Made available in DSpace on 2015-03-26T13:37:44Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1333692 bytes, checksum: 9bcd2dfa7f5cb679627a7e4240a82858 (MD5) Previous issue date: 2011-02-24 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The coffee leaf rust caused by the biotrophic fungus Hemileia vastatrix, it is the most important fungal disease of coffee, causing productivity losses and its control increases the costs of coffee production. This study aimed to identify genes of H. vastatrix that encode secreted proteins that may function as effectors needed for the establishment of the biotrophic interaction and / or as triggers of resistance responses, by analyzing a database consisting of 9828 expressed sequence tags (ESTs) from an susceptible interaction. These ESTs were generated by sequencing the 5' end of cDNA clones from a library constructed from mRNA isolated from coffee leaves collected 12 days after inoculation with single pustule isolation HV-01. It was obtained 1004 contigs and 3301 singlets after clustering the sequences using the CAP3 program. From 890 unique transcripts that encode proteins without similarity to sequences of non-redundant database of GenBank / NCBI, which had no identity to sequences of Coffea spp., also deposited in this database, were identified 46 ORFs that encode peptides over 60 amino acids with positive prediction from five or more parameters of algorithms used to predict secretion signal sequences. We selected five genes which were amplified from the H. vastatrix genome, generating fragments equal or higher than those amplified from cDNAs. These genes, unique to H. vastatrix showed no identity with cDNA sequences derived from germination spores, demonstrating that their expression may occur within the infected tissue. The secretion of proteins encoded by four genes was demonstrated using the Yeast Secretion System. Functional studies should be conducted to confirm the effector activity of the genes characterized, as well as from other genes identified in this study, whose origin and secretion of fungal the predicted proteins in yeast has been demonstrated. / A ferrugem causada pelo fungo biotrófico Hemileia vastatrix é a doença mais importante do cafeeiro, pois atinge, com gravidade, grandes áreas de lavouras, onde causa prejuízos na produtividade e seu controle aumenta os custos de produção. O presente trabalho teve por objetivo identificar genes de H. vastatrix que codificam proteínas secretadas que possam funcionar como efetores necessários para o estabelecimento da interação biotrófica e, ou, como desencadeadores de respostas de resistência, por meio da análise de um banco constituídos por 9828 etiquetas de sequências expressas (ESTs) em uma interação suscetível. Essas ESTs foram geradas pelo sequenciamento da extremidade 5´de clones de cDNA de uma biblioteca construída a partir de mRNA isolado de folhas de cafeeiro coletadas 12 dias após a inoculação com o isolado monopustular HV-01. Foram obtidos 1004 contíguos e 3301 singletos após o alinhamento das sequências pelo programa CAP3. A partir de 890 transcritos únicos que codificam proteínas sem similaridade a sequências do banco não redundante do GenBank/NCBI e que não possuíam identidade a sequências de Coffea spp. também depositadas nesse banco de dados, foram obtidas 46 ORFs que codificam peptídeos com mais de 60 aminoácidos e predição positiva em cinco ou mais parâmetros do algoritmo de predição de sequências sinal de exportação SignalP. Foram selecionados cinco genes que tiveram a região da ORF amplificada do genoma de H. vastatrix, gerando fragmentos iguais ou maiores aos amplificados a partir do cDNA. Esses genes, exclusivos de H. vastatrix, não mostraram identidade com sequências únicas derivadas de esporos germinados desse patógeno, demonstrando que a sua expressão ocorre no interior do tecido infectado. A secreção das proteínas codificadas por quatro genes foi confirmada em levedura. Estudos funcionais deverão ser realizados para comprovar a atividade efetora dos genes caracterizados, assim como dos demais genes identificados nesse estudo, cuja origem fúngica e secreção das proteínas preditas em levedura seja demonstrada.

Thesis (M. Sc. Agriculture (Plant Protection)) -- University of Limpopo, 2021 / Mimusops zeyheri tree groves made up of seventeen trees collected from communities in Southern Africa were used in this study. The trees had high morphological variations in terms of growth rate, fruit (size and taste) and leaf (shapes and sizes) and their identification was made by communal people from where the trees were collected using their morphological characteristics. Generally, this evergreen tree is drought‒tolerant, salt‒tolerant and highly resistant to root-knot (Meloidogyne spp.) nematodes, along with various other pests. This could probably be attributed to high concentration of latex in aboveground organs. Some typical fungal rust symptoms have been observed believed to be the cause of high leaf abscission in certain groves. Currently, there is no report of leaf rust disease on M. zeyheri plants and the mechanism of resistance to other pests is not documented. The objective of this study was to (i) identify the pathogen associated with M. zeyheri leaf rust symptoms using morphological technique and to (ii) determine levels and types of potential defence chemicals and endophytes in M. zeyheri. Samples of M. zeyheri leaves showing rust like symptoms were collected from University of Limpopo, South Africa (23°53”10’S, 29°44”15’E) during summer in September 2018. Light compound microscope and electron microscope were used in the identification of the leaf rust spores. The species identity of the seventeen M. zeyheri trees that form a grove collection at University of Limpopo was confirmed using internal transcribed spacer (ITS) of ribosomal nuclear DNA. DNA extraction and sequencing was done with the help of Inqaba Biotechnologies. Obtained DNA sequences were aligned using CLUSTALX (2.0), with the phylogenetic tree constructed through the neighbour-joining method (NJM) in MEGA v. 5.1 programme. Evolutionary distances were computed using the Juke– Cantor method. Phytochemicals in leaves were identified and quantified using Liquid chromatography–mass spectrometry (LC–MS) at ARC-VOP. The pustules on the collected leaves contained reddish brown spores. The uredospores were oval and ellipsoidal under a light microscope. The size of spores ranged between 35-37 × 24-26 µm. The cell walls showed bilaminate structures with the outer layer hyaline. The warts were rod shaped with one subequatorial germ pore. The most common identified endophytic fungi observed in all M. zeyheri leaves were Teratosphaeria species, Zeloasperium species, Pezizomycotina. In addition, endophytes such as Cladosporium species, Aspergillus species, Phyllosticta species and Epicoccum species were also identified to be associated with some M. zeyheri trees. There were significant differences on the level of tannins, flavonoids, proteins and phenolics among the M. zeyheri trees. The highest level of tannins was 7.2151 mg/g and the lowest being 2.7232 mg/g. The highest level of flavonoids was 1.1537 mg/g the lowest being 0.0123 mg/g. The level of phenolics among the trees ranged from 2.4749 mg/g to 1.5788 mg/g. Protein content ranged from 5.3100% to 2.7967% among the trees. Very high levels of tannins, flavonoids, phenolics among the trees when compared with others studies indicate the potential role of these metabolites in previously reported resistance of M. zeyheri to a number of pests. The morphological characteristics of the identified leaf rust pathogen causing rust symptoms on M. zeyheri in South Africa is more similar to Maravalia species previously isolated from M. caffra. And as such, this finding is paramount, as it is the first report of association between the pathogen and the plant. Teratosphaeria species has been associated with stem cancer in Eucalyptus trees, its presence in M. zeyheri species in asymptomatic plants is a major find since the tree is common in Limpopo and Mpumalanga, Provinces well known for forestry production making M. zeyheri a potential host for pathogens of commercial forestry production. Other endophytes observed such as Aspergillus species, Phyllosticta species and Cladosporium species have been shown to protect plants against some pests and pathogenic organism. Further studies to determine the direct role of identified phytochemicals and endophytes in the resistance of M. zeyheri to pests needs to be conducted

Made available in DSpace on 2015-03-26T13:39:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2656828 bytes, checksum: 8bc70c0429aa62c04a9679cf9f1486b7 (MD5) Previous issue date: 2010-07-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In order to support the breeding programs coffee that to aim to obtain cultivars resistant to rust (Hemileia vastatrix), this work aimed to study the resistance inheritance of Híbrido de Timor UFV 443-03 and identify molecular markers linked characterized genes. To study the inheritance were evaluated three populations originated from cross the Híbrido de Timor UFV 443-03 resistant parent and Catuaí Amarelo (UFV 2148-57) susceptible cultivar. These plants analyzed, 243 correspond to an F2 population of 114 susceptible backcrossing (BCs) and 87 resistant backcrossing (BCr). Inoculation was performed with a spore suspension (2mg/mL) isolated from the Race I of H. vastatrix in leaf discs, with three replicates for the F2 and RCs, and two for RCr. Plants of the Catuaí Amarelo UFV 2148-57 were susceptible in all inoculations, while the Híbrido de Timor UFV 443-03, the plant F1 and the BCr plants were resistant. Segregation analysis of F2 populations obtained two significant cases, the resistance of a coffee Híbrido de Timor UFV 443-03 the H. vastatrix was governed by two independent and dominant genes (15:1, P = 63.10) and the other three genes is conferred by two dominant and one recessive (61:3, P = 8.87). Plants of the BCs were used to confirm the segregation patterns, which segregated in a rate of 3:1 (P = 74.56), expected segregation for two and three genes. This result demonstrate that the resistance of the Híbrido de Timor is conditioned by two dominant independent genes or three independent genes (two dominant and one recessive). As was found oligogenic inheritance (two or three genes), for confirmation and identification of molecular markers linked to genes, they were treated as QTL, and then located in link genetic map. For this, subjects in the F2 population were analyzed with a total of 110 molecular markers. As 16 markers showed distortion of Mendelian segregation expected rate, the map was constructed with 94 markers (62 AFLP, 28 SSR and 4 RAPD). Whereas a LOD score minimum of three and of 40% recombination maximum, the markers were grouped into 11 linkage groups covering a distance 964.31 cM of the genome, with 13 markers not in any of the linkage groups formed. The longest interval between two markers was 32.1 cM and 68.57% of the markers did not exceed 20 cM. The characterization and identification of QTLs were performed by simple mark and simple interval methods. Methodology of the simple mark was possible to identify markers five associated with QTLs by the methods of ANOVA, regression and maximum likelihood. These QTLs were confirmed by simple interval methodology by means of regression and maximum likelihood. Two QTLs were identified in a 2 linkage group the 0 cM marker 21a, explaining 9.6% of the phenotypic variation. The other was located in 3 linkage group to 13 cM marker 43a, explaining 9.3% of the phenotypic variation. These two QTLs confirm the number and position in the coffee resistance Híbrido de Timor UFV 443-03 H. vastatrix is governed by two independent and dominant genes, thus demonstrating the importance of genomics to identify them. It should be noted that the information obtained in this study are unique to coffee, which may be useful in breeding programs based on assisted selection and positional cloning of the gene for rust resistance. So, should provide support for future breeding populations to obtain more productive and resistance. / Com o intuito de dar suporte aos programas de melhoramento do cafeeiro que visam à obtenção de cultivares resistentes à ferrugem (Hemileia vastatrix), neste trabalho objetivou-se estudar a herança da resistência do Híbrido de Timor UFV 443-03 e identificar marcadores moleculares ligados aos genes caracterizados. Para o estudo de herança foram avaliadas três populações originadas do cruzamento entre o progenitor resistente Híbrido de Timor UFV 443-03 e o cultivar suscetível Catuaí Amarelo (UFV 2148-57). Dessas plantas analisadas, 243 correspondem a uma população F2, 114 a retrocruzamento suscetível (RCs) e 87 a retrocruzamento resistente (RCr). A inoculação foi realizada com a suspensão de esporos (2 mg/mL) do isolado da Raça I de H. vastatrix, em discos foliares, com três repetições para as populações F2 e RCs e duas para RCr. As plantas do Catuaí Amarelo UFV 2148-57 foram suscetíveis em todas as inoculações, enquanto o Híbrido de Timor UFV 443-03, a planta F1 e as plantas do RCr foram resistentes. Pela análise de segregação das populações F2, obtiveram-se duas hipóteses significativas: uma de que a resistência do cafeeiro Híbrido de Timor UFV 443-03 a H. vastatrix era governada por dois genes dominantes e independentes (15:1; P = 63,10) e a outra de que era conferida por três genes, sendo dois dominantes e um recessivo (61:3; P = 8,87). Plantas do RCs foram utilizadas para a confirmação dos padrões de segregação e segregaram na proporção de 3:1 (P = 74,56), o que era esperado para dois e para três genes. Esse resultado indicou que a resistência desse Híbrido de Timor é condicionada por dois genes dominantes independentes ou três genes independentes (dois dominantes e um recessivo). Como a herança encontrada foi oligogênica (dois ou três genes), para a confirmação e identificação de marcadores moleculares ligados aos genes, estes foram tratados como QTL e, então, localizados em mapa genético de ligação. Para isso, os indivíduos da população F2 foram analisados com um total de 110 marcadores moleculares. Como 16 marcadores apresentaram distorção da razão de segregação mendeliana esperada, o mapa foi construído com 94 marcadores (62 AFLP, 28 SSR e 4 RAPD). Considerando um LOD score mínimo de 3 e máxima recombinação de 40%, os marcadores ficaram agrupados em 11 grupos de ligação, cobrindo uma distância de 964,31 cM do genoma, e 13 marcadores não se ligaram a nenhum dos grupos formados. O maior intervalo entre dois marcadores foi de 32,1 cM, e 68,57% dos marcadores não excederam 20 cM. A caracterização e identificação de QTLs foram realizadas com o auxílio de metodologias de marca simples e intervalo simples. Pela metodologia da marca simples foi possível identificar cinco marcadores associados aos QTLs pelos métodos da ANOVA, regressão e máxima verossimilhança. Esses QTLs foram confirmados pela metodologia de intervalo simples por meio da regressão e máxima verossimilhança. Dois QTLs foram identificados, um deles no grupo de ligação 2 a 0 cM do marcador 21a, explicando 9,6% da variação fenotípica. O outro ficou localizado no grupo de ligação 3 a 13 cM do marcador 43a, explicando 9,3% da variação fenotípica. Esses dois QTLs confirmam, em número e posição, que a resistência do cafeeiro Híbrido de Timor UFV 443-03 a H. vastatrix é governada por dois genes dominantes e independentes, mostrando, assim, a importância da genômica para a identificação destes genes. Cabe salientar que as informações deste trabalho são inéditas para o caso do cafeeiro e podem ser úteis em programas de melhoramento baseado em seleção assistida e na clonagem posicional do gene de resistência à ferrugem. Assim, os dados deste estudo deverão fornecer subsídios para futuros trabalhos de melhoramento visando à obtenção de populações mais resistentes e produtivas.