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| Author(s): |
Maria Gabriela Fontanetti Rodrigues
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2015-03-25 |
| Examining board members: |
Silvana Giuliatti;
Luiz de Souza Correa;
Ana Lilia Alzate Marin;
Ester Silveira Ramos;
Claudio Jose von Zuben
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| Advisor: | Silvana Giuliatti |
| Abstract | |
The fig tree (Ficus carica L.) breeding programs by conventional methods such as directed crosses, in order to obtain new cultivars, are unworkable in many countries, as in Brazil, mainly by small genetic variability found, and by the difficulty for obtaining plants originated from the fusion of gametes, since the wasp Blastophaga psenes, responsible for natural pollination, doesn`t exist in the country. In this way, the genetic breeding, with the use of mutagenic, becomes an important research line for the improvement of culture, being necessary to gather information about this species, mainly in relation to its genetic variability, for perform propagation projects and appropriate management. Given the above, The objective of this study was to verify the existence of epigenetic variability due to DNA methylation in irradiated fig selections, with each other and when compared to the main commercial cultivar, Roxo-de-Valinhos, using MSAP and ELISA techniques, and subsequent DNA sequencing, treated with sodium bisulfite, for detection of the position of the polymorphic regions, analyzed by bioinformatics tools. Samples of genomic DNA were double-digested with the HpaII enzyme (sensitive to methylation) with its isoschizomer MspI (insensitive to methylation), together with the EcoRI enzyme. Fourteen primer combinations were tested and it was obtained 87.9%, 10.1% e 2.0%, respectively, unmethylated CCGG, methylated CmCGG and hemimethylated regions hmCCGG, from a total of 553 amplification products, displaying, the MSAP technique, efficient for detection of differentially methylated sites in the genomic material studied, demonstrating their epigenetic divergence. With the sequencing of DNA isolated of these differentially methylated sites, it was possible to verify different patterns of methylation in them by sequencing the DNA treated with sodium bisulfite, in coding regions of regulatory genes of the development and fruits ripening, besides they have been found in the mitochondrial DNA of treatments, which regulates the supply of energy in ATP form for the plants, being closely related to their development, justifying the different phenotypes found in both fruits and plant growth that suffered stress due to exposure to gamma radiation. By the technique of immunochemistry (ELISA), using 5-mC antibodies, significant differences were observed by Tukey test, at 95% of trustworthiness, in the global content methylation of the treatments DNA, indicating that this abiotic factor was responsible for the changes in the epigenome of the plants. Since the material used as control was found also methylated, a supposed demethylation of the genomic material may be responsible for phenotypic variation among treatments. Considering this, future study of gene expression between treatments becomes an extreme important strategy for understanding the complex regulatory systems, leading to the identification of genes with agronomic interest for the fig culture, allowing its subsequent manipulation and propagation of improved cultivars for commercial purposes. (AU) | |
| FAPESP's process: | 10/05763-4 - Analysis of fig selections DNA methylation by MSAP and sequencing. |
| Grantee: | Maria Gabriela Fontanetti Rodrigues |
| Support Opportunities: | Scholarships in Brazil - Doctorate |