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FINAL 2 FINAL MESMO ESSE é VERDADE.Xlsx UNIVERSIDADE ESTADUAL DE PONTA GROSSA SETOR DE CIENCIAS AGRÁRIAS E DE TECNOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM COMPUTAÇÃO APLICADA BARBARA SCHAEDLER FIDELIS SCHNEIDER ANÁLISE METAGENÔMICA DE BACTÉRIAS DIAZOTRÓFICAS DE SOLOS ORGÂNICOS DO ESTADO DO PARANÁ PONTA GROSSA 2016 BARBARA SCHAEDLER FIDELIS SCHNEIDER ANÁLISE METAGENÔMICA DE BACTÉRIAS DIAZOTRÓFICAS DE SOLOS ORGÂNICOS DO ESTADO DO PARANÁ Dissertação de Mestrado apresentada ao Programa de Pós-Graduação em Computação Aplicada da Universidade Estadual de Ponta Grossa, como parte dos requisitos para obtenção do título de mestre em Computação Aplicada na Área de Tecnologias para Agricultura. Orientador: Prof. Dr. Rafael Mazer Etto PONTA GROSSA 2016 Dedico este trabalho à minha filha Clara, ao meu esposo Diogo, aos meus pais Rita e Jorge e aos meus irmãos Debora e Ronaldo, por terem me apoiado e por serem minha fortaleza e inspiração. AGRADECIMENTOS À Deus, por ter me dado saúde e forças para realizar este trabalho. À minha família, por ser minha fortaleza, me proporcionar amor e carinho nos momentos mais difíceis que vivenciei durante esta fase, principalmente por acreditarem em mim e me alegrarem com seus abraços apertados e suas palavras de amor. À CAPES, pela bolsa de estudos. Ao meu orientador Prof. Dr Rafael Mazer Etto, por toda paciência e todos os ensinamentos. À toda a equipe do Laboratório de Biologia Molecular Microbiana da Universidade Estadual de Ponta Grossa, pelo coleguismo e ensinamentos que recebi durante esta fase. A todos os professores e colegas do Programa de Pós-Graduação em Computação Aplicada da Universidade Estadual de Ponta Grossa, por todos os ensinamentos, incentivo, conversas e apoio. Aos professores, componentes da banca examinadora, Dr. Leonardo Magalhães Cruz e Drª Jesiane Stefania da Silva Batista, pela avaliação e sugestões para enriquecimento deste trabalho. “Eu não disse que seria fácil, mas que valeria a pena!” (São João Bosco) RESUMO Os solos orgânicos dos Campos de Altitude paranaense caracterizam-se por serem grandes reservatórios de água, fixarem carbono, terem baixo pH e serem pobres em nutrientes. Embora sejam essenciais para a Mata Atlântica, há pouca informação sobre os microrganismos envolvidos na sua manutenção. Nesse trabalho, foram analisados dados microbiológicos de solos orgânicos de três regiões distintas, durante as estações de seca e chuva. Utilizando ferramentas de bioinformática e estatística foram analisadas pirossequencias do gene nifH para determinar a estrutura das comunidades diazotróficas e para identificar os fatores ambientais que afetam a sua diversidade. As sequências do gene nifH mostraram a prevalência de Proteobactéria (35,25%) seguida por Spirochaetes (1,27%), Firmicutes (0,80%), Cyanobacteria (0,76%), Chlorobi (0,41%), Actinobacteria (0,10%) e Bacteriodetes (0,04%). Bradyrhizobium foi o gênero mais abundante nas seis bibliotecas analisadas, apresentando correlação positiva com o aumento do pH dos solos. A maioria das OTU do gene nifH foram cosmopolitas e o endemismo está relacionado às espécies raras. As OTU classificadas como Proteobacteria, Calothrix, Spirochaeta, Halorhodospira, Rhizobiales, Alfaproteobacteria e Bradyrhizobium apresentaram significativa correlação com os índices pluviométricos. Palavras Chave: Bioinformática, Fixação Biológica de Nitrogênio, nifH, Organossolo, Ecologia Microbiana. ABSTRACT The organic soils from High-Elevation Grasslands of the Parana State are characterized by being large water tanks which fix carbon, have low pH and are poor in nutrients. Though are essential to the Atlantic Forest, there is little information on the microorganisms involved in its maintenance. In this work, microbiological data of organic soils in three different regions, during the dry and rainy seasons were analyzed. Using bioinformatics and statistic tools, nifH gene pirosequences were analyzed to determine the structure of diazotrophic communities and to identify the environmental factors that affect their diversity. The sequences of the nifH gene showed the prevalence of Proteobacteria (35.25%) followed by Spirochaetes (1.27%), Firmicutes (0.80%), Cyanobacteria (0.76%), Chlorobi (0.41%) Actinobacteria (0.10%) and Bacteriodetes (0.04%). Bradyrhizobium was the most abundant genus in the six libraries analyzed, presenting a positive correlation with the increase in the soil pH. Most of nifH gene OTUs showed to be cosmopolitan and the endemism was related to rare species. The OTUs classified as Proteobacteria, Calothrix, Spirochaeta, Halorhodospira, Rhizobiales, Alphaproteobacteria and Bradyrhizobium presented a significant correlation with the rainfall indexes. Keywords: Bioinformatics, Biological Nitrogen Fixation, nifH, Histosol, Microbial Ecology. LISTA DE ILUSTRAÇÕES Figura 1 - Principais processos do ciclo do nitrogênio no solo. ................................. 17 Figura 2 - Distribuição dos organismos detentores de genes nif encontrados no domínio bactéria ........................................................................................................ 19 Figura 3 - Evolução do custo e da quantidade de dados gerados. ............................ 24 Figura 4 - Organograma da metodologia utilizada neste trabalho. ............................ 26 Figura 6 – Classificação taxonômica das sequências em nível de filo para a amostra SMseca ..................................................................................................................... 32 Figura 7 - Classificação taxonômica das sequências em nível de filo para a amostra SMchuvosa. ............................................................................................................... 33 Figura 8 - Classificação taxonômica das sequências em nível de Filo para a amostra SPseca ...................................................................................................................... 34 Figura 9 - Classificação taxonômica das sequências em nível de Filo para a amostra SPchuvosa ................................................................................................................ 35 Figura 10 - Classificação taxonômica das sequências em nível de Filo para a amostra TPseca. ....................................................................................................... 37 Figura 11 - Classificação taxonômica das sequências em nível de filo para a amostra TPchuvosa. ............................................................................................................... 38 Figura 12 - Classificação taxonômica das OTU0.08 da Serra do Mar em nível de filo. .................................................................................................................................. 41 Figura 13 - Classificação taxonômica das OTU0.08 do Segundo Planalto em nível de filo. compartilhadas nas duas estações. .................................................................... 43 Figura 14 - Classificação taxonômica das OTU0.08 do Terceiro Planalto em nível de filo. ............................................................................................................................. 45 Figura 15 - Distribuição das OTU0.08 nas estações seca e chuvosa.......................... 46 Figura 16 - Classificação taxonômica das OTU0.08 nas estações seca e chuvosa em nível de filo ................................................................................................................ 48 Figura 17 - Distribuição das OTU0.08 considerando a distribuição geográfica das três bibliotecas. ................................................................................................................ 50 Figura 18 - Classificação taxonômica das OTU0.08 nas três unidades fisiográficas em nível de filo .......................................................................................................... 52 Figura 19 - Rede de Interações entre as OTU0.08 compartilhadas e as bibliotecas SM, SMrainy, SP, SPrainy, TP e TPrainy. ................................................................. 54 Figura 20 - Ordenação das bibliotecas de NifH com base nos atributos químicos do solo, por Análise de Componentes Principais (PCA .................................................. 60 Figura 21 - Ordenação dos locais com base na abundância de OTU0.08 de bactérias diazotróficas (gene nifH)............................................................................................ 61 Figura 22 – Comparação dos perfis de diversidade de Renyi, com base nas OTU0.08. .................................................................................................................................. 64 Figura 23 - Composição taxonômica das pirosequencias de 16S rRNA e NifH. ....... 68 LISTA DE TABELAS Tabela 1 - Produtos dos genes Nif e suas funções, conhecidas ou propostas no processo de fixação do nitrogênio. ............................................................................ 18 Tabela 2 - Coordenadas geográficas das amostras. ................................................. 25 Tabela 4 –Número de OTU0.08 compartilhadas e específicas de cada uma das bibliotecas, nas duas estações. ................................................................................. 39 Tabela 5 - Número de OTU classificadas com as bases de dados NCBI e RDP. ..... 46 Tabela 6 - Classificação taxonômica das OTU0.08 compartilhadas entre as bibliotecas. ................................................................................................................ 51 Tabela 7- Resultados do Teste de Mantel entre a matriz de OTU0.08
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