Caracterização funcional e identificação de SNPs e de genes de resistência a doenças do cafeeiro

• Ano de defesa: 2011
• Autor(a) principal: Alvarenga, Samuel Mazzinghy
• Orientador(a): Sakiyama, Ney Sussumu
• Banca de defesa: Siqueira, Cláudio Lísias Mafra de , Zambolim, Laércio , Oliveira, Antônio Carlos Baião de
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Genética e Melhoramento
• Departamento: Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
• País: BR
• Palavras-chave em Português: Café; Genoma; Bioinformática; Marcadores moleculares
• Palavras-chave em Inglês: Coffee; Genome; Bioinformatics; Molecular markers
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL
• Link de acesso: http://locus.ufv.br/handle/123456789/1332

The Brazilian Coffee Genome Project has generated a database of 200,000 ESTs (Expressed Sequence Tags). These sequences are stored in the Project database, the CafEST. In silico analysis of the CafEST sequences was performed in previous work. It allowed the identification of 14,060 sequences related to the process of plant defense against diseases. Of the 14,060, 1,855 were sequences whose predicted products were chitinases. Given the importance of chitinases, both in plant-pathogen interaction, as in other plant processes, the chitinases ESTs were thoroughly analyzed in this study. For that, a functional characterization was carried out and an in silico expression profile was built. The functional categorization was accomplished using the Blast2GO software, and the in silico gene expression profile was determined by virtual Northern Blot Analysis. The results of functional characterization showed the versatility of chitinases, which have enzymatic activity and are involved in important biological processes and molecular functions in the plant cells. The in silico expression profile analysis has shown that chitinases are more expressed especially in libraries that have some component of stress. To select among the 14,060 sequences mined, those which are involved in the resistance of coffee to rust, a set of 40 pairs of primers were designed in previous work. The primers were tested on 24 coffee trees: 12 resistant and 12 susceptible to Hemileia vastatrix, fungus that causes leaf rust. Twenty-nine primers resulted in unique and well defined bands, one of which was polymorphic between resistant and susceptible trees. Of these primers, 15 were chosen for Single Nucleotide Polymorphisms (SNPs) analysis in DNA sequences between 12 H. vastatrix resistant trees and 12 susceptible ones. The sequences obtained were analyzed with the Sequencher® v.4.10, ORF Finder, BLAST and ClustalW programs. Of the 15 genes, four were monomorphic. Five genes showed polymorphism only for the Coffea liberica var. dewevrei species clone. The other six genes were polymorphic among the sampled genotypes. Seventy one SNPs were detected, of which 34 were transitions (47.89%) and 37 (52.11%) were transversions. The rate of transitions/ transversions was 0.9189. Of the 71 substitutions, 27 (38.02%) occurred in the first codon position, 15 (21.12%) in the second position and 29 (40.84%) in the third. Eleven (15.49%) synonymous and 60 (84.51%) nonsynonymous substitutions were detected. The ratio of synonymous/ nonsynonymous substitutions was 0.1833. A high level of non-synonymous mutations, as was found in this study, may reflect positive selection. An example is the antagonist scenario of the host-pathogen coevolution, where the host genes are engaged in an "arms race" and under strong evolutionary selection pressure to change and adapt. As a result, they evolve at a faster rate than other genes. This scenario can be applied to this work, given the high level of nonsynonymous changes detected and the fact that the analyzed genes somehow act in the defense against coffee pathogens. A frequency of 0.1029 SNPs per amplicon was observed. In the 119,061 bp analyzed, an extremely low frequency of 1 SNP every 1,676.91 bp or 0.0596 SNPs per 100 bp was detected. The low polymorphism level observed can be attributed to the low diversity of the C. arabica genome. The gene products analysis has shown that the nonsynonymous mutations do not lead to formation of distinct predicted proteins. Also, in the present work, the cloning of a gene potentially involved in resistance to coffee rust was initiated. In previous work, a resistance gene fragment was identified. The fragment, amplified by primer CARF 005, is present in the resistant individuals and absent in the coffee leaf rust susceptible genotypes. This primer was used in this study to perform a screening of a BAC (Bacterial Artificial Chromosome) library containing 56,832 clones. The pools fragmentation method was used to identify the positive clone(s). Two positive clones (i.e. two clones containing the resistance gene fragment amplified by marker CARF 005) were identified. The sequencing data from the two identified clones may provide important information about the structure of disease resistance genes in Coffea.