detected throughout the Udzungwas, genetic diversity in Abbott’s duiker was very low in comparison to other species. These results suggest several promising research directions but also have significant conservation implications that will be disseminated to the Tanzanian wildlife authorities and the wider conservation community.

Recent metagenomic studies have shown that there is a higher diversity of ssDNA viruses in the environment than previously thought. While some viral families are well characterised, novel ssDNA isolates discovered with sequence-independent molecular techniques are often too divergent to fit within the currently established viral taxonomy. Several factors have contributed to the gap in knowledge, including: the (previously) high cost of sequencing, the disproportionate amount of research that occurs after a threat is identified, and the use of sequence-based molecular techniques to isolate viral sequences. Recent studies have begun to explore viral diversity in the environment, however, most of these studies have occurred outside New Zealand. Several benefits would come from uncovering the true ssDNA viral diversity and global distribution including improving the resolution of the current taxonomic structure for identifying unknown isolates and inferring possible virus-host relationships, and providing baseline data for the development of disease prevention and monitoring strategies. Studies specific to the New Zealand environment are essential. With its geographical isolation and Gondwana ancestry, New Zealand will possess a unique viral sequence space. Studies on local viral diversity and the spread of ssDNA viruses are going to be most relevant if they are conducted within the established ecosystems in New Zealand. In this dissertation, a novel protocol was developed for exploring viral diversity in the New Zealand environment using basic molecular techniques and animal faecal samples. Design considerations included: identifying highly novel small circular viral sequences with DNA genomes without the use of specific primers, inflicting as little environmental impact as possible, and keeping the cost low. The faecal sampling approach does not require animal handling and therefore incorporates the use of viral reservoirs while remaining non-invasive. The molecular techniques in this protocol used non-specific rolling circle amplification (RCA) followed by restriction enzyme (RE) digests, cloning, and sequencing of the cloned genomes via sanger sequencing. This inexpensive exploratory method provided preliminary sequence information from which primers were designed for recovery of full viral genomes. The success of this protocol was demonstrated by the recovery and molecular characterisation of a novel ssDNA virus isolate from a pig faecal sample, which was tentatively named porcine stool-associated circular virus (PoSCV). This protocol was then applied to sample viruses in the faecal matter from variety of domesticated, wild, and farmed animals in New Zealand. The faecal samples were collected from the North and South Island of New Zealand as well as South East Island of the Chatham Islands (Rangatira). Several putative gemycircularviral isolates (novel viruses with similarities to geminiviruses and the recently discovered ssDNA virus infecting Sclerotinia sclerotiorum) were identified in the sequencing results based on BLASTx similarities to viral sequences available in public databases (GenBank). The full genomes of these isolates were recovered and characterised. Identification was based on genome organization, phylogenetic analysis of the replication associated protein (Rep), and full genome nucleotide pairwise identities. Fourteen novel ssDNA virus sequences relating to gemycircularviruses were discovered, of which ten were representative of new species (FaSCV-1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) and three were identified as strains of the same species (FasGCV-1). Two additional isolates were discovered to be distantly related to these viruses: Ostrich faecal associated ssDNA virus (OfaV) and Rabbit faecal associated ssDNA virus (RfaV). Additionally, this protocol was used to recover novel ssDNA viruses from the nesting material of a dead Yellow-crowned Parakeet chick found in the Poulter Valley in the South Island of New Zealand. The nesting material likely contained faecal matter and thus represented another approach strategy for exploring ssDNA viruses in the environment. Two novel ssDNA isolates were discovered and molecularly characterised: Cyanoramphus nest-associated circular X virus (CynNCXV), and Cyanoramphus nest-associated circular K virus CynNCKV.

A ampliação do conhecimento sobre as formas de comunicação, controle e regulação existentes em bactérias traz luz aos avanços no combate das infecções hospitalares que são responsáveis por inúmeros prejuízos relacionados à saúde pública em todo planeta. DUMOULIN et al (2013), descreveram o regulador transcricional (RT) ElrR, que regula positivamente a transcrição do gene elrA, um fator de virulência de Enterococcus faecalis. ElrA apresenta grande similaridade com as internalinas de Listeria monocytogenes, que facilitam a invasão da bactéria ao hospedeiro. ElrR é considerada como pertencente à família Rgg-like de RT exclusivo de bactérias Gram positivas. Por vários motivos a família Rgg foi inserida à superfamília RNPP, gerando a superfamília RRNPP de RT. Os RRNPP fazem parte de um sistema de regulação por quorum sensing (QS), um sistema de comunicação célula-célula dependente de densidade celular, com função associada na ativação ou inibição da expressão de proteínas relacionadas, dentre outros, à virulência, formação de biofilme e esporulação. Para a melhor compreensão do mecanismo de como ocorre a ativação da transcrição do fator de virulência ElrA, este trabalho apresenta resultados de expressão heteróloga em E. coli e purificação das proteínas ElrR e ElrA, bem como resultados de experimentos biofísicos que caracterizam algumas propriedades estruturais e biológicas destas proteínas. Utilizando técnicas de cromatografia, espectroscopia de dicroísmo circular (CD), anisotropia de fluorescência, espalhamento dinâmico de luz (DLS), cristalografia de raios X e ressonância plasmônica de superfície (SPR), foi possível a obtenção da estrutura tridimensional de ElrR e de indícios da interação com uma região de 25bp do DNA. Realizou-se ainda, em colaboração com Dra. Pascale Serror, a tentativa de obtenção da molécula autoindutora (AI) de ElrR. São apresentados primeiros resultados da obtenção heteróloga de ElrA, sua purificação e cristalização, com importantes características que permitirão a continuação da investigação deste fator de virulência. ElrR é composta somente por alfa-hélices e apresenta-se dimérico em solução. Apesar da similaridade estrutural dos RRNPP, a identidade da sequência entre ElrR e os outros membros é extremamente baixa, o que motivou a resolução das fases cristalográficas experimentalmente. A estrutura de ElrR apresenta-se similar às homólogas, porém, com maior interface de interação entre os protômeros, que formam o dímero. O sítio de ligação do AI, em ElrR, apresenta-se mais amplo, com cavidade maior que as demais estruturas estudadas, conservando vários dos resíduos apresentados nos homólogos que realizam a estabilização do AI. Os altos fatores de temperatura dos cristais de ElrR, adicionado a anisotropia dos átomos, de uma das estruturas obtidas, apresenta a grande flexibilidade desse RT. Os indícios de interação entre ElrR e DNA aqui apresentados, obtidos por SPR e anisotropia de fluorescência, apresentam que ElrR liga especificamente ao fragmento proposto do DNA, ainda na ausência do AI. A não cristalização do complexo (ElrR-DNA), adicionada a alta flexibilidade apresentada na estrutura e a instabilidade observada na ligação ao DNA (por SPR) apontam para a obrigatoriedade da molécula de regulação (AI) para que o complexo ElrR-DNA seja estável.
The enhancing of the knowledge about communication, control and regulation in bacteria bring possibilities on the advance of hospital-acquired infections control responsible for various prejudices related to public health worldwide. DUMOULIN, et al (2013) described ElrR, a transcriptional regulator (TR), that positively regulates transcription of the elrA gene, which codifies a virulence factor of Enterococcus faecalis. ElrA shows high similarity with Listeria monocytogeneses internalins, which facilitates host invasion by these bacteria. ElrR are considered belonging to Rgg-like TR family exclusive of Gram positive bacteria. Several reasons include the Rgg family into the RNPP superfamily, generating the RRNPP superfamily of TR. The RRNPP are controlled by a quorum sensing (QS) regulation system, a cell-cell communication system based on cellular density that activates or inhibits the expression of proteins related with virulence, biofilm formation, sporulation, and others. For a better understanding of the transcription activation mechanism of ElrA, this work shows ElrR and ElrA heterologous expression in E. coli and purification of these proteins, as well as biophysics assays to characterize some structural and biological features of both proteins. Using chromatography, circular dichroism (CD), fluorescence anisotropy, dynamic light scattering (DLS), X-ray crystallography and surface plasmon resonance (SPR) technics, it was possible to obtain the tridimensional structure of ElrR, and evidences of ElrR-DNA complex formation, confirming DNA interaction site of ElrR with a 25 bp fragment. In collaboration with Dr. Pascale Serror, we attempted to achieve the ElrR auto-induction (AI) molecule. Also, results of the heterologous obtainment of ElrA are presented, as well as ElrA purification and crystallization, presenting important characteristics which will allow the further investigation of this virulence factor in near future. ElrR is composed by alpha-helices presenting dimeric fold in solution. Despite the similarity between the RRNPP members, the low identity of ElrR to the other members motivates the experimental crystallographic phases solution. ElrR structure is very similar to the homologous structures, presenting a higher interface between the protomers that compose the dimer. Its AI binding site is wider than the other structures studied, conserving several amino acid residues presented at the homologous proteins, that stabilizes the AI molecule. High temperature factors of the amino acid residues showed in all the obtained ElrR crystallographic structures plus the anisotropy of the atoms in one of those structures show the high flexibility of this TR. The evidence of the ElrR-DNA complex presented in this study, obtained by SPR and fluorescence anisotropy, indicates that ElrR binds at the proposed DNA site even in the absence of the AI molecule. The failure to obtain the ElrR-DNA complex crystals added to the high flexibility presented at some places of the structure and the observed instability at the formed complex (observed at SPR) suggest the mandatory need of the AI molecule to create a stable ElrR-DNA complex.

Syftet med litteraturstudien är att sammanfatta vilken gensekvens som används vid genetisk barcoding av växter och hur väl metoden i fråga tillämpas i tre biologiska yrkesområden: dietanalyser i ekologin, analys av pollensporer i forensisk biologi samt analys av uråldrigt DNA (ancient DNA) i paleontologin. Vidare var det även av intresse att se hur genetisk barcoding kan användas i den gymnasiala undervisningen och hur väl den passar in med de svenska styrdokumenten för skolan. Hur elever har gynnats av den valda metoden samt vilka begränsningar som har uppstått har också berörts. Litteraturstudien baseras på vetenskapliga artiklar som har sökts fram med de nedan listade nyckelorden. Resultaten visar att en kombination av gensekvenser, däribland rbcL, matK, trnH-psbA och ITS, fungerar bäst vid identifiering av växter. I dagsläget är genetisk barcoding fortfarande i utvecklingsfasen, där metoden begränsas av antalet referenssekvenser i databaserna, vilket gör det svårt att utesluta morfologiska identifieringsmetoder i de tre yrkesområdena. Vid användning av barcoding i den gymnasiala undervisningen visar det sig att det stämmer väl överens med de svenska styrdokumenten och ökar elevers intresse för de naturvetenskapliga ämnena, då de kan bidra med värdefull forskning genom tillägg av referenssekvenser i databaserna. De största begränsningarna är att det blir ett stort arbetslass för läraren, att läraren i fråga måste vara bekväm med de olika laboratiska momenten och att skolan ska ha tillgång till nödvändig apparatur.
The purpose of the literature study is to conclude which gene sequences are being used in DNA barcoding on plants and how the method in question is being used in three different biological occupations: diet analysis in ecology, analysis of pollen in forensics and analysis of ancient DNA (aDNA) in paleontology. Further it was also of interest to study how DNA barcoding can be used in high school settings and how the method correlates with the Swedish curriculum. How pupils have benefited from the chosen method and what limitations have arisen have also been touched upon. This literature study is based on scientific articles that have been sought with the keywords listed below. The results show that a combination of gene sequences, including rbcL, matK, trnH-psbA and ITS, works best in plant identification. At present, genetic barcoding is still in the developmental phase, where the method is limited by the number of reference sequences in the databases, which makes it difficult to exclude morphological-based methods in the three occupational fields. When using barcoding in upper secondary education it turns out that it’s in good agreement with the Swedish curriculum and increases the students' interest in the scientific subjects, since they can contribute with genuine research when adding reference sequences in the databases. The main limitations are the workload for the teacher, the teacher in question must be comfortable with the different laboratory steps and that the school must have access to necessary equipment.

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Sequ?ncias de DNA usadas na identifica??o de material biol?gico t?m alcan?ado consider?vel popularidade nos ?ltimos anos, especialmente no contexto dos c?digos de barras de DNA. Aferir a esp?cie de origem em amostras de pelos, penas, peles e particularmente fezes ? um passo fundamental para quem estuda a ecologia e evolu??o de diversos animais com este tipo de amostra. Este ? o caso em carn?voros, cujos h?bitos furtivos e baixas densidades populacionais de algumas esp?cies evidenciam a import?ncia de estudos baseados em amostras n?o-invasivas. Entretanto a atual escassez de ensaios padronizados de identifica??o de carn?voros freq?entemente dificulta a aplica??o dessas amostras em larga escala e compara??es de resultados entre diferentes localidades. No presente estudo n?s avaliamos dois segmentos curtos (<250 pb) de DNA mitochondrial (mtDNA) localizados nos genes ATP sintase 6 e citocromo oxidase I com potencial de servirem como marcadores-padr?o para identifica??o de carn?voros. Entre um e 11 indiv?duos de 66 esp?cies de carn?voros foram seq?enciados para um ou ambos os segmentos do mtDNA e analisados usando tr?s diferentes m?todos (?rvore de dist?ncia, dist?ncia gen?tica e an?lise de caracteres). Em geral, indiv?duos conspec?ficos apresentaram menor dist?ncia gen?tica entre si do que em rela??o a outras esp?cies, formando agrupamentos monofil?ticos. Exce??es foram algumas esp?cies que divergiram recentemente, algumas das quais ainda puderam ser identificadas pelo m?todo de caracteres, hapl?tipos esp?cie-espec?ficos, ou reduzindo a abrang?ncia geogr?fica das compara??es (restringindo a an?lise a uma regi?o zoogeogr?fica). An?lises in silico, usando um segmento curto do citocromo b freq?entemente empregado em carn?voros, tamb?m foram realizadas para comparar o desempenho deste segmento em rela??o aos outros dois propostos. N?s ent?o testamos o desempenho destes segmentos na identifica??o de fezes de carn?voros por meio de tr?s estudos de caso: (i) fezes de felinos de zool?gico, objetivando-se verificar o potencial de contamina??o das seq?encias com DNA da presa (coelho); (ii) fezes coletadas no Cerrado brasileiro contendo restos de presas (p?los, ossos, penas), supostamente proveniente de lobo-guar?, objetivando-se investigar a efici?ncia de identifica??o do predador e ocorr?ncia de interfer?ncia do DNA da presa na identifica??o; e (iii) fezes coletadas em uma reserva na Mata Atl?ntica, tamb?m com o objetivo de avaliar a efici?ncia de identifica??o. Apesar de diferen?as em alguns aspectos de sua performance, nossos resultados indicam que os dois segmentos propostos t?m um bom potencial de servir como marcadores moleculares eficientes para identifica??o acurada de amostras de carn?voros ao n?vel de esp?cie.
DNA sequences for species-level identification of biological materials have achieved considerable popularity in the last few years, especially in the context of the DNA barcoding initiative. Species assignment of biological samples such as hairs, feathers, pelts and particularly faeces is a crucial step for those interested in studying ecology and evolution of many species with these samples. This is especially the case for carnivores, whose elusive habits and low densities highlight the importance of studies based on noninvasive samples. However, the current lack of standardized assays for carnivore identification often poses challenges to the large-scale application of this approach, as well as the cross-comparison of results among sites. Here we evaluate the potential of two short (<250 pb) mitochondrial DNA (mtDNA) segments located within the genes ATP synthase 6 and cytochrome oxidase I as standardized markers for carnivore identification. Between one and eleven individuals of 66 carnivore species were sequenced for one or both of these mtDNA segments and analyzed using three different approaches (tree-based, distance-based and character-based), in conjunction with sequences retrieved from public databases. In most cases, conspecific individuals had lower genetic distances from each other relative to other species, resulting in diagnosable monophyletic clusters. Notable exceptions were the more recently diverged species, some of which could still be identified using diagnostic character attributes, species-specific haplotypes, or by reducing the geographic scope of the comparison (restricting the analysis to a single zoogeographic region). Additional in silico analyses using a short cytochrome b segment frequently employed in carnivore identification were also performed aiming to compare performance to that of our two focal markers. We then tested the performance of these segments in the identification of carnivore faeces via three case studies: (i) felid faeces collected in a controlled zoo experiment, aimed at assessing whether DNA from rabbit prey would contaminate the resulting sequences; (ii) field-collected faeces from the Brazilian Cerrado presumed to be from maned wolves and containing prey remains (hairs, bones, feathers), aimed at investigating the efficiency of predator identification and occurrence of prey DNA interference; and (iii) field-collected scats from an Atlantic Forest study site, also addressing the issue of PCR success rate and identification efficiency. In spite of some relevant differences in some aspects of their performance, our results indicate that both of our focal segments have a good potential to serve as efficient molecular markers for accurate species-level identification of carnivore samples.

碩士
國立中興大學
昆蟲學系所
104
The amplification of “DNA barcode” in terms of the diet studies by faeces sample has a more powerful method, no need to identify prey remains by visual in faeces and have higher identification resolution. Combining the high-throughput ability of next generation sequencing (NGS), we evaluated the efficiency of this molecular method to identify the prey in river bird’s faeces. Brown dipper (Cinclus pallasii Temminck, 1820) has an ability to dive into water for foraging, and mainly feeds on aquatic insects, and sometimes on fishes. This research investigated the diet of the dipper in the breeding season (February 2015) and non-breeding season (October 2014 and June 2015), sampling throughout the Cijawan Stream basin. After extracting DNA from faeces and running the NGS analysis, we identified sequences by BOLD (Barcode of Life Database) database to get the prey composition of brown dipper in different periods. We sampled and analyzed at certain sites in breeding season as well. No matter in breeding or non-breeding seasons, brown dipper consumed higher proportion of Ephemeroptera. However they consumed more Diptera in the non-breeding season and more Trichoptera in the breeding season. This may be the cost benefit trade-off strategy. Comparing the results of certain sites, nearly no Trichoptera’s sequences appeared, we still found that brown dipper’s adult tended to feed their nestlings large prey, consumed other prey for themselves. Result also showed that using NGS on diet study, we can use few samples to get nearly complete prey composition of brown dipper. However there’re almost half of sequences not identified by BOLD database. The need of local reference database is urgent to get more accurate prey identification, and it will be helpful for the development of relative diet studies.