Dissertations / Theses on the topic 'ITS2 rDNA'

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O gênero Cyathus Haller: Pers., junto com mais quatro gêneros, formou, por muito tempo, um grupo conhecido como “Bird’s nest fungi” devido a morfologia cônica com estrutura lenticulares em seu interior, lembrando ovos em seus ninhos. Os estudos morfológicos, durante muito tempo, forneceram a base para as diferentes construções de classificação infragenérica e, como muitas espécies se diferenciavam de forma tênue, isso gerava muita polêmica. Sequências de DNA vêm sendo usadas para esclarecer melhor a história evolutiva do grupo. No Brasil, o grupo é pouquíssimo estudado, de forma que mais trabalhos envolvendo a união da taxonomia molecular com a tradicional são necessários para uma melhor compreensão do gênero. Assim, o presente trabalho teve como objetivos inferir a filogenia de espécimes de Cyathus oriundas do nordeste brasileiro e, com a associação dos caracteres morfológicos, propor uma classificação melhor para o táxon. Para isso, foram examinadas 46 exsicatas, das quais seis eram do herbário JPB, cinco do Herbário HUEFS e 37 do herbário de Fungos UFRN, sendo que este último apresenta o maior número de exsicatas de Cyathus no Nordeste, somando um total de 19 espécies. Dos 46 espécimes, 33 foram utilizados para extração de DNA, resultando no sequenciamento da região ITS de seis espécies e da região LSU de oito espécies. As árvores filogenéticas mostram que essas espécies se encontram em clados separados das demais espécies oriundas de regiões não tropicais, no entanto, não altera a topologia geral dos principais clados sugeridos em trabalhos anteriores. De acordo com os estudos taxonômicos tradicionais, temos duas espécies que constituem segundo registro para o mundo: C. pedicelatus e C. setosus; uma primeira referencia para o Brasil de C. olivaceobrunneus, e a primeira referência para o Nordeste de C. bekerleyanus. A análise dos dados moleculares aponta para a delimitação de cinco novas espécies para a ciência. Todas as sequências de DNA geradas foram depositadas no GenBank e são novas para o banco de dados.
The genus Cyathus Haller: Pers., together with other four genera, formed a group known as bird's nest fungi due to their conic morphology with lenticular structures inside resembling bird’s eggs in their nests. Morphological studies, for a long time, provided the basis to the different constructions of infrageneric classification and, as many species differed weakly from one another, that generated much controversy. DNA sequences have been used to better clarify the evolutionary history of the group. In Brazil, the group is as yet poorly studied, hence, more work involving the union of the traditional and molecular taxonomy is needed for better understanding of the genus. Therefore, this work had the objectives of inferring the phylogeny of specimens of Cyathus from the Brazilian Northeast and, in association with morphological characters, to propose a better classification for the taxon. Forty six exsiccates were examined, being six from JPB herbarium, five from HUEFS herbarium and 37 of UFRN-Fungos herbarium. The latter has the largest number of exsiccates of this genus in the Northeast, totaling 19 species. Thirty-three specimens were chosen for DNA extraction, resulting in the sequencing of the ITS region of six species and the LSU region of eight species. The phylogenetic trees show that these species sampled in this work are positioned in clades separated from those reported from non-tropical regions, however, this does not change the general topology of the main clades suggested in the works published previously. According to the traditional taxonomy studies we have two species that constitute the second record to the world, C. pedicelatus and C. setosus; one first record to Brazil, C. olivaceobrunneus; and one first record to the Northeast region of C. bekerleyanus. Analysis of the molecular data suggests five new species to Science. All DNA sequences of the tropical species sampled were deposited in the GenBank and are new records for the database.

U radu su analizirani nukleotidni diverzitet COI mtDNK i fenotipska varijabilnost  veličinskih komponenti krila taksona roda  Cheilosia. Dobijeni podaci su korišćeni u sagledavanju filogenetskih i  evolucionih odnosa odabranih taksona. Amplificiran je i sekvenciran 3' kraj gena COI mtDNK 119 jedinki 14 vrsta roda Cheilosiasakupljenih na 8 lokaliteta Balkanskog poluostrva i u Laponiji, Finska (vrsta C. albitarsis). Analizom su bile obuhvaćene i sekvence COI mtDNK devet vrsta C. melanuragrupe i tri vrste C.  canicularis grupe preuzete iz Banke Gena. Geometrijsko morfometrijskom metodom analizirane su veličinske komponente desnog krila (oblik i veličina) 4717 jedinki 29 vrsta roda Cheilosia poreklom sa 21 područja Balkanskog poluostrva.U radu su utvrđeni diferencijalni fenotipovi veličine i oblika krila i specijes-specifični haplotipovi COI mtDNK koji su omogućili identifikaciju i razdvajanje blisko srodnih vrsta roda Cheilosia. Analizom parametara krila kod većine analiziranih taksona utvrđene su značajne razlike između konspecifičkih populacija većine analiziranih taksona, kod je jasan   polni dimorfizam u obliku krila uočen kod svih analiziranih vrsta.Usaglašeno filogenetsko stablo na osnovu sekvenci 3' COI mtDNK ukazuje na monofiletski rod Cheilosiau okviru kojeg se izdvajaju četiri jasno odvojene klade koje odgovaraju podrodovima Convocheila,  Taeniochilosia,  Eucartosyrphusi Cheilosias. str. definisanim na osnovu morfoloških karaktera tradicionalnom  taksonomijom (((Cheilosia s.  str. + Eucartosyrphus) + Taeniochilosia) + Convocheila). Unutar klade Cheilosias. str. sve analizirane vrste su formirale monofiletske klastere sa  njima blisko srodnim vrstama. Fenogrami evolucionih odnosa konstruisani su UPGMA metodom na osnovu oblika krila su bili podudarni sa topologijom filogenetskih stabla analiziranih grupa vrsta.
Nucleotide COI mtDNA diversity and phenotypic variation of wing parameters (size and shape) of taxa of the genus Cheilosiawere analysed. Obtained data were used to solve phylogenetic and evolutionary relationships of these taxa. A total of 119 specimens from 14 Cheilosiaspecies collected from eight localities on the Balkan Peninsula and one from Finnish Lapland (specimens of C. albitarsis) were used for DNA sequencing. Amplification was attempted for 3' end of COI mtDNA gene (and 5' COI mtDNA and ITS2 rDNA in C. laticornis species group). COI mtDNA sequences from nine species of the C. melanura group and three species of the C. canicularis group were obtained from GenBank. Geometric morphometric analysis of wing size and shape was conducted on 4717 specimens from 29 species collected from 21 localities on the Balkan Peninsula.Based on differential phenotypes of wing size and shape and species-specific COI mtDNA haplotypes it was possible to identify and delimitate closely related species of genus  Cheilosia. It was estimated that size and shape variation occurred among conspecific populations. A consistent sexual wing shape dimorphism was revealed in all analyzed species.Strict consensus cladogram based on COI mtDNA data revealed monophyletic genus  Cheilosia and subgeneric divisions that are congruent with subgenera described based on traditional morphological character (((Cheilosia s. str. + Eucartosyrphus) + Taeniochilosia) + Convocheila). Within the clade Cheilosias. str. closely related species group were supported as monophyletic. UPGMA phenograms of evolutionary relationships based od wing traits produced the same topology as the phylogenetictrees constructed using molecular data.

U  okviru  ove  doktorske  disertacije  vršeno  je istraživanje  zajednica  makrogljiva  u  okviru  5 šumskih  staništa  na  Vidliču,  Kopaoniku  i  Tari. Ispitivan  je  mikodiverzitet  sa  morfološkog, funkcionalnog i genetskog stanovišta. U istraživanju morološkog  i  funkcionalnog  diverziteta,  korišćene su  različite  klasične  metode  čiji  rezultati  suomogućili procenu stanja posmatranih mikocenoza, kao  i  samih  šumskih  staništa.  Za  analizu  sastava vrsta  u  okviru  mikocenoza,  kao  i  procenu  uticaja abiotičkih faktora na brojnost i sastav vrsta u okviru različitih funkcionalnih grupa, korišćeno je nekolikostatističkih  metoda  (PCA,  PLS,  CA  i  CCA).  Osam vrsta,  koje  su  pripadale  najrasprostranjenijim  i najzastupljenijim  vrstama  su  odabrane  za molekularne  analize,  koje  su  podrazumevale sekvenciranje  ITS  regiona  rDNK,  analizu  njihovihpolimorfizama  kao  i  filogenetske  analize  u  okviru vrste/roda.  U  cilju  procene  zagađenja  staništa,  u plodnim telima makrogljiva i njihovom supstratu je određen  sadržaj  metala  (atomskom  apsorpcionom spektrofotometrijom)  i  radionuklida(gamaspektrometrijom).  Dobijeni  rezultati  ukazuju na  to  da  diverzitet  makrogljiva  oslikava  stanje samog  staništa  i  da  dugoročnim  monitoringom mogu ukazati na promene u njemu.
Within the framework of this doctoral dissertation, monitoring  of  macrofungal  communities,  within  5 forest habitats on  Vidlič, Kopaonik and Tara, was done.   Mycodiversity  was  investigated  from  the morphological,  functional  and  genetic  point  of view. Various classical methods  used,  enabled the assessment  of  the  condition  of  macrofungal communities,  as  well  as  the  observed  forest habitats.   Several  statistical  methods  (PCA,  PLS, CA  and  CCA)  were  used  to  analyze  the composition of  species within the  mycocenosis, as well  as  the  assessment  of  the  effects  of  abiotic factors  on  the  species  richness  and  species composition  within  different  functional  groups.Some  of  the  most  represented  species  have  been selected  for  molecular  analyzes,  which  includedsequencing  of  the  ITS  region,  the  analysis  of polymorphisms,  as  well  as  phylogenetic  analyzes within  the  species/genus.  In  order  to  assess  the pollution of habitats, the content of metals (atomic absorption  spectrophotometry)  and  radionuclides (gamma  spectrometry)  was  determined  in  the sporocarps  of   macrofungi  and  their  substrate.  The obtained  results  indicate  that  diversity  of macrofungi  reflects  the  state  of  the  habitat  itself and that long-term monitoring can indicate changes in it.

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The determination of the prevailing species responsible for American Tegumentary Leishmaniasis (ATL) and the detection at species level of the varieties of the protozoan parasite agent of this endemic disease at the Amazon region are important for determination of the clinical evolution and to better indicate the therapeutic conduct due to the different responses to chemotherapeutic drugs by the Leishmania species. By now we are not aware of an available test to Leishmania (Viannia) guyanensis capable of detecting its intra-specific varieties obtained from human clinical presentations or from vertebrate and invertebrate hosts. We succeed to discriminate Leishmania (V.) guyanensis from Leishmania (V.) braziliensis and also from Leishmania (V.) panamensis through a polimerase chain reaction direct genomic DNA amplification aimed at the transcribed internal spacer (ITS) of the ribosomal RNA gene (rDNA). The positive reaction is visualized at the agarose gel electrophoresis by the presence of an aproximatedly 229 nucleotides size band for L. guyanensis that is absent for the two other species L. braziliensis and L. panamensis. The diagnosis can be done using culture material, skin biopsy or squashing the sand fly vector and having the DNA extracted. In a second step of the investigation, the ITS rDNA was sequenced in L. lainsoni; L. naiffi and four L. guyanensis strains, two of them presenting a muco-cutaneous form of Leishmaniasis. These sequences were compared to others available at the Genbank Database, confirming the previous data from multilocus enzyme electrophoresis and ITS restriction fragment lenght polymorphisms analysis, positioning L. lainsoni and L. naiffi as more divergent species as compared to L. braziliensis, L. panamensis and L. guyanensis species inside de Viannia sub-genus. A correlation was observed in grouping nearer the two L. guyanensis strains, respectively IM4243 and IM4235, responsible for the muco-cutaneous form. This protozoan belonging to sub-genus Viannia show clinical and epidemiological importance in South America north region, particularly the Amazon region and this study can be considered an advance in providing a tool to better understand the parasite species silvatic cycle and in providing tools to characterize the main species responsible for the clinical form presentation of tegumentary leishmaniasis in the human population at this region.
A determinação da espécie responsável pela Leishmaniose Tegumentar Americana e a detecção específica das variedades do protozoário agente dessa doença endêmica na Amazônia, é importante para determinação do comportamento clínico e indicação da conduta terapêutica, devido as diversas respostas aos quimioterápicos pelas espécies de Leishmania. No momento se desconhece um teste disponível para a Leishmania (Viannia) guyanensis, que seja capaz também de incluir suas variedades intra-específicas obtidas de casos humanos e de hospedeiros e vetores silvestres. Neste trabalho se conseguiu discriminar alguns isolados de Leishmania (Viannia) guyanensis, de Leishmania (Viannia) braziliensis e também de Leishmania (Viannia) panamensis através da reação em cadeia da polimerase direcionada para a amplificação do espaçador transcrito interno (ITS) do gene do RNA ribossomal. A reação positiva é dada pela visualização de um fragmento amplificado de cerca de 229 nucleotídeos, para L. (V.) guyanensis e ausente para as outras duas espécies. O diagnóstico pode ser feito com o DNA extraído de cultivo do parasita, diretamente das amastigotas da borda de lesão cutânea ulcerada ou do vetor infectado pela promastigota de Leishmania. Numa etapa seguinte a região ITS do rDNA foi sequenciada para L. lainsoni, L. naiffi e quatro cepas de L. guyanensis em que duas apresentavam forma muco-cutânea e duas outras a forma cutânea da leishmaniose. Essas sequências foram comparadas com as disponíveis em banco de dados como o Genbank, resultando na confirmação dos dados obtidos de variabilidade genética por outros autores, quando utilizada a técnica de isoenzimas e ITS-RFLP. O posicionamento de L. lainsoni e L. naiffi se mostrou mais divergente que L. guyanensis, L. braziliensis e L. panamensis, dentro do subgênero Viannia. Foi também encontrada uma relação de proximidade entre os isolados de L. guyanensis IM4243 e IM4235 apresentando lesão cutâneo-mucosa. As leishmânias do sub-gênero Viannia apresentam importância epidemiológica na América do Sul, sobretudo na região amazônica e esse estudo poder ser considerado importante ao desenvolver meios de diagnóstico específico do parasita, detectando sua presença em vetores e reservatórios silvestres em certas áreas e servindo como marcador para a principal espécie responsável pelas formas clínicas de leishmaniose nessa região.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
The apple is a temperate clime fruit with larger dispersion, consumption and the more marketed all over the world as fresh fruit. The apple production has been committed by the incidence of diseases, in which Apple Leaf Spot (ALS), whose causal agent is the Colletotrichum spp. Nowadays this disease is one of the largest concerns of the producers and researchers. The origin of this disease in apple tree plants is not still well known. Studies based on the phylogeny allow to relate evolutionarily the organisms and characterize possible divergence mechanisms origin. This will contribute in disease control and prevention of new races sprout. The objective of this work was to study the variation of the ITSs rDNA among apple tree pathogenic isolates of Colletotrichum spp. from apple, citrus and feijoa. All isolates were first cultivated on PDA, for one week at 24oC. In the PCR amplification of the rDNA was used initiators ITS1 and ITS4. The amplified fragment was digested with restriction enzymes (Rsa I, Alu I, Hinf I, Hpa II, Hha I, Xho I, Acc I, Eco RI and Taq I). The revelation was in 2% agarose gel. It was obtained a great variation with products of the cleaved PCR products, from 90 to 500 pb. The amplified fragment was purified (QIAquick Gel Extraction Kit 250 - Unisciense of Brazil) and the fragment (ITS of the rDNA) was sequenced for all isolates. The sequences were aligned with the ClustalW software and the phylogenetic tree was constructed in the Mega 3.1 software. The products obtained in the PCR amplification of the rDNA region (ITS1-5,8S-ITS2), with the initiators pair ITS1 and ITS4 revealed a fragment with approximately 600 pb, for all the isolated analyzed. Through the analysis for the ARDRA was possible to separate isolates groups in haplotypes, which was efficient for the correlation between haplotypes and hosts, containing in a same haplotypes of the same host. It was possible to group the isolates of Colletotrichum in species, using the phylogenetic tree, independent of his host. All of the obtained sequences presented more than 93% of similarity, fact that reinforces the hypothesis that the Colletotrichum spp., causal agent of ALS, is nearly related with citrus and feijoa Colletotrichum
A maçã é a fruta de clima temperado de maior dispersão, consumo e a mais comercializada como fruta fresca em todo o mundo. A produção de maçã no Brasil tem sido comprometida pela incidência de várias doenças, das quais a Mancha Foliar da Gala (MFG), cujo agente causal são espécies de Colletotrichum spp., tem sido, atualmente, uma das maiores preocupações dos produtores e pesquisadores. A origem desta doença em macieira ainda não está bem esclarecida e a utilização de estudos baseados na filogenia permitem relacionar os organismos evolutivamente, caracterizando possíveis mecanismos de divergência evolutiva e a origem do patógeno, contribuindo na adoção de medidas de controle e prevenção quanto ao surgimento de novas raças. O presente trabalho objetivou estudar a variação do DNA ribossomal, (rDNA) entre isolados de Colletotrichum spp. patogênicos em macieira, em citros e goiaba serrana. Todos os isolados foram cultivados em meio BDA, por uma semana a 24oC. A amplificação do rDNA foi realizada utilizando-se iniciadores ITS1 e ITS4, seguida pela digestão da região ITS (espaçador interno transcrito) com enzimas de restrição (Rsa I, Alu I, Hinf I, Hpa II, Hha I, Xho I, Acc I, Eco RI e Taq I). Os produtos obtidos na amplificação da região ITS1-5,8S-ITS2 do rDNA revelaram um fragmento de aproximadamente 600 pares de bases (pb), para todos os isolados analisados. Todos os fragmentos amplificados foram clivados, obtendo uma grande variação, a qual foi de 90 a 500 pb. Utilizando a técnica de ARDRA (Amplified Ribosomal DNA Restriction Analysis) foi possível separar e agrupar os isolados em haplótipos, a qual foi eficiente para a correlação entre haplótipo e hospedeiros, agrupando em um mesmo haplótipo isolados do mesmo hospedeiro. O fragmento amplificado foi purificado com kit QIAquick Gel Extraction Kit 250 (Qiagen, Hilden, Germany) e as amostras foram seqüenciadas na região ITS do rDNA de todos os isolados. As seqüências foram alinhadas no software ClustalW e a árvore filogenética foi construída no software Mega 3.1. Com o seqüenciamento de DNA foi possível inferir sobre a possível espécie de fungo que pertence o segmento de DNA analisado. A região ITS do rDNA mostrou-se muito eficaz para estudos de filogenia. A partir da árvore filogenética foi possível agrupar os isolados de Colletotrichum por espécies, independente do seu hospedeiro. Todas as seqüências obtidas apresentaram valores superiores a 93% de similaridade, fato esse, que reforça que o Colletotrichum spp., causador da MFG, possui relações de parentesco com o isolados de Colletotrichum spp. isolados de citros e de goiaba serrana

Zwei Populus-Pfropfchimären (MA und AI), die aus P. x canadensis ‘Marilandica’ (M), P. maximowizcii x P. trichocarpa ‘Androscoggin’ (A) and P. nigra L. ’Italica’ (I) aufgebaut sind, wurden für Untersuchungen zur Laub- und Blütenblattentwicklung genutzt. In MA bildet M die äußere Lage (L1) und ihr Derivat, die Epidermis, während die inneren Lagen (L2, L3 etc.) von A gebildet werden. Bei AI stammt die L1 von A und L2, L3 etc. werden von I gebildet. Die genotypisch andersartige Epidermis bedingt bei Periklinal-Chimären morphologische Effekte wie zum Beispiel einen Fruchtknoten in einigen MA-Blüten. Morphologische Besonderheiten verschiedener Gewebe sowohl von M und A als auch von MA wurden verglichen, um festzustellen, wie sie durch die Gewebetransplantation verändert oder beeinflusst wurden und, um mögliche Genotyp- Interaktionen oder -Wechselwirkungen in einem Gewebe ausfindig zu machen. Für die Genotypidentifizierung in verschiedenen Organen wurde die RAPD-PCR getestet. Einer von 20 getesteten 10mer Zufallsprimern (GGAGTGGACA) ermöglichte bei der Verwendung von DNA aus Blattmaterial die Erzeugung verschiedener Bandenmuster für M und A. Bei der Verwendung von MA-Blattmaterial zeigte sich eine Kombination der Muster von M und A, sodass ein Chimärennachweis für das MA-Blattmaterial erbracht wurde. Für ein übertragbares System wurde die spezifische PCR getestet. Unter Verwendung spezifischer Primer für die 16s-rDNA zeigten die PCR-Produkte einheitliche Banden und nach anschließender Sequenzierung eine weitgehende Übereinstimmung der phylogenetischen Verwandtschaft von I, M und A. Weiterhin wurden die kernkodierten rDNA Bereiche ITS 1 und ITS 2 zwischen 18S und 25S getestet. Für I, M und A konnten jeweils zwei Banden von unterschiedlicher Größe und Sequenz ermittelt werden, die vermutlich auf funktionierende rDNA aber auch auf Pseudogene (beschnitten) in niedriger Kopienzahl hinweisen. Die ITS-Regionen von I, M und A wurden charakterisiert, um einen Einblick in die Struktur und Phylogenie der Salicacaee-Familie zu erhalten. Aus den Sequenzunterschieden konnten für I und A spezifische Primerpaare abgeleitet werden, die für die Identifizierung von I und A in AI und MA verwendet werden können. Mittels A-Marker konnte nachgewiesen werden, dass Fruchtknoten aus MA-Blüten neben M-Gewebe auch den A-Genotyp enthalten.
Two Populus graft chimeras (MA and AI) produced of P. x canadensis ‘Marilandica’ (M), P. maximowizcii x P. trichocarpa ‘Androscoggin’ (A) and P. nigra L. ’Italica’ (I) were used for investigations of leaf and flower development. In MA the exogenous layer (L1) forms the epidermis and is derived from M while inner layers (L2, L3 etc.) descend from A whereas in AI L1 is formed by A while L2, L3 etc. descend from I. The exogenous epidermis of the periclinal chimeras imposes morphological effects such as an extra female sex in some of the MA flowers. The morphological characteristics of different plant tissues of parents and chimera were compared to determine how they were modified or altered by the tissue transplantation and possibly identify co-existing or interacting genotypes in one tissue. RAPD-PCR was tested for its usefulness to amplify polymorphic fingerprints including donor specific DNA fragments. One random 10mer primer (GGAGTGGACA) out of 20 tested revealed the amplification of patterns including donor specific DNA bands using extracts from leaf tissues of the M and A parents that were combined using extract from leaf tissue of the MA chimera. This indicates that the leaves of the MA chimera are formed by tissues of M and A. However, the inherent disadvantage of RAPD-PCR is the reproducibility of PCR product generation. Therefore the discriminative potential of the ITS region located between the rRNA genes was investigated. The application of specific 16S ribosomal DNA (rDNA) primers for amplification and sequencing of PCR products revealed a closely phylogenetic relationship between I, M and A. Consequently the ITS1 and ITS2 of nuclear rDNA between 18S and 25S were used. The amplified fragments were purified, cloned in E. coli and sequenced. Analyses of multiple clones demonstrated extensive paralogy within and between I, M and A ITS operons. For each parent were at least two rDNA operons as well as multiple paralogous sequences within operons identified. The use of PCR and sequence analyses showed that one of the operons encodes a putative expressed (functional) rDNA whereas the second encodes a pseudogen (truncated) in low copy number. We also characterized the ITS regions of I, M and A to gain insights into structure and phylogeny of the Salicacaee family. Based on sequence divergence primers were designed for A and I and used for the identification of A in MA carpels.

O gênero Sargassum C. Agardh (Sargassaceae) constitui um dos mais representativos dentre os 41 gêneros da ordem Fucales (Phaeophyceae, Heterokontophyta), é amplamente distribuído nas regiões tropicais e subtropicais do globo e é considerado um importante componente da flora marinha. Devido ampla variabilidade fenotípica é considerado um dos gêneros de taxonomia mais complexa dentre as algas pardas, como são popularmente conhecidos os representantes da classe Phaeophyceae. A filogenia da ordem Fucales é bastante discutida para gêneros do hemisfério norte, mas ainda pouco elucidada. Os estudos de filogenia referentes ao gênero Sargassum são escassos, limitando-se a poucos marcadores moleculares, com baixa resolução no âmbito inter-específico e limitados à espécies de ocorrência nos oceanos Indo-Pacífico e Atlântico Norte. Nenhum estudo filogenético incluí espécies do Atlântico Sul para este gênero. Este estudo é pioneiro na análise de seqüências de diferentes marcadores moleculares para espécies do Atlântico Sul. Neste estudo, foram seqüenciados completamente os marcadores moleculares nucleares SSU rDNA e ITS2 para os táxons infra-genéricos Sargassum cymosum var. cymosum, S. cymosum var. nanum, S. furcatum, S. stenophyllum e S. vulgare. Todas as seqüências obtidas para ambos os marcadores apresentaram 100% de identidade entre os táxons analisados. Foram feitas seqüências também para o marcador molecular plastidial rbcL (parcial) e espaçador rbcLS para as espécies S. filipendula, S. stenophyllum e S. vulgare que resultaram também em 100% de identidade. Análises filogenéticas de cada um dos marcadores moleculares, incluindo nossas seqüências e aquelas disponíveis no Genbank e geradas pelos métodos de inferência \"Neigbour-joining\", máxima parcimônia e máxima verossimilhança se apresentaram robustas e corroboram outros resultados descritos na bibliografia referente a ordem Fucales e ao gênero Sargassum. Entretanto, tais resultados fornecem um forte indício da necessidade de busca de marcadores moleculares eficientes, devidamente respaldados por estudos de hibridação in vitro, dados ecológicos e de biogeografia para um melhor entendimento acerca das espécies ocorrentes na costa brasileira.
The genus Sargassum C. Agardh (Sargassaceae) is one of the most conspicuous among the 41 genera of the order Fucales (Phaeophyceae, Heterokontophyta). The genus has a broad distribution in the tropical and subtropical regions of the world, and is considered an important component of the marine flora. Due to a high phenotipic variation, the taxonomy of the genus is considered of the most complex among the brown seaweeds, as are known the representatives of the class Phaeophyceae. The phylogeny of the order Fucales was studied for the North Hemisphere genera, but is still not well understood. The phylogenetic studies of the genus Sargassum are scarce and limited to a few molecular markers, presenting low resolution for inter-specific analysis and are available only for species from the Indo-Pacific and the North Atlantic. There are no phylogenetic studies including species from the South Atlantic for the genus. This study is the first to analyze sequences from different molecular markers for species from the South Atlantic. In this study, the nuclear SSU rDNA and ITS2 were completely sequenced for the infra-generic taxa Sargassum cymosum var. cymosum, S. cymosum var. nanum, S. furcatum, S. stenophyllum and S. vulgare. All the sequences for both markers presented 100% identity among analyzed taxa. Sequences were also obtained for the chloroplast marker rbcLS, including parcial rbcL and the spacer region rbcLS for the species S. filipendula, S. stenophyllum and S. vulgar. These sequences also presented 100% identity among analyzed taxa. Phylogenetic analysis of each of the molecular markers, including our sequences together with other sequences available in the Genbank and generated by the inference methods Neigbour-joining, maximum parsimony and maximum likelihood were robust and similar to other results described in the literature for the order Fucales and the genus Sargassum. Nonetheless, these results are an indicative of the need for more efficient molecular markers, associated with data from in vitro hibridization, ecology and biogeography for a better understanding about the taxa occurring on the brazilian coast.

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A antracnose causada pelo fungo Colletotrichum gloeosporioides é uma das doenças mais graves da mangueira, sendo um fator limitante à produção de frutos sadios e comercializáveis. O presente trabalho objetivou analisar a variabilidade de isolados de C. gloeosporioides, quanto à patogenicidade a frutos de mangueira, marcadores RAPD e região ITS do rDNA. Foram utilizados cinco isolados obtidos de cebola e 10 de mangueiras de diferentes regiões do estado de Pernambuco. O teste de patogenicidade foi realizado em frutos das variedades Rosa e Espada. Para a análise de RAPD foram utilizados os iniciadores OPX-15, OPX-02, OPX-06, OPA-11 e OPA-02 e para a amplificação do locus ITS1-5.8S-ITS2 do rDNA, os iniciadores ITS1 e ITS4. Os isolados oriundos de mangueiras com sintomas de antracnose foram mais agressivos para as duas variedades estudadas do que os isolados de cebola. Do dendrograma gerado pela análise do RAPD, dois grupos foram delineados, separando todos os isolados de mangueiras dos isolados de cebola. Não houve polimorfismo entre os isolados de C. gloeosporioides pela análise dos produtos de amplificação da região ITS1-5.8S-ITS2 do rDNA, porém, a digestão do produto com a enzima Msp I evidenciou diferença para o isolado URM 4596 (Mangifera indica L./Ilha de Itamaracá-PE)

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
The investigation of intra-individual variation of the rDNA cistron ITS in Carapichea ipecacuanha was made from direct sequencing of 26 individuals and from sequencing of 240 clones, representing another 51 individuals. The secondary structures of 5.8S and ITS2 regions show the typical configurations present in angiosperms respectively for 20 and 48 haplotypes. The comparison with the conserved motifs in angiosperms and the secondary structures obtained for the 5.8S region, the most reliable markers of functional sequences in C. ipecacuanha, allowed the identification of 13 pseudogenes among 26 haplotypes of 5.8S. The analysis of the secondary structure of ITS2 added one haplotype to the set of pseudogenes. The loss of functionality in some haplotypes has occurred prior to the others copies due to the accumulation of a larger number of substitutions and the formation of groups of pseudogenes that belong to distinct populations.
A investigação da variação intra-individual do cistron de rDNA ITS em Carapichea ipecacuanha foi realizada a partir de seqüenciamento direto de fragmentos de PCR de 26 indivíduos e do seqüenciamento de 240 clones, representando outros 51 indivíduos. As estruturas secundárias das regiões 5.8S e ITS2 apresentam as configurações típicas presentes em angiospermas respectivamente em 20 e 48 haplótipos encontrados. A comparação com os motivos conservados em angiospermas e as estruturas secundárias obtidas para a região 5.8S, indicadores mais confiáveis da funcionalidade das seqüências em C. ipecacuanha, permitiram a identificação de 13 pseudogenes entre os 26 haplótipos de 5.8S encontrados. A análise da estrutura secundária do ITS2 adicionou um haplótipo ao conjunto de pseudogenes. A perda de funcionalidade em alguns haplótipos deve ser mais antiga do que em outros em razão do acúmulo de maior número de substituições e da formação de grupos de pseudogenes pertencentes a populações distintas.

L'utilisation des coquilles carbonatées de foraminifères planctoniques comme marqueurs paléocéanographiques repose sur l'hypothèse fondamentale que chaque espèce morphologique correspond à une espèce biologique caractéristique d'un habitat spécifique. Cette relation empirique a été récemment remise en cause par des analyses moléculaires qui ont révélé la présence systématique de plusieurs espèces génétiques (espèces cryptiques) au sein des morpho-espèces actuelles. Il a été suggéré que ces espèces cryptiques ou génotypes, présentaient (1) des préférences biogéographiques et écologiques restreintes par rapport à leur morpho-espèce respective et (2) des différences morphologiques. Dans ce travail, nous avons caractérisé la diversité génétique, morphologique et écologique de 4 morpho-espèces clefs de la paléocéanographie, i.e. Orbulina universa, Truncorotalia truncatulinoides, Globoconella inflata et Globigerina bulloides. Cette étude repose sur le développement d'un protocole d'extraction ADN non destructif pour la coquille calcaire, permettant l'analyse conjointe de la variabilité génétique et morphologique d'un même individu. Les variations de forme ou de porosité de chacun des génotypes des morphoespèces ont été quantifiées. Il apparaît que le fort degré de plasticité morphologique largement documenté chez les foraminifères planctonique et jusqu'alors interprété comme écophénotypique, est au moins en partie la conséquence du regroupement de plusieurs génotypes présentant des morphologies et préférences écologique particulières. Sur la base de ces observations, des modèles de reconnaissance morphologique permettant d'identifier les génotypes à partir de la morphologie de la coquille, utilisables à l'échelle populationnelle, ont été développés. Afin de quantifier l'impact de l'intégration de la diversité cryptique dans les reconstitutions paléocéanographiques basées sur les assemblages de foraminifères planctoniques, les fonctions de transfert couramment utilisées ont été re-calibrées en intégrant les distributions restreintes des génotypes d'O. universa, T. truncatulinoides, G. inflata et G. bulloides. Ces re-calibrations conduisent à un degré de précision jusqu'alors jamais atteint dans les reconstructions paléocéanographiques basées sur les assemblages de foraminifères planctoniques.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Fungi represent an extremely important group of organisms in terrestrial ecosystems. Among their several important ecological roles, is the mutualistic association with plant roots, forming different types of mycorrhiza. Most studies carried out so far focused on epigeous ectomycorrhizal fungi, which occur above ground. On the other hand, below ground, hypogeous ectomycorrhizal fungi, are still poorly studied. This research aimed to study the diversity of hypogeous ectomycorrhizal fungi (Basidiomycetes) collected in Eucalyptus spp. plantations in the Central Region of Rio Grande do Sul, Brazil, based on morphological and molecular characters. Samples were taken between May 2009 and July 2010. A total of five species belonging to three families were identified. The material was analyzed to determine the morphological and molecular characters. Among the species identified are: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway and Setchelliogaster tenuipes (Setch.) Pouzar. Additionally a second species of Chondrogaster sp. was collected. However, no identity was determined for this species. Furthermore, it is believed it represents a new undescribed species to science. Among the species studied, Descomyces albus is reported for the first time in the State of Rio Grande do Sul. Hysterangium affine and H. inflatum are newly recorded species from Brazil while Chondrogaster pachysporus is recorded for the first in South America.
Os fungos representam um grupo de organismos extremamente importante nos ecossistemas terrestres. Entre as muitas funções por eles desempenhadas está a associação mutualística com as raízes dos vegetais, formando diferentes tipos de micorrizas. A maioria dos estudos realizados até o momento está principalmente focada nos fungos ectomicorrízicos epígeos, os quais ocorrem acima do solo. Por outro lado, os fungos ectomicorrízicos hipógeos, que vivem abaixo da superfície do solo, são ainda pouco estudados. A presente pesquisa tem como objetivo estudar a diversidade de fungos ectomicorrízicos hipógeos (Basidiomycetes) coletados em plantações de Eucalyptus spp. na Região Central do Rio Grande do Sul, Brasil, com base em características morfológicas e moleculares. O período de coleta dos fungos foi de maio de 2009 a julho de 2010. Um total de cinco espécies pertencentes a três famílias foram identificadas. Este material foi analisado para verificar suas características morfológicas e moleculares. Dentre as espécies identificadas estão: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway e Setchelliogaster tenuipes (Setch.) Pouzar. Adicionalmente identificou-se uma segunda espécie de Chondrogaster sp. porém não se chegou a uma espécie conhecida, acreditando tratar-se de uma espécie ainda desconhecida para a ciência. Dentre as espécies estudadas, destacam-se a Descomyces albus que é citada pela primeira vez para o Estado do Rio Grande do Sul, Hysterangium affine e H. inflatum que têm sua ocorrência registrada pela primeira vez no Brasil, além de Chondrogaster packysporus citado pela primeira vez para a América do Sul.

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Fungos endofíticos são micro-organismos que habitam o interior da planta hospedeira, sem causar danos aparentes ou estruturas externas visíveis. O coqueiro (Cocos nucifera L.) é uma espécie tropical, largamente distribuída na Ásia, África, América Latina e regiões do Pacífico, sendo o Brasil o quarto maior produtor mundial. Devido à importância agroeconômica do coco, este trabalho teve por objetivo determinar a micobiota endofítica de folhas sadias de C. nucifera em duas cultivares (Anão-amarelo e Anão-verde) e um híbrido (PB 121) em Goiana, Pernambuco. No laboratório, fragmentos de raque e folíolo foram lavados com água corrente e sabão neutro, e com auxílio de um furador esterilizado foram feitos discos foliares (6 mm), posteriormente desinfestados em álcool 70% por 1 minuto, em hipoclorito de sódio (NaOCl) a 2% por 2 minutos e 30 segundos, em álcool 70% por 30 segundos e finalmente duas lavagens com água destilada esterilizada. Os fragmentos foram tranferidos para placa de Petri, em triplicata, contendo Batata-Dextrose-Ágar (BDA) acrescido de cloranfenicol (50 mg.L-1). Foram sequenciados a região ITS do rDNA dos isolados e em seguida verificada a similaridade Blastn e realizada análises filogenéticas. Índices de diversidade, curva de acumulação de espécies, similaridade e frequência de ocorrência foram calculados. O total de 1296 fragmentos de folhas/raques foram analizados e 474 espécimes de fungos endofíticos foram isolados e distribuídos em 31 gêneros e 37 espécies. A espécies identificadas são representantes do filo Ascomycota, pertencentes às classes Sordariomycetes, Dothideomycetes e Eurotiomycetes, além de Occultifur externus, Rhodotorula marina e Pseudozyma hubeiensis anamorfos do filo Basidiomycota e Syncephalastrum racemosum do filo Zygomycota. Arthrinium xenocordella, Ascotricha guamenses, Phlyctaeniella sp., Pyrenochaetopsis decipiens, P. leptospora e Stenella musae são citadas pela primeira vez como endofíticos. Fusarium proliferatum, F. solani e Paecilomyces lilacinus são reportadas pela primeira vez como endofíticos em Cocos nucifera.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
This study aimed to select and characterize strains of entomopathogenic fungi in order to control Gyropsylla spegazziniana. For this, the fifth instar nymphs were transferred to Paraguay tea seedlings, followed by spraying conidial suspensions of 48 strains of Beauveria spp., Metarhizium anisopliae, Isaria spp. and Lecanicillium spp. at a concentration of 1 × 109 conidia/mL. The seedlings were placed in PVC cages kept in a climatized room (26 ± 1°C; 12 h photophase and 60 ± 10% R.H.), and the insect mortality was evaluated daily for 10 days. The most active isolates were compared by means of vegetative growth, conidial production in culture media, insecticidal activity and molecular analyses, by sequencing the region rDNA-ITS and RAPD. The genus 22 Beauveria spp. was more efficient among the entomopathogenic fungi, especially for strain Unioeste 44 that showed the best performance, demonstrating its potential to control the pest. Molecular analysis of rDNA-ITS region allowed the identification of isolates as B. bassiana e B. brongniartii and RAPD markes were associated with virulence
O objetivo desse trabalho foi selecionar e caracterizar isolados de fungos entomopatogênicos, visando o controle dessa praga. Para tal, ninfas de 5o ínstar foram transferidas para mudas de erva-mate, seguido da pulverização de suspensões de conídios (1 × 109 conídios/mL) de 48 isolados de Beauveria spp., Metarhizium anisopliae, Isaria spp. e Lecanicillium spp. As mudas foram acondicionadas em gaiolas de PVC, e mantidas em sala climatizada (26 ± 1°C; 12 h de fotofase e U.R. 60 ± 10%). 21 A mortalidade dos insetos foi avaliada diariamente, por 10 dias e os isolados mais ativos foram comparados entre si por meio do crescimento vegetativo, produção de conídios em meios de cultura, atividade inseticida e análises moleculares, por meio do seqüenciamento da região rDNA-ITS e marcadores RAPD. O gênero Beauveria spp. foi mais eficiente dentre os fungos entomopatogênicos, em especial o isolado Unioeste 44 que apresentou o melhor desempenho, evidenciando seu potencial para o controle da praga. As análises moleculares da região rDNA-ITS possibilitou a identificação dos isolados como B. bassiana e B. brongniartii e os marcadores RAPD foram associados à virulência

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Foram analisadas vinte linhagens de Beauveria bassiana isoladas de diferentes regiões e hospedeiros, quanto ao perfil de DNA, através da análise da região ITS do rDNA, de RAPD e Intron, para avaliação da diversidade genética e auxílio na caracterização. A região ITS1-5.8s-ITS2 do rDNA foram digeridas com as enzimas Eco RI, Dra I, Msp I e Hae III, onde a Eco RI e Dra I não apresentaram sítio de restrição e a Hae III e Msp I não apresentaram polimorfismo entre as linhagens, confirmando a espécie estudada. Para RAPD foram selecionados cinco primers OPX17, OPW4, OPW7, OPW19, 0PW20, que produziram um total de 442 bandas. Os dados obtidos foram submetidos à análise estatística pelo programa NTSYS.PC e construída uma matriz de similaridade utilizando o coeficiente de Jaccard, que gerou um dendrograma através do método de agrupamento UPGMA. A similaridade foi em torno de 70%. A análise do dendrograma mostrou a formação de quatro grupos, onde não houve correlação com o hospedeiro ou origem geográfica, embora duas linhagens isoladas de Nezara viridula e duas de Deois flavopicta tenham mostrado maior nível de similaridade. Os dados obtidos pela análise de Intron, mostraram o aparecimento de cinco grupos

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Foram analisadas 20 linhagens de C. gloeosporioides quanto às características genéticas e citomorfológicas. Os marcadores moleculares, RAPD, microssatélites, Intron Spice Site Primer e região ITS do DNA ribossomal, foram utilizados para avaliar a diversidade genética entre as linhagens. A análise de agrupamento através do método UPGMA confirmou a diversidade genética intraespecífica reconhecida em C. gloeosporioides. Com a técnica de RAPD foi detectada uma maior similaridade genética entre as linhagens. As regiões de microssatélites investigadas, demonstraram alto polimorfismo genético e os introns discriminaram todos as linhagens apenas com o primer (EI-1), e revelaram maior diversidade genética em relação aos outros marcadores moleculares utilizados. As três enzimas de restrição testadas, HaeIII, DraI e MspI evidenciaram a diversidade genética entre as linhagens nos produtos de amplificação dos loci ITS1-5.8S-ITS2 do rDNA com os primers ITS1 e ITS4. Todos os marcadores empregados, foram eficientes em demonstrar o alto grau de polimorfismo genético, constatado pela formação de grupos altamente diversificados, sem apresentar correlação entre os hospedeiros. Os aspectos macroscópicos exibiram uma variação na coloração, textura e segregação de setores nas colônias, e as observações microscópicas demonstraram a formação de estruturas vegetativas e reprodutivas peculiares da espécie. A condição nuclear investigada através da técnica de HCl-Giemsa, evidenciou conídios 100% uninucleados

Made available in DSpace on 2014-06-12T15:05:47Z (GMT). No. of bitstreams: 2 arquivo4602_1.pdf: 941507 bytes, checksum: c0ae7f823963e2cbd711e367c4dd1d23 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006
Conselho Nacional de Desenvolvimento Científico e Tecnológico
Foram analisadas as linhagens Metahizium anisopliae var. acridum e Metarhizium anisopliae var. anisopliae quanto à patogênicidade sobre Zaprinus indianus, utilizando as concentrações 104, 105, 106, 107, 108 conídios/mL considerando o percentual de emergência de adultos. De acordo com a metodologia empregada verificou-se que as duas linhagens apresentaram ação contra Z. indianus. Os marcadores moleculares ITS (Internal Trancride Spacer) do rDNA, Intron Splice Site Primer e Microssatélite (SSR- Simple Sequence Repeats), foram utilizados para avaliar a diversidade genética entre as linhagens antes e após a passagem pela mosca. A análise de agrupamento usando o método de UPGMA baseada nas distâncias genéticas dos marcadores moleculares confirmou a diversidade genética reconhecida no gênero Metarhizium. O microssatélite (GTG)5 e o intron do grupo mRNA nuclear tiveram a mesma sensibilidade em detectar a variabilidade genética entre as linhagens de Metarhizium . Os produtos de amplificação dos loci ITS1-5.8-ITS2 do rDNA com os iniciadores ITS4 e ITS5 foram eficientes em demonstrar que as linhagens estudadas pertence à espécie Metarhizium anisopliae, apesar da diversidade genética demonstrada pelos marcadores (GTG)5 e EI1. Os perfis de amplificações da região microssatélite, intron e ITS após a passagem por Z. indianus comprovaram que as linhagens reisoladas foram às mesmas que foram utilizadas para infectar

Made available in DSpace on 2014-06-12T15:03:44Z (GMT). No. of bitstreams: 2 arquivo4555_1.pdf: 1910369 bytes, checksum: 5a0109c9538c737e8440a1c2af5b2757 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005
Foram analisadas 15 linhagens de Metarhizium isoladas de diferentes regiões e hospedeiros quanto às características genéticas e 7 linhagens quanto a patogenicidade sobre Diatraea saccharalis. Os marcadores moleculares ITS (Internal Transcrided Spacer) do rDNA, Intron splice site primer, RAPD e Microssatélites (SSR-Simple Sequence Repeats) foram utilizados para avaliar a diversidade genética entre as linhagens. A análise de agrupamento usando o método UPGMA baseada nas distâncias genéticas dos quatro marcadores moleculares confirmou a diversidade genética reconhecida no gênero Metarhizium. As enzimas de restrição, HaeIII e MspI, evidenciaram a diversidade genética entre as linhagens ao digerirem os produtos de amplificação do locus ITS1-5.8S-ITS2 do rDNA com os iniciadores ITS4 e ITS5, a enzima DraI não apresentou sítios de restrição. Os introns do grupo mRNA nuclear discriminaram as linhagens de Metarhizium apenas com a utilização do iniciador EI1. As técnicas de RAPD e regiões de Microssatélite foram eficientes em demonstrar a diversidade entre as linhagens. Porém o microssatélite (GACA)4 foi mais sensível em detectar a variabilidade intra e interespecífica entre as diferentes linhagens de Metarhizium. Não houve correlação entre grupos e regiões geográficas. As linhagens 4415, 4400 e 4897 causaram maior percentual de mortalidade das larvas de Diatraea saccharalis. Também não houve correlação entre os agrupamentos gerados pelas técnicas moleculares e percentual de mortalidade de larvas de D. saccharalis

Fascioliasis is a worldwide emerging snail-borne zoonotic trematodiasis with a great spreading capacity linked to animal and human movements, climate change, and anthropogenic modifications of freshwater environments. South America is the continent with more human endemic areas caused by Fasciola hepatica, mainly in high altitude areas of Andean regions. The Peruvian Cajamarca area presents the highest human prevalences reported, only lower than those in the Bolivian Altiplano. Sequencing of the complete rDNA ITS-2 allowed for the specific and haplotype classification of lymnaeid snails collected in seasonal field surveys along a transect including 2007–3473 m altitudes. The species Galba truncatula (one haplotype preferentially in higher altitudes) and Pseudosuccinea columella (one haplotype in an isolated population), and the non-transmitting species Lymnaea schirazensis (two haplotypes mainly in lower altitudes) were found. Climatic seasonality proved to influence G. truncatula populations in temporarily dried habitats, whereas L. schirazensis appeared to be more climatologically independent due to its extreme amphibious ecology. Along the southeastern transect from Cajamarca city, G. truncatula and L. schirazensis shared the same site in 7 localities (46.7% of the water collections studied). The detection of G. truncatula in 11 new foci (73.3%), predominantly in northern localities closer to the city, demonstrate that the Cajamarca transmission risk area is markedly wider than previously considered. Lymnaea schirazensis progressively increases its presence when moving away from the city. Results highlight the usefulness of lymnaeid surveys to assess borders of the endemic area and inner distribution of transmission foci. Similar lymnaeid surveys are still in need to be performed in the wide northern and western zones of the Cajamarca city. The coexistence of more than one lymnaeid transmitting species, together with a morphologically indistinguishable non-transmitting species and livestock movements inside the area, conform a complex scenario which poses difficulties for the needed One Health control intervention.
Ministerio de Economía y Competitividad
Revisión por pares

This project represents an initial examination of the evolutionary history of the genus Pilobolus, and provides a starting point for developing a method to accurately classify members of this genus using molecular genetics. The data analysis presented in this paper suggests a potential evolutionary path in which P. umbonatus and P. sphaerosporus diverged (along a common path) from an original generic ancestor early, but independently, from the evolutionary course taken by P. kleinii. Both Maximum Likelihood and parsimony analyses concur in the branch patterns. Additionally, formation of species-specific clades between samples of P. sphaerosporus, P. kleinii and P. umbonatus suggests that there may be markers present in the ITS sequence that can be used for the development of a molecular test for the classification of species within the genus Pilobolus.Ball State UniversityMuncie, IN 47306
Department of Biology

Orientadora : Profª. Drª. Graciela Inés Bolzon de Muñiz
Co-orientadora: Profª. Drª. Silvana Nisgoski
Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Engenharia Florestal. Defesa: Curitiba, 10/02/2015
Inclui referências (fls. 106-118)
Área de concentração : Tecnologia e utilização de produtos florestais
Resumo: A identificação taxonômica da madeira é de fundamental importância para a sua correta aplicação tecnológica, controle de fraudes e controle de atividades do comércio ilegal de espécies protegidas. No Paraná, a Mata Atlântica, que já ocupou 99% da área do estado, hoje ocupa apenas 13% e continua ameaçada pelas atividades madeireiras ilegais, avanço das áreas agrícolas e urbanas. Metodologias de identificação da madeira podem contribuir para a preservação desse bioma. A metodologia tradicional de identificação de madeiras baseia-se nos conhecimentos da anatomia da madeira, que vem sendo complementada por técnicas alternativas. Uma dessas técnicas é o DNA-barcode que permite a identificação de espécie baseada em regiões padronizadas do DNA. Uma região com promissora aplicação como marcador de barcode é a região do espaçador interno transcrito (ITS) do DNA ribossômico. Essa região tem consagrada aplicação como ferramenta de inferência evolutiva e filogenética e vem sendo indicada como candidata ao DNA-barcode. Esse trabalho teve como objetivo a identificação de amostras de madeira de apreensão utilizando a anatomia da madeira associada à região do ITS do rDNA. Oito amostras de madeira foram caracterizadas anatomicamente, e tiveram seu DNA extraído segundo dois protocolos, amplificado e sequenciado. Os dados anatômicos foram aplicados em chaves de identificação e as sequências do rDNA foram procuradas utilizando-se da ferramenta BLAST no banco de dados GENBANK. Foi possível extrair DNA genômico de todas as amostras, tendo o protocolo de grande escala rendido melhores resultados. Quatro das amostras tiveram a região do ITS completa sequenciada e uma a região do ITS1. As amostras foram identificadas como Hovenia dulcis, um integrante da família Lauraceae, Protium spp., Cedrela spp., Chrysophyllum spp. e Nectandra spp. Do isolado da amostra L3 foi possível a amplificação, sequenciamento da região do ITS1 e identificação de um fungo. O GENBANK se mostrou uma boa fonte de sequências para espécies madeireiras e a anatomia da madeira conciliada com a região do ITS foi eficaz identificando a maioria das amostras, porém se faz necessário o conhecimento da sequência de um número maior de indivíduos para se obter resoluções maiores. Palavras-chave: Identificação da madeira, Barcoding, Anatomia da Madeira.
Abstract: The timber's taxonomic identification is of fundamental importance for its proper technological application, fraud control and control of illegal protected species trade activities. In Paraná State, the Atlantic Forest that has held 99% of the state area today occupies only 13% and still threatened by illegal logging, increase of agricultural and urban areas. Wood identification methodologies can contribute to the preservation of this biome. The traditional methodology for wood identification is based on the knowledge of wood anatomy, which has been supplemented by alternative techniques. One of these techniques is the DNA barcode, that allows species identification based on DNA standardized regions. A region with promising application as barcode marker is the internal transcribed spacer (ITS) region of the ribosomal DNA. This region has usual application as evolutionary and phylogenetic inference tool and has been recommended as a candidate for DNA barcode. This study aimed to identify seizure wood samples using the wood anatomy associated with the rDNA ITS region. Eight wood samples were characterized anatomically, and had their DNA extracted, according to two protocols, amplified and sequenced. The anatomical results were applied in identification keys and the rDNA sequences were searched using the BLAST tool in GENBANK database. It was possible to extract genomic DNA from all samples, the large-scale protocol yielded the best results. Four of the samples had the complete ITS region sequenced and one the ITS1 region. The samples were identified as Hovenia dulcis, a member of the Lauraceae family, Protium spp., Cedrela spp., Chrysophyllum spp. and Nectandra spp. The sample L3 isolate was amplified and sequenced, the ITS1 sequence was identified as a fungus. The GENBANK proved to be a good source of sequences for wood species and wood anatomy combined with the ITS region was effective identifying samples, however more sequences for a larger number of individuals are necessary to obtain higher resolutions. Keywords: Wood Identification, Barcoding, Wood Anatomy.

Made available in DSpace on 2014-06-12T15:04:56Z (GMT). No. of bitstreams: 2 arquivo4513_1.pdf: 733474 bytes, checksum: 96d9ff147d1527b544a0ce02910e2af1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004
A antracnose foliar ou mal-das-sete-voltas na cebola, causada pelo fungo Colletotrichum gloeosporioides, destaca-se como uma das principais doenças em locais de produção da cebola no estado de Pernambuco. O presente trabalho objetivou estudar a patogenicidade na cebola de isolados de C. gloeosporioides e analisar a variabilidade genética pelo uso dos marcadores moleculares RAPD e a região ITS do rDNA. No trabalho foram utilizados 11 isolados do fungo, obtidos de diferentes substratos e hospedeiros e 5 isolados obtidos de cebola de diferentes regiões de Pernambuco e um do Amazonas. O teste de patogenicidade foi realizado em mudas e bulbos da cebola Os isolados mais agressivos foram quatro provenientes de cebola nos dois testes. Nas análises de RAPD foram utilizados sete primers de seqüências arbitrárias. Os produtos de amplificação foram separados em gel de agarose a 1,4%. e mostraram bandas polimórficas que permitiram avaliar a distância genética entre os 15 isolados. Estes foram classificados em quatro grupos distintos. Os produtos de amplificação dos lócus ITS1-5.8S-ITS2 do rDNA com primer ITS1 e ITS4 também apresentaram polimorfismo e foram digeridos com três enzimas de restrição, Dra I, Hae III e Msp I. Apenas as duas últimas foram eficientes em mostrar variações genéticas entre os isolados. Os dois marcadores utilizados foram capazes de diferenciar o isolado do Amazonas dos demais de Pernambuco. Os resultados obtidos com os marcadores moleculares não mostraram relação com o grau de patogenicidade dos mesmos à cebola

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
As cianobactérias apresentam uma ampla variabilidade fenotípica e ecológica. Porém, esta variabilidade, muitas vezes, não corresponde à sua diversidade genética. Assim, o uso de marcadores moleculares é fundamental para os estudos de filogenia neste grupo. Entretanto, a filogenia molecular enfrenta um desafio na seleção dos marcadores devido à ocorrência relativamente frequente da transferência de genes de forma lateral entre os procariotos. Em cianobactérias os marcadores dos espaçadores dos genes ribossomais (16S-23S-ITS rDNA) e do operon da ficocianina (PC-IGS) estão entre os mais utilizados nestes estudos. Contudo, alguns trabalhos sugerem que o PC-IGS possa ter sito transferido lateralmente em sua história evolutiva. A identificação de morfoespécies dos gêneros Microcystis e Geitlerinema é baseada em caracteres morfológicos que em geral não correspondem à sua variabilidade genética. Com o objetivo de investigar a transferência lateral do operon da ficocianina em Geitlerinema e Microcystis, foram obtidas e comparadas árvores filogenéticas de ambas espécies baseadas nos marcadores PC-IGS e 16S-23S-ITS rDNA. As topologias das árvores obtidas para ambos os marcadores foram muito semelhantes e indicaram que o PC-IGS é estável e indicado para os estudos de taxonomia e filogenia de linhagens de Geitlerinema e Microcystis. Assim, hipótese inicial de transferência lateral foi refutada. Algumas linhagens tiveram seu posicionamento divergente entre um marcador e outro, o que ressalta a importância do uso de mais de um marcador em estudos de filogenia. O marcador PC-IGS apresentou melhor desempenho que 16S-23S-ITS rDNA. As árvores filogenéticas de Geitlerinema baseadas em ambos os marcadores indicaram a ocorrência de espécies crípticas dentre as linhagens estudadas e corroboraram que G. amphibium e G. unigranulatum devem ser consideradas sinonímias...
Cyanobacteria show a wide phenotypic and ecological variability, but frequently this variability does not correspond to their genetic variation. Therefore, the use of molecular markes is critical for phylogenetic studies in this group. At the same time, the selection of molecular markers represents a challenge for the molecular phylogeny due to the horizontal gene transfer, witch is a relatively common process among the prokaryotes. In cyanobacteria, makers for the ribosomal genes spacer (16S-23S-ITS rDNA) and for the phycocyanin operon spacer (PC-IGS) are among of the most used for phylogeny. However, some studies suggest that the PC-IGS marker may have been horizontally transferred during its evolutionary history. The identification of the morphospecies from the genus Microcystis and Geitlerinema is based in their morphological characters, but they generally do not correspond to genetic variability. In order to investigate the possibility of horizontal transfer of the phycocyanin operon in Microcystis and Geitlerinema, phylogenetic trees based on the PC-IGS and 16S- 23S-ITS rDNA were generated and compared. The topologies obtained for both markers were very similar, indicating that the PC-IGS marker is stable and suitable for taxonomical and phylogenetic studies in Microcystis and Geitlerinema. Therefore, the initial hypothesis of horizontal transfer was rejected. Some strains were found to have divergent positions between the trees based on the two molecular markes, witch highlights the importance of using more than one marker in phylogenetic studies. The PC-IGS marker performed better than 16S-23SITS rDNA. The Geitlerinema phylogenetic trees based on both markers indicated the occurrence of cryptic species among the strains and corroborated that G. amphibium and G. unigranulatum should be treated as synonyms. The phylogenetic tree based on PC-IGS formed a monophyletic clade... (Complete abstract click electronic access below)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
The phylogeny of the family Selenastraceae was investigated by light microscopy, molecular analysis of 18S rDNA, rbcL, ITS1-5.8S-ITS2 and ITS-2 markers. Several morphological features traditionally used for identification of genera and species were investigated. All Selenastraceae strains studied presented naked pyrenoids within the chloroplast, except for Chlorolobion, which presented starched pyrenoid. Molecular analysis showed that no isolated morphological criteria considered so far is significant for the systematic of Selenastraceae, but a set of characteristics may be appropriate to identify species of genera Ankistrodesmus, Chlorolobion, Kirchneriella, Raphidocelis and Tetranephris. Phylogenetic analyses showed that genera Monoraphidium, Kirchneriella and Selenastrum are polyphyletic and not distinguishable as separate genera. The Selenastrum morphotype revealed three different molecular lineages, leading to the description of two new genera, Curvastrum gen. nov and Messastrum gen. nov. In addition, molecular phylogenetic analysis revealed four lineages assigned to Kirchneriella orphotype, leading to the description of five new species: Genus 1 sp. nov. 1, Genus 2 sp. nov. 1, Genus 2 sp. nov. 2, Raphidocelis sp. nov. and Tetranephris sp. nov.
A filogenia da família Selenastraceae foi investigada por microscopia ótica, análises moleculares dos marcadores 18S rDNA, rbcL, ITS1-5,8S-ITS2 e ITS-2. Várias características morfológicas tradicionalmente utilizadas para identificação de gêneros e espécies foram investigadas. Todas as cepas de Selenastraceae estudadas têm pirenóides nus dentro do cloroplasto, exceto o gênero Chlorolobion, que apresentou pirenóide amilóide. As análises moleculares mostraram que nenhum critério morfológico isolado considerado até agora é significativo para a sistemática do Selenastraceae, mas o uso de um conjunto de características morfológicas pode ser adequado para identificar espécies dos gêneros Ankistrodesmus, Chlorolobion, Kirchneriella, Raphidocelis e Tetranephris. As análises filogenéticas moleculares mostraram que os gêneros Monoraphidium, Kirchneriella e Selenastrum são polifiléticos e não distinguíveis como gêneros. O morfotipo de Selenastrum revelou três linhagens moleculares diferentes, levando à descrição de dois novos gêneros, Curvastrum gen. nov. e Messastrum gen. nov. Além disso, as análises filogenéticas revelaram quatro linhagens moleculares atribuídas ao morfotipo de Kirchneriella, levando à descrição de cinco espécies novas: Gênero 1 sp. nov. 1, Gênero 2 sp. nov. 1, Gênero 2 sp. nov. 2, Raphidocelis sp. nov. e Tetranephris sp. nov.
FAPESP: 2012/19520-1

Orientador: Maria do Carmo Bittencourt de Oliveira
Banca: Luiz Henrique Zanini Branco
Banca: Mariana Cabral de Oliveira
Resumo: As cianobactérias apresentam uma ampla variabilidade fenotípica e ecológica. Porém, esta variabilidade, muitas vezes, não corresponde à sua diversidade genética. Assim, o uso de marcadores moleculares é fundamental para os estudos de filogenia neste grupo. Entretanto, a filogenia molecular enfrenta um desafio na seleção dos marcadores devido à ocorrência relativamente frequente da transferência de genes de forma lateral entre os procariotos. Em cianobactérias os marcadores dos espaçadores dos genes ribossomais (16S-23S-ITS rDNA) e do operon da ficocianina (PC-IGS) estão entre os mais utilizados nestes estudos. Contudo, alguns trabalhos sugerem que o PC-IGS possa ter sito transferido lateralmente em sua história evolutiva. A identificação de morfoespécies dos gêneros Microcystis e Geitlerinema é baseada em caracteres morfológicos que em geral não correspondem à sua variabilidade genética. Com o objetivo de investigar a transferência lateral do operon da ficocianina em Geitlerinema e Microcystis, foram obtidas e comparadas árvores filogenéticas de ambas espécies baseadas nos marcadores PC-IGS e 16S-23S-ITS rDNA. As topologias das árvores obtidas para ambos os marcadores foram muito semelhantes e indicaram que o PC-IGS é estável e indicado para os estudos de taxonomia e filogenia de linhagens de Geitlerinema e Microcystis. Assim, hipótese inicial de transferência lateral foi refutada. Algumas linhagens tiveram seu posicionamento divergente entre um marcador e outro, o que ressalta a importância do uso de mais de um marcador em estudos de filogenia. O marcador PC-IGS apresentou melhor desempenho que 16S-23S-ITS rDNA. As árvores filogenéticas de Geitlerinema baseadas em ambos os marcadores indicaram a ocorrência de espécies crípticas dentre as linhagens estudadas e corroboraram que G. amphibium e G. unigranulatum devem ser consideradas sinonímias... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Cyanobacteria show a wide phenotypic and ecological variability, but frequently this variability does not correspond to their genetic variation. Therefore, the use of molecular markes is critical for phylogenetic studies in this group. At the same time, the selection of molecular markers represents a challenge for the molecular phylogeny due to the horizontal gene transfer, witch is a relatively common process among the prokaryotes. In cyanobacteria, makers for the ribosomal genes spacer (16S-23S-ITS rDNA) and for the phycocyanin operon spacer (PC-IGS) are among of the most used for phylogeny. However, some studies suggest that the PC-IGS marker may have been horizontally transferred during its evolutionary history. The identification of the morphospecies from the genus Microcystis and Geitlerinema is based in their morphological characters, but they generally do not correspond to genetic variability. In order to investigate the possibility of horizontal transfer of the phycocyanin operon in Microcystis and Geitlerinema, phylogenetic trees based on the PC-IGS and 16S- 23S-ITS rDNA were generated and compared. The topologies obtained for both markers were very similar, indicating that the PC-IGS marker is stable and suitable for taxonomical and phylogenetic studies in Microcystis and Geitlerinema. Therefore, the initial hypothesis of horizontal transfer was rejected. Some strains were found to have divergent positions between the trees based on the two molecular markes, witch highlights the importance of using more than one marker in phylogenetic studies. The PC-IGS marker performed better than 16S-23SITS rDNA. The Geitlerinema phylogenetic trees based on both markers indicated the occurrence of cryptic species among the strains and corroborated that G. amphibium and G. unigranulatum should be treated as synonyms. The phylogenetic tree based on PC-IGS formed a monophyletic clade... (Complete abstract click electronic access below)
Mestre

Orientador: Carlos Roberto Ceron
Banca: Cláudia Maria Aparecida Carareto
Banca: Maura Manfrin
Banca: Rogério Pincela Mateus
Banca: Ana Silvia Lapenda
Resumo: As espécies de Drosophila pertencentes ao cluster buzzatii se distribuem pelo território sul americano, na área compreendida desde a Amazônia brasileira até o chaco argentino. São espécies tipicamente cactofílicas, por utilizarem cladódios de cactos em decomposição para realizarem seu ciclo de vida. Neste trabalho investigamos as relações filogenéticas entre as seguintes espécies pertencentes ao cluster buzzatii: D. richardson (cluster stalkeri), D. koepferae (linhagem B26D2), D. seriema (linhagens D54M e D40F1M), D. serido (linhagens 1431.3 e H49F1M) e D. borborema (linhagem 1282.2). Dentre as 690 posições analisadas, foram encontrados 152 sítios conservados e 173 sítios informativos para parcimônia quando todas as seqüências foram alinhadas. O número médio total de nucleotídeos obtido para alinhamento foi de 493.4, sendo que nestes foi encontrada uma porcentagem superior no conteúdo de A-T, cerca de ~70%, em relação ao conteúdo de G-C, cerca de ~30%. Os métodos da máxima parcimônia e de distância foram utilizados para o estabelecimento das relações filogenéticas entre as seqüências analisadas e demonstraram topologias semelhantes. Nossos resultados mostram que as espécies analisadas do cluster buzzatii constituem um grupo monofilético, e que D. richardsoni (cluster stalkeri) apresenta-se como uma espécie irmã colocada Resumo em uma posição basal em relação às outras espécies. Ainda com base em nossos resultados verificamos que a análise das seqüências do espaçador intergênico ITS-1 forneceu relações filogenéticas resolvidas, sem quaisquer politomias, embora o grupo de espécies analisadas seja constituído por espécies com baixa divergência genética.
Abstract: Drosophila species belonging to cluster buzzatii are cactophilic species found in South América with a distribution from Amazonian region until Argentina. In this work we investigate the phylogenetic relationship among four species of the cluster buzzatii: D. koepferae (linhagem B26D2), D. seriema (linhagens D54M e D40F1M), D. serido (linhagens 1431.3 e H49F1M) e D. borborema (linhagem 1282.2) as well as D. richardsoni (stalkeri cluster), analised as outgroup species by comparison of intergenic ITS-1 sequences of ribosomal DNA. This spacer sequences presented a length of ~550 bp in all of analysed species. It was not found any restriction site for Eco RI, Alu I, Hind III, Sal I e Hae III inside ITS-1 amplified fragments, indicating a similarity among them. Higher proportion of A-T content (~70%) was found for all analysed ITS-1 fragments, as expected for non-coding sequences. In respect to phylogenetic relation among species belonging to cluster buzzatii our data suggest that this cluster is monophyletic. However, was observed that D. serido lineages presented polymorphism higher than other species.
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Universidade Estadual Paulista (UNESP)
As espécies de Drosophila pertencentes ao cluster buzzatii se distribuem pelo território sul americano, na área compreendida desde a Amazônia brasileira até o chaco argentino. São espécies tipicamente cactofílicas, por utilizarem cladódios de cactos em decomposição para realizarem seu ciclo de vida. Neste trabalho investigamos as relações filogenéticas entre as seguintes espécies pertencentes ao cluster buzzatii: D. richardson (cluster stalkeri), D. koepferae (linhagem B26D2), D. seriema (linhagens D54M e D40F1M), D. serido (linhagens 1431.3 e H49F1M) e D. borborema (linhagem 1282.2). Dentre as 690 posições analisadas, foram encontrados 152 sítios conservados e 173 sítios informativos para parcimônia quando todas as seqüências foram alinhadas. O número médio total de nucleotídeos obtido para alinhamento foi de 493.4, sendo que nestes foi encontrada uma porcentagem superior no conteúdo de A-T, cerca de ~70%, em relação ao conteúdo de G-C, cerca de ~30%. Os métodos da máxima parcimônia e de distância foram utilizados para o estabelecimento das relações filogenéticas entre as seqüências analisadas e demonstraram topologias semelhantes. Nossos resultados mostram que as espécies analisadas do cluster buzzatii constituem um grupo monofilético, e que D. richardsoni (cluster stalkeri) apresenta-se como uma espécie irmã colocada Resumo em uma posição basal em relação às outras espécies. Ainda com base em nossos resultados verificamos que a análise das seqüências do espaçador intergênico ITS-1 forneceu relações filogenéticas resolvidas, sem quaisquer politomias, embora o grupo de espécies analisadas seja constituído por espécies com baixa divergência genética.
Drosophila species belonging to cluster buzzatii are cactophilic species found in South América with a distribution from Amazonian region until Argentina. In this work we investigate the phylogenetic relationship among four species of the cluster buzzatii: D. koepferae (linhagem B26D2), D. seriema (linhagens D54M e D40F1M), D. serido (linhagens 1431.3 e H49F1M) e D. borborema (linhagem 1282.2) as well as D. richardsoni (stalkeri cluster), analised as outgroup species by comparison of intergenic ITS-1 sequences of ribosomal DNA. This spacer sequences presented a length of ~550 bp in all of analysed species. It was not found any restriction site for Eco RI, Alu I, Hind III, Sal I e Hae III inside ITS-1 amplified fragments, indicating a similarity among them. Higher proportion of A-T content (~70%) was found for all analysed ITS-1 fragments, as expected for non-coding sequences. In respect to phylogenetic relation among species belonging to cluster buzzatii our data suggest that this cluster is monophyletic. However, was observed that D. serido lineages presented polymorphism higher than other species.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A diversidade genética de vinte e sete isolados de leveduras coletadas em laticínios, utilizando como referências dos gêneros Kluyveromyces e Debaryomyces, foi averiguada por meio de RAPD (Randomly Amplified Polymorphic DNA). A amplificação resultou em um total de oitenta e oito fragmentos polimórfico de DNA, utilizando treze oligonucleotídeos decâmeros aleatórios. As distâncias genéticas variaram de 6,5 a 71%, gerando na análise gráfica, cinco grupos geneticamente divergentes. Nas avaliações da região ITS do rDNA foi observado um polimorfismo de tamanho que variou de 380 a 710 pb. A análise de agrupamento utilizando valores da distância genética resultou na formação de oito grupos, sugerindo a existência de pelo menos oito espécies de leveduras. Na análise por PCR-RFLP da região ITS do rDNA, os produtos das amplificações foram hidrolisados com diferentes endonucleases de restrição evidenciando o padrão polimórfico, e os valores das distâncias genéticas foram utilizados para o agrupamento, resultando na formação de quinze grupos. Os agrupamentos obtidos com os marcadores moleculares possibilitaram a diferenciação genética dos isolados. Os resultados sugerem que quatro dos vinte e sete isolados pertencem à espécie Kluyveromyces lactis.
The genetic diversity of twenty-seven yeasts collected at dairies, was evaluated by RAPD using Kluyveromyces and Debaryomyces reference genera. Amplification using thirteen random oligonucleotides resulted in eighty-eight DNA polymorphic fragments. The Genetic distances varied from 6,5 to 71%, and generated a Dendrogram with five different genetic groups. The amplification of the ITS 18SrDNA region from the yeast resulted in DNA fragments with length polymorphism (380 and 710 bp). The clustering analysis using the genetic distance value produced eight groups, reflecting at least eight yeasts species. The amplification products of the rDNA ITS region using PCR-RFLP analyses were cleaved with different restriction endonucleases showing polymorphic patterns. The values of the genetic distances were used for the clustering resulted in fifteen groups. The clusters obtained using the molecular markers showed genetic difference between you isolates. The results suggest that four of the twenty-seven isolates can be identified as the yeast Kluyveromyces lactis.

Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent R. solani consists of several genetically different anastomosis groups (AGs) and subgroups. Though anastomosis or hyphal fusion reactions have been used to group Rhizoctonia species, they are time consuming and sometimes difficult to interpret. Anastomosis reactions are incapable of identifying isolates belonging to different AG subgroups within an AG. This study evaluated molecular techniques in comparison with traditional anastomosis grouping (AG) to identify and group isolates of Rhizoctonia. More than 400 Rhizoctonia isolates were collected from diseased turfgrass leaves from eight geographic areas in Virginia and Maryland. A random sample of 86 isolates was selected and initially characterized by colony morphology, nuclei staining and anastomosis grouping. Molecular identification was performed by analysis of rDNA-ITS region and DNA fingerprinting techniques universally primed PCR (UP-PCR) and amplified fragment length polymorphism (AFLP). The cladistic analysis of ITS sequences and UP-PCR fragments supported seven clusters. Isolates of R. solani AG 1-IB (n=18), AG 2-2IIIB (n=30) and AG 5 (n=1) clustered separately. Waitea circinata var. zeae (n=11), and var. circinata (n=4) grouped separately. A cluster of six isolates (UWC) did not fall into any known Waitea group. Most of the binucleate Rhizoctonia-like fungi (BNR) (n=16) grouped separately. AFLP grouping also largely agreed with the above results. However, UWC isolates clustered into two groups. Molecular analyses corresponded well with traditional anastomosis grouping by clustering isolates within an AG or AG subgroup together. UP-PCR cross-hybridization could distinguish closely related Rhizoctonia isolates to their infraspecies level. Genetically related isolates belonging to the same AG subgroups cross-hybridized strongly, while isolates of different AGs did not cross-hybridize or did so weakly. Sequence-characterized amplified region (SCAR) markers were generated from UP-PCR products to identify isolates of major pathogenic groups AG 1-IB and AG 2-2IIIB. Specific primer pairs successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from isolates of other AGs. Sensitivity of Rhizoctonia species and AGs was tested in vitro to commercial formulations of iprodione, triticonazole and pyraclostrobin. W. circinata isolates were moderately sensitive to iprodione while isolates of R. solani and BNR were extremely sensitive. Isolates of AG 2-2IIIB showed less sensitivity to triticonazole than other Rhizoctonia isolates. W. circinata var. zeae isolates were moderately sensitive to pyraclostrobin while most of the other isolates were extremely sensitive.
Ph. D.

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases.
L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP.
Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn.
S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes.
Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies.
El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempelts
La caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri.
Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP.
Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades.
Many bacteria pertaining to the fluorescent Pseudomonas group are able to control plant diseases caused by bacterial and fungal plant pathogens (BCAs). Also, some of them exhibit activity as plant growth promoting bacteria (PGPBs). Several metabolites like 2,4-diacetylphloroglucinol (PHL), phenazin-1-carboxilic acid (PCA), pyrrolnitrin (Prn), hydrogen cyanide (HCN), indol-3-acetic acid, siderophores and chitinases has been described accounting for their ability of being BCAs or PBPBs.
The aim of the present work was to compare the phenotypic and genotypic characteristics of a selected group of fluorescent Pseudomonas by means of a polyphasic approach in order to establish relationships with the ability of being BCA and PGPB.
Due to the importance of production of metabolites like Phl, PCA and Prn in biocontrol, the preliminary objective of this work was to search and isolate of metabolite producing strains. To achieve this objective a molecular approach was used based on the detection of biosynthetic genes, which are implicated in the metabolite production, instead of direct detection of the metabolites. The procedure was performed to avoid the effect of culture conditions on induction or repression of metabolite synthesis.
Two procedures were used (i) assisted search of metabolite producing strains by means of phenotypic markers and subsequent confirmation by PCR analysis, (2) direct use of PCR for gene detection in direct extracts or enrichments from samples and subsequent colony-hybridization for assistance in strain isolation. The best method for isolation of Phl producers in the type of samples processed in the present work, where the frequency of Phl producers is very low, was the assisted search by means of phenotypic markers followed of a confirmation by PCR amplification of phlD gene. Four strains harboring the PCA genes, 15 strains with Phl genes, and one with Prn genes were isolated.
A collection of 72 strains of Pseudomonas pertaining to the fluorescent group was made including 18 isolates derived from the present work which harbor the biosynthetic genes for PhL, PCA and Prn production; 6 strains from reference collections; 14 strains producing several antibiotics which were supplied by other colleagues; and a selection of 34 strains from a previous work performed by our research group. Within the collection there are many strains with promising potential as BCAs and PGPBs of several diseases and plant hosts.
The collection was characterized phenotypically and genotypically. The phenotypic characterization included identification at the species level with the aid of API20NE kit and several specific biochemical tests; the production of metabolites such as PCA, Phl, Prn, IAA, HCN, chitinases and siderophores; in vitro antagonism in several culture media against two fungi (Stemphylium vesicarium and Penicillium expansum) and three bacterial pathogens (Erwinia amylovora, Pseudomonas syringae pv. syringae and Xanthomonas arboricola pv. juglandis); efficacy in biossays of inhibition of infection of plant material by P. expansum on apple fruit tissue and S. vesicarium on pear leaves, E. amylovora on immature pear fruit, and of growth promotion in two commercial Prunus rootstocks. P. expansum causes blue mold rot of apple and pear in postharvest, S. vesicarium cause brown spot of pear and E. amylovora is the causal agent of fire blight of rosaceous plants.
The number of strains, over a total of 72 studied, producing IAA and chitinases was very low (4 producing IAA and 6 producing chitinases), whereas a high number of HCN producing strains (32) was detected which was associated to Phl production. In vitro antagonism against bacteria was highly dependent on indicator pathogen and growth medium, but the presence or absence of iron did not seem to affect. However, in the case of antagonism against fungi, no influence of the medium composition was observed. Among the collection a low frequency of strains exhibiting antagonism against 3 or 4 pathogens a total of 7 strains was observed in all tested media. Only two out of the seven strains were effective in infection inhibition bioassays caused by two of the three pathogens tested. Some of the effective strains in the in vivo assays were not antagonist for the indicator pathogen in any of the media tested. In the case of plant growth promoting strains, the growth in rootstock Marianna 2624 was more stimulated than in GF677, and there was a strain-host specificity.
Genotypic characterization of strains was performed by means of restriction fragment length polymorphism analysis of the ribosomal DNA (RFLP-rDNA) and macrorestriction fragment length polymorphism analysis of chromosomal DNA separated by pulsed field gel electrophoresis (MRFLP-PFGE). Both techniques showed a high level of genotypic diversity within the collection of strains, and no relationships were observed between clusters and phenotypic characteristics or ability to be BCA or PGPB. Patterns of MRFLP-PFGE for the model bacterium P. fluorescens EPS288 were found stable within time and independent of cultivation conditions in the laboratory or under natural conditions. Therefore the method is highly suitable for identification of strains reisolated from natural samples previously inoculated with the bacterium.
A further selection of strains which produce phloroglucinol were characterized by RFLP analysis and phlD sequencing. The groups observed were consistent among the RFLP-rDNA, RFLP-phlD and gene sequence data. Phylogenetic analysis obtained using phlD sequences showed a high level of polymorphism revealing three main clusters. These clusters appear to be related with metabolite production: a first cluster producing Phl, HCN and Prn, a second producing Phl and HCN, and a third producing only Phl. However, this groups were related neither to the geographic origin of strains nor to the activity as BCA or PGPB.
A multivariate analysis (correspondence, pairwise Spearman rank correlation, and frequency analysis)was performed using data of phenotypic and genotypic characterization of strains. The results emphasize the importance of discarding from the database those strains being genetically identical because skew the final results. The three types of analysis did not revealed a significant and consistent relationship between the activity of infection inhibition or growth promotion of the strains and their characteristics.

Subject of information entity is one of the fundamental concepts in the field of information science. Subject of any document represents its intellectual potential -- 'aboutness' of the document. Traditionally, subject (along with title and author) is the one of three major ways to access information, so subject metadata plays a central role in this process and the role is constantly growing. Previous research concluded that the larger bibliographic database is, the richer subject vocabularies and classification schemes are needed to support information discovery. Further, a high proportion of information objects are unretrievable without subject headings in metadata records. This exploratory study provides the analysis of the subject metadata in MARC 21 bibliographic records created in 2020; and develops understanding of the level and patterns of 'aboutness' representation in the MARC 21 bibliographic records. Study also examines how these records apply the recent RDA and MARC21 guidelines and features intended to support functionality in a Linked Data environment. Methods of Social Network Analysis were applied along with content analysis, to answer research questions of this study. Suggestions for future research, implications for education, and practical recommendations for library metadata creation and management are discussed.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 ìm2) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

Relatório de projecto de estágio para obtenção de grau de Mestre em Biologia Molecular em Saúde
"No presente trabalho, estudaram-se aspectos morfológicos, histoquímicos e moleculares da medusa Catostylus tagi. As abordagens morfológica e histoquímica focalizaram-se nos nematocistos do animal. Através da análise por microscopia óptica e electrónica de varrimento, determinaram-se parâmetros em variedades de isorhizas e euryteles. Utilizando colorações histoquímicas constatou-se que as proteínas NOWA e “spinalin”, detectadas em Hydras, podem estar presentes na C. tagi, a primeira na membrana exterior da cápsula e a segunda integrando o mecanismo de ejecção. Ainda em relação às macromoléculas, verificou-se que a membrana da cápsula pode conter mucinas ácidas, com polissacáridos semelhantes à quitina, enquanto os componentes azotados, distribuídos no interior do nematocisto, assemelham-se a materiais colagénicos. Pela ausência de cobre, foi descartada a ocorrência de proteínas antioxidantes do tipo superóxido dismutase. Em relação ao cálcio, observou-se a sua presença na membrana envolvente dos nematocistos e no sistema contráctil, provavelmente complexado com as macromoléculas aniónicas. A propósito das relações genéticas, compararam-se exemplares de Catostylus que ocorrem nos estuários do Tejo e do Sado, e estes com outras três medusas, nomeadamente Catostylus mosaicus, Cyanea capillata e Aurelia aurita, através das sequenciações parciais dos genes que codificam para os RNAs ribossomal 18S e 28S, além da sequenciação total do espaçador interno transcrito 1 (ITS1). A análise cladística, baseada no método da máxima verosimilhança, confirmou os resultados macroscópicos que indicam que os exemplares do Sado e Tejo pertencem à mesma espécie, denominada C. tagi. Na comparação da C. tagi com as outras medusas, comprovou-se a sua relação monofilética com a C.mosaicus e parafilética com a C. capillata e a A. aurita. Os resultados deste estudo elucidaram, pela primeira vez, a tipologia e a composição química parcial dos nematocistos da C.tagi, e comprovaram que a recolha para análise pode ser feita indistintamente no Sado ou no Tejo."

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados
Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)

Orientadores: Shirlei Maria Recco Pimentel, Luciana Bolsoni Lourenço
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-26T06:02:48Z (GMT). No. of bitstreams: 1 Vittorazzi_StenioEder_D.pdf: 3469046 bytes, checksum: 548f412477dbed97bd45f19e26cea793 (MD5) Previous issue date: 2014
Resumo: Os genomas dos eucariotos são ricos em sequências repetitivas, as quais compreendem sequências dispersas e em tandem. Entre as sequências em tandem, incluem-se os DNA satélites, os DNA ribossomais e cópias parálogas. Os DNA satélites são os principais constituintes da heterocromatina constitutiva e os genes ribossomais transcrevem os RNAr para compor os ribossomos, que dividem-se em duas famílias, DNAr 45S e 5S. Em um mesmo organismo, diferentes tipos de DNAr 5S já foram reconhecidos, mostrando a existência de uma diversidade quanto a esse tipo de sequência. No anuro Physalaemus cuvieri, uma forma de DNA satélite denominada PcP190 foi caracterizada e teve sua origem atribuída a uma derivação do DNAr 5S em uma espécie ancestral. Em diferentes populações de P. cuvieri estudadas previamente com as ferramentas da citogenética convencional, uma acentuada variação interpopulacional nos cromossomos portadores da NOR pôde ser observada, e nas demais espécies do grupo de P. cuvieri, peculiaridades interespecíficas foram evidenciadas, porém, os cariótipos entre essas espécies são muito semelhantes. O objetivo nessa tese foi ampliar o estudo citogenético de espécies do grupo de P. cuvieri que possuíam carência na descrição cromossômica, como P. kroyeri e P. cicada. Objetivou-se também analisar o DNA satélite PcP190 em nível cromossômico e molecular, assim como estudar a organização molecular e a diversidade de sequências do DNAr 5S no genoma das espécies de Physalaemus e espécies de gêneros relacionados. Com os resultados da pesquisa, três capítulos são apresentados, sendo eles: (i) "Long-time evolution and highly dynamic satellite DNA in leptodactylid and hylodid frogs", o qual mostra que o DNA satélite PcP190 é amplamente conservado e pode ser reconhecido em representantes de duas famílias de anuros, Leptodactylidae e Hylodidae; além disso, mostra também que essas sequências são altamente dinâmicas nos cromossomos das espécies do grupo de P. cuvieri. (ii) "Diversidade do DNAr 5S em Leiuperinae (Anura)", os resultados mostram que no genoma desses anuros existe uma diversidade dessa classe de família multigênica maior do que proposto até então, e que essas sequências do DNAr 5S permanecem conservadas desde a divergência evolutiva entre os Actinopterigio e Sarcopterigio. (iii) "Cariótipo e mapeamento de DNA repetitivo em Physalaemus kroyeri e P. cicada (Anura Leptodactylidae)", onde é apresentado que a banda 5p de P. kroyeri tem se mostrado um bom marcador cromossômico para as espécies do grupo de P. cuvieri, o que indica se tratar de uma sinapomorfia cromossômica. Contrariamente, a ausência dessa banda 5p em P. cicada não fornece evidência para a inclusão de P. cicada no grupo de P. cuvieri ou em qualquer outro grupo de espécies
Abstract: The genomes of eukaryotic organisms are rich in repetitive DNA sequences, which can be dispersed or arrayed in tandem. The tandem repeat sequences include the satellite DNA, the ribosomal DNA, and paralogous copies. Satellite DNA is the main component of constitutive heterochromatin, while the ribosomal genes encode the rRNAs that make up the ribosomes; they are divided into two families, the 45S and 5S rRNA. Different types of 5S rDNA have been identified in a single organism, proving that there is diversity in this type of DNA sequence. In the anuran Physalaemus cuvieri, a satellite DNA family called PcP190 has been identified, which is thought to have derived from the 5S rDNA of an ancestor species. In different populations of P. cuvieri that were previously studied with conventional cytogenetic tools, an accentuated interpopulational variation among chromosomes harboring the NOR was observed, while in other species of the P. cuvieri group, interspecific traits were found. However, the karyotypes in these species are very similar. The aim of this thesis was to expand the cytogenetic studies on P. cuvieri species that needed further chromosomal description, such as P. kroyeri and P. cicada. Another objective was to analyze the PcP190 satellite DNA at the chromosomal and molecular level, as well as to study the molecular organization and the diversity of 5S rDNA sequences in the genomes of the Physalaemus species and other species of related genera. We present three chapters as a result of this research: (i) "Long-time evolution and highly dynamic satellite DNA in frogs," which demonstrates that the PcP190 satellite DNA is widely conserved and was recognized in representatives of two anurans families, Leptodactylidae and Hylodidae. Moreover, it also demonstrates that these sequences are highly dynamic within the chromosomes of the P. cuvieri species group. (ii) "5S rDNA in Leiuperinae (Anura): new insights on its evolution." The results show that in the genomes of these anurans there is wider diversity among this multigenic family than previously assumed and that these 5S rDNA sequences have remained conserved since the evolutionary divergence of Actinopterygii and Sarcopterygii. (iii) "Karyotype and repetitive DNA mapping of the Physalaemus kroyeri and P. cicada (Anura Leptodactylidae)," which demonstrates that the 5p chromosomal band of P. kroyeri is a good chromosomal marker for species from the P. cuvieri group, indicating that it is a chromosomal synapomorphy. Conversely, the absence of this 5p band in P. cicada does not provide evidence for the inclusion of this species in the P. cuvieri group or any other species group
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Η πραγματική ταξινομική κατάσταση των πληθυσμών του γένους Ligidium, κυρίως αυτών που κατανέμονται στη νότια Ελλάδα, δεν είναι ξεκάθαρη. Αυτό συμβαίνει γιατί η απόληξη του δεύτερου ενδοποδίτη του πλεοποδίου των αρσενικών, που αποτελεί σημαντικό διαγνωστικό χαρακτήρα, ποικίλλει και η εύθραυστη δομή της μπορεί εύκολα να καταστραφεί και κατ’ επέκταση να οδηγήσει σε μια μη ακριβή περιγραφή της κατάστασης αυτού του χαρακτήρα. Οι παλαιότερες μελέτες των φυλογενετικών σχέσεων των ισοπόδων του γένους Ligidium, με χρήση μοριακών δεικτών είναι ολιγάριθμες. Πιο συγκεκριμένα, για τη μελέτη πληθυσμών του γένους Ligidium από τον ελλαδικό χώρο έχει γίνει μόνο μια μελέτη στην οποία χρησιμοποιήθηκε η τεχνική της αλληλούχισης του DNA. Η παρούσα μεταπτυχιακή εργασία αποτελεί την πιο εκτεταμένη φυλογενετική ανάλυση μέσα στο γένος Ligidium. H έρευνα επιλέχθηκε να γίνει σε επίπεδο ατόμων συλλέγοντας πολλούς πληθυσμούς από διαφορετικές περιοχές, οι οποίοι ανήκουν είτε στο ίδιο είτε σε διαφορετικά ποτάμια συστήματα. Συγκεκριμένα χρησιμοποιήθηκαν μοριακοί δείκτες από 26 πληθυσμούς ισοπόδων, οι οποίοι καλύπτουν σχεδόν όλο το φάσμα των ειδών, που ανήκουν στo γένος Ligidium και διαβιούν στον ελλαδικό χώρο. Η πειραματική προσέγγιση περιελάμβανε τον πολλαπλασιασμό αλληλουχιών με τη μέθοδο της αλυσιδωτής αντίδρασης πολυμεράσης (PCR), τον προσδιορισμό της αλληλουχίας τους, και τη στατιστική και φυλογενετική ανάλυσή τους. Οι αλληλουχίες που χρησιμοποιήθηκαν προέρχονται από τους πυρηνικούς γονιδιακούς τόπους ITS1, 5.8s, ITS2 οι οποίοι είναι από τους πιο ευρέως διαδεδομένους μοριακούς δείκτες για μελέτες σε επίπεδο πληθυσμών και ειδών και δεν έχουν προηγουμένως μελετηθεί για άλλα χερσαία ισόποδα. Οι αλληλουχίες επεξεργάστηκαν με τρεις μεθόδους φυλογενετικής ανάλυσης οι οποίες είναι η μέθοδος σύνδεσης γειτόνων (Neighbor-Joining), η Μέθοδος Μέγιστης Φειδωλότητας (Maximum Parsimony) και η Μπεϊεσιανή Συμπερασματολογία (Bayesian Inference). To τελικό σύνολο των στοιχισμένων αλληλουχιών, μετά την αφαίρεση τμημάτων από την αρχή και το τέλος τους, είχε μήκος 614 βάσεων. Σε γενικές γραμμές τα αποτελέσματα που προέκυψαν από τη χρήση των εν λόγω πυρηνικών δεικτών και των τριών φυλογενετικών μεθόδων (BI, MP, NJ), τόσο ως προς τα δέντρα όσο και ως προς τις γενετικές αποστάσεις, δεν θεωρούνται ασφαλή για την εξαγωγή ικανοποιητικών συμπερασμάτων. Επίσης πραγματοποιήθηκε σύγκριση αποτελεσμάτων προηγούμενων μελετών με αυτά που προκύπτουν από τη δική μας έρευνα, καθώς και αξιολόγηση της αξιοπιστίας των μοριακών δεικτών που χρησιμοποιήθηκαν.
Terrestrial isopods (suborder Oniscidea) form a monophyletic group within the crustacean order Isopoda that has conquered almost all terrestrial environments. The family Ligiidae, forming the sister group to all the other Oniscidea (with the possible exception of Tylidae) consists of five genera. The genus Ligidium is the most species-rich genus within Ligiidae and consists of 47 species distributed throughout the northern hemisphere, from western Europe to Japan and North America. The taxonomy of the genus has hitherto been based on a few morphological characters most of which exhibit high intrapopulation variation. As a consequence, the current taxonomy of Ligidium is problematic and the validity of described species cannot be taken for granted. Molecular data for only a few species have been used in broader phylogenetic analyses within terrestrial isopods In the present study we perform the most extensive phylogenetic analysis within the genus Ligidium. We attempt to clarify the taxonomic status of the various populations and try to identify geographical patterns in genealogical affinities that might help us reconstruct the evolutionary history of the respective species. We used three nuclear ribosomal gene segments (ITS1, 5.8s, ITS2) for the phylogenetic analysis of Ligidium populations, which were collected from several locations in Greece, representing almost all species reported from this region. For comparative reasons, we have also included one population from each of the two widespread European species, L. hypnorum, Cuvier, 1792 and L. germanicum, Verhoeff, 1901. Since the taxonomy within the genus is still ambiguous, and because most morphological characters are not dependable for species identification, we chose to perform the analysis at the individual level, sampling several populations from different locations that belong either to the same or to different river systems. Fragments of the three rDNA genes (ITS1, 5.8s, ITS2) were amplified using the polymerase chain reaction (PCR) technique and then individual sequences were determined via automated sequencing. We compare the results which occurred from our study with those of previous ones and we evaluate the plylogenetic utility of the molecular markers that we used. In general the results that came out by the analyses of three following phylogenetic methods Neighbor joining (NJ), Maximum parsimony (MP) and Bayesian Inference (BI), are not considered to be safe for making generalized conclusions.

We can find a lot of microorganisms living in snow including psychrophilic snow algae from the order Chlamydomonadales (Chlorophyta). They are adapted to the extreme conditions in this habitat and can cause the phenomenon of coloured snow. The species Chlamydomonas nivalis (Bauer) Wille is the most commonly associated with red snow in alpine and polar regions during summer season worldwide. In the field material, we can find red spherical cells without flagella and any morphological characteristics suitable for species determination. Until now, this species has not been isolated into laboratory culture and its life cycle is unclear. Furthermore it has been shown that red coloured snow can be caused by more species which used to be determined as Chlamydomonas nivalis. The aim of this study was to collect samples of red snow from different parts of Europe, to describe the morphological variability of Chlamydomonas nivalis-like snow algae in relation to region of origin, to try to isolate laboratory strain of this species and to describe its position and distribution by phylogenetic analysis of laboratory strains and field samples. Red snow samples were collected from 30 European localities in Slovenian Alps, Romania, Dolomites, Ötztal, Wallis and Sarntal Alps, High Tauern, Ortler massif, in Norway,...

Since the human inclination to estimate and trace natural diversity, usable species definitions as well as taxonomical systems are required. As a consequence, the first proposed classification schemes assigned the filamentous and parenchymatous taxa to the green algal order Chaetophorales sensu Wille. The introduction of ultrastructural and molecular methods provided novel insight into algal evolution and generated taxonomic revisions based on phylogenetic inference. However, until now, the number of molecular phylogenetic studies focusing on the Chaetophorales s.s. is surprisingly low. To enhance knowledge about phylogenetic relationships among taxa within the order, the nuclear?encoded SSU rDNA sequences from 30 strains covering all three chaetophoralean families have been investigated. All revealed monophyletic groupings were further screened for molecular non-homoplasious synapomorphies within the Viridiplantae. To address the question of the correspondence between morphological characters traditionally used for taxonomical delimitation of the Chaetophorales and the tree topology favored by molecular data, the list of morphological/ ultrastructural/ecological characters was elaborated and further analyzed. In addition, to obtain a close-up view into the evolution of Compensatory Base Changes (CBCs) of the second internal transcribed spacer (ITS2) which is currently often used to delimit putative biological species, 86 newly obtained/published sequences of ITS2 for five families of the order Ulvales were analyzed. Furthermore, a detailed comparative study of all ITS2 substitutions has been done. Subsequently all revealed CBCs and hemi- CBCs have been mapped upon the ITS2 phylogenetic tree topology. Finally, CBCs/hCBCs taxonomic inference in the Ulvales has been discussed.

碩士
國立臺灣大學
植物學研究所
91
17~18S-5.8S-25~28S rDNA are organized into clusters of tandem repeat with each repeat consisting of a transcribed region, two small ITS, and an IGS. The ITS have sequences diverged more than the transcribed region, besides the small size and the presence of highly conserved sequences flanking each of ITS make it easy to amplify and useful for phylogenetic analyses of interspecies. In this study, ITSs of 28 Phalaenopsis species were amplified by the polymerase chain reaction which use specific primers devised by 17~18S and 25~28S transcribed region of P. ahrodite. Phylogenetic analysis of ITS consensus sequences could be divided into six groups. The second group contains P. stobartiana and P. parishii and the third group contains P. lued-pulchra, P. pallens, and P. fasciata. These two groups were both consistent with 5S rDNA IGS analysis. P. amboinensis, P. venosa and P. violacea were grouped together which was consistent with cytogenetic data, RAPD analysis, and 5S rDNA IGS analysis. The P. equestris, P. amabilis, P. aphrodite, P. sanderiana, P. stuartiana, and P. schilleriana were divided into three groups, which differ from the phylogenies based on 5S rDNA IGS analysis.

碩士
大仁科技大學
生物科技研究所
101
The ninety percent of Chinese herbal medicine in Taiwan has been imported from China. It is said that the quality of Chinese herbal medicine imported is not uniform with overdose heavy metal and pesticide residue. Therefore, the control of quality in Chinese herbal medicine is a significant issue for compatriots’ health. First of all, the Chinese herbal medicine strain is confirmed from the sources in the control of quality. The traditional identification in Chinese herbal medicine gives priority to appearances, tissue slice, and thin layer chromatography. In recent years, some scholars have assisted in the origin identification of Chinese herbal medicine by means of the molecular biology method. Analysis in this thesis, compared with the nucleotide sequences of the internal transcribed spacers (ITS) of the ribosomal, among several kinds of Chinese herbal medicine produced in Taiwan, is to be aware if this sequence can be taken as the auxiliary tool for the origin identification. The layouts in different Chinese herbal medicine primers, during the experiment, are to compare with the ITS sequence from the different species of the same genus in Chinese herbal medicine NCBI GenBank, including Lonicera spp., Salvia miltiorrhiza, Andrographis paniculata, Dioscorea spp., Taraxacum spp., and etc. After proceeding as the multiple sequences alignment in utilization of software, we design diverse pairs of conserved primers; then make good use of liquid nitrogen to grind Chinese herbal medicine for its chromosome DNA. To amplify its sequence in ITS rDNA by Polymerase Chain Reaction (PCR) method, to connect the PCR product and the TA cloning vector, to deliver clone to Biotechnology company for the sequencing, as well as to make sequence alignment from the sequence we got by NCBI GenBank are required. The identity of sequence in Salvia miltiorrhiza, Dioscorea spp., and Taraxacum spp. between grown in Taiwan and gotten in GenBank reaches more than ninety-nine percent. Again, the diversity among other different species of the same genus is much higher. It is a proof that these three sorts of Chinese herbal medicine can be made into the sequence identification by ITS rDNA. The identity of sequences in Lonicera spp. collected from distinct habitants in Taiwan reaches ninety-nine percent; however, the identity of Lonicera spp. in China or USA GenBank reaches merely eighty-three percent. Two possibilities are as follows: one, Lonicera spp. grown in Taiwan is endemic species, and the other, the amplifying sequences in Lonicera spp, is another set of ITS. Therefore, the sequence in GenBank is much far from the one grown in Taiwan. Owing to the result from those experiments, it is known that the ITS sequences analysis method cannot be identified effectively as five kinds of Chinese herbal medicine origin but possibly as the auxiliary tool. Keywords: Chinese herbal medicine, internal transcribed spacers, identification

碩士
國立嘉義大學
園藝學系研究所
94
In order to establish pomegranate cultivation in Taiwan, selected pomegranate lines which were introduced from Mainland China, Singapore and United States, had been assayed for their growth and physiological characteristics, phylogenetic relation by contents of polyphenolic compounds, ITS sequence of rDNA, ISSR and RAPD markers. The investigation was performed using four large-fruited lines of three-year-old plants. Gradual increase of the fresh fruit weight, width, length, and volume was observed during the experimental period. Twenty days post-fruit setting, fruits grew more in width than in length. Thirty days post-fruit setting, its pericarp weight was greater than seed weight. However, fifty days post-fruit setting, the seed weight was greater than the pericarp weight. The result showed that four pomegranate lines with different shapes were affected mainly by genetic components. On the other hand, soluble solid contents increased but accompanied decrease in acidity during fruit development. At hundred days post-fruit setting, soluble solid juice contents of A-01 and B-02 lines were reached the maximum. For the period of maximal full bloom, A-01 and A-02 lines flowered approximately at September whereas B-01 and B-02 lines reached in December. The analysis of fruit setting percentage, quality, yield and full bloom suggests that pomegranate will be harvested for marketing and breeding at hundred day post-fruit setting. Polyphenol contents of pericarp from 31.8% to 45.6% were measured by HPLC. B-01 line had the highest content among 4 Singapore lines. Based on cluster analysis of polyphenol data, five pomegranate lines could be divided into two groups: one group included A-02, B-01 and B-02, the others were A-01 and the commercial fruit line. To use the molecular technology to differential these accessions, sequence analysis of 5.8S rDNA and internal transcribed spacer (ITS) in eight samples of five lines. It showed no differences among 5 lines. In RAPD analysis twenty-four polymorphic bands were amplified by six primers, The polymorphism rate was 28.37%. A clone-specific band was only found in Singapore lines. Using UPGMA clustering analysis of RAPD polymorphic bands, the pomegranate could be divided into five groups with their similarities of 71%. Although many polymorphic bands were produced amongst different lines none showed any correlation with fruit color or growth characters. The cophenetic correlation coefficient of Nei/Li's and Jaccard's similarity coefficient of RAPD was 0.995. Dinucleotides motif: (GA)n sequence presented major variations in pomegranate genome through ISSR analysis. Based on UPGMA clustering analysis of ISSR polymorphic bands, the pomegranate could be divided into three groups with their similarities of 87% to 88%. Pomegranate lines of 3 origins could be distinguished by seven polymorphic bands. The result of ISSR clustering analysis coincided with the origins of pomegranate lines. The cophenetic correlation coefficients of RAPD and ISSR were 0.467 and 0.351, respectively. It suggested that two molecular markers could be used in identifying part of pomegranate lines. Key words: Pomegranate; Flowering; Fruit growth and development; polyphenolic compounds; ITS; RAPD; ISSR; Similarity; Identification

碩士
國立中興大學
昆蟲學系
93
The development, longevity, fecundity and population parameters of the cowpea aphid, Aphis craccivora, were studied at six constant temperatures (10, 15, 20, 25, 30 and 35℃) on the asparagus bean, Vigna sesquipedalis, in the laboratory. The results showed that the development time of immature stage shortened from 28.1 days at 10℃ to 4.2 days at 30℃. According to the linear regression analysis of development rate and temperature between 10 and 30℃, the overall immature development required 97.2 day-degrees above 7.4℃. The adult longevity shortened from 42.2 days at 15℃ to 6.2 days with increasing temperature to 35℃. At 15 to 25℃, the fecundity was more than 82 (offspring/♀/life). The intrinsic rate of increase (r) and the finite rate of increase (λ) were the lowest at 10℃ (r = 0.0437/d, λ= 1.0447/d), and highest at 30℃ (r = 0.4330/d, λ= 1.5419/d). The population reared at 25℃ had the highest net reproductive rate (R0 = 97.1 offspring/♀/generation). The mean generation time (T) shortened as the temperature increased from 41.1 days at 10℃ to 8.3 days at 35℃. The sequence of bacterial 16S rDNA of A. craccivora revealed that it belongs to primary symbionts, Buchnera sp. According to the RFLP maps of Buchnera 16S rDNA, cowpea aphid seems not to have secondary symbionts. We designed specific primers, AcPS128F and AcPS453R, based on non-conserved regions of 16S rDNA, to quantify the Buchnera 16S rDNA of A. craccivora by real-time PCR technique. The result indicated that the related contents of Buchnera 16S rDNA of older nymphs were higher than those of young nymphs at 10 to 30℃. When temperature was lower, the tendency of such phenomenon was more obvious. In the whole reproduction stage of A. craccivora, the related contents of Buchnera 16S rDNA was higher at 10-25℃than at 30℃ and 35℃.

碩士
臺灣大學
園藝學研究所
98
In order to establish genetic relationship of native lily in Matsu, Random Amplified Polymorphic DNA（RAPD）markers and rDNA Internal Transcribed Spacer（ITS）were analyzed. In RAPD analysis, 10 of 60 primers screened to provide polymorphic and reproducible bands. A total of 35 strong polymorphic bands were scored for 25 individuals of native lily collected in Matsu (21), Kinmen (3) and Guangxi China (1), and 15 individuals of outgroup. Principle coordinate analysis and unweighted pair group method with arithmetic mean（UPGMA）cluster analysis based on these RAPD profiles were performed. A clear distinction was found between the native lily in Matsu and outgroups. The results of analysis of molecular variance（AMOVA）of native lily in Matsu and Kinmen population indicated that 13％ of the total variation was attributable to the differences among population while 87％ was due to variation among individuals within population. The results of direct sequencing of PCR products and sequencing by cloning PCR products of the ITS and the 5.8S coding regions of nuclear ribosomal DNA in native lily in Matsu, L. longiflorum and L. formosanum were compared and phylogenetically analyzed. The length of ITS region was 626 to 627 bp in native lily in Matsu, including 229 bp of ITS1, 164 bp of 5.8S rRNA gene, and 233 to 234 bp of ITS2. The GC content was about 55.49%~66.09%. Variable sites mainly occurred in the ITS1 and ITS2. A dendrogram generated by neighbor-joining（NJ）and UPGMA cluster analysis. According to the dendrogram, two main clusters were generated: (1) native lily in Matsu and Kinmen, (2) L. longiflorum and L. formosanum. In ITS sequence analysis, direct sequencing data analyzed by UPGMA method would be more reasonable than NJ method, while the NJ method could get a better interpretation with cloning sequencing data. The results showed an explicit line of demarcation between native lily in Matsu and outgroup. Because the native lily in Matsu and other Lilium brownii were classified in the same group, we concluded that native lily in Matsu should be L. brownii. Owing to the sample number of L. brownii were too few, it needs further study to confirm that the native lily in Matsu is really unique.

Green algae are quite important primary producers in fresh waters. The genus Chlorella represents one of algae most frequently utilized in algal biotechnologies to produce biomass, using either autotrophic or heterotrophic cultivation systems. It is than exploited as a food supplement for humans or animals. However, particular species within the genus are morphologically indistinguishable and molecular markers should be used to characterize production strains. This work is aimed to molecularly characterize three production strains of Chlorella for patent protection purposes and to specify their phylogenetic and taxonomic position.