Estudo da expressão e Análise de poliformismo nucléicosde genes candidatos para a tolerância à seca em cafeeiro

The coffee is a perennial tree which belongs to the Rubiaceae family and teh genus Coffea. Among the cultivated species, Coffea canephora (Robusta) and Coffea arabica (Arabica) are the most important economically, representing respectively 30% and 70% of the world trade production. One major problem that affects this culture is it susceptibility to soil water deficiency, which can lead to important economic, ecological and social consequences - such as the massive migration of unemployed rural workers to urban centers one of the objectives of coffee genetic improvement is to produce drought-tolerance coffe hybrids. At the molecular level, some projects are being carried out in order to identify candidate genes (CG) for drought tolerance, either by the evaluation of differential gene expression on both the transcriptional (transcriptome) and the translation levels (proteomics). The first objective of this work consisted in the evaluation by the real-time PCR (qPCR) technique, of the differential expression of drought-tolerance CGs, such as DREB, NAC-R26, RD29, GC10, CCoAMT, OEC, A.C, M6FR, as well as the A and B isoforms of the RBCS gene, in several C.arabica cultivars grown under different field conditions odf drought intensy. The CG were previously identified and validated in experiments with cultivars of C. canephora grown in a greenhouse. The second objective was to analyze the in vivo nucleotide diversity of some CGs (DREB, RD29 and RD26-NAC) present in several varieties of coffee, and to identify SNPS (Single nucleotide polymorphisms) related to the drought tolerance phenotype. The genomic DNAs (gDNAs) from different species, clones and cultivars of coffee were extracted and submitted to specific amplification with two CGs primers carrying fowards and reverse M13 adapters in the extremities, wich allowed us to evaluate the presence of SNPs by sequencing the PCR products without cloning. To identify the allelic forms present in each genoma, regular primers were utilized to amplified and to sequence the same portions of gDNA after proper cloning. The resulting allelic sequence of ten independent clones were aligned in silico and different polymorphic regions were properly identified. (Résumé d'auteur)