Desenvolvimento e validação de marcadores microssatélites para o feijão-comum

• Autor(a) principal: Tanure, Janaína Paula Marques
• Data de Publicação: 2009
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/4710
• topic: Microssatélites; Feijão-comum; Microsatellite; Common bean; CNPQ::CIENCIAS BIOLOGICAS::GENETICA

Common bean (Phaseolus vulgaris L.) is a crop of great nutritional, economical and social importance. Breeding programs use molecular markers as important auxiliary tools for various types of genetic studies. Different classes of molecular markers have been developed, and among them microsatellites highlight. Microsatellites are DNA simple sequence repeats (SSR) distributed in tandem along the genome, forming highly variable polymorphic sites, enabling their use as molecular markers. SSRs are codominant, multiallelic, and thus can be used in several types of studies, mainly for genetic mapping. Primers flanking microsatellite sequences are commonly developed from genomic libraries, enriched genomic libraries, sequences obtained from databases and, alternatively, from internal simple sequence repeats (ISSR-PCR). Common bean breeding molecular geneticists have developed SSR markers for mapping specific traits of interest. However, a saturated consensus genetic map for common bean has not been established so far that can be used as a reference for the development of specific maps. Therefore, the BIOAGRO/UFV common bean breeding program developed a population of recombinant inbred lines (RILs) which is suggested to be used to integrate, in one single map, all microsatellite markers that have been developed so far. However, to saturate the map, there must be a great number of markers available. The objective of the present study was to develop and validate primers that amplify microsatellites sequences obtained from enriched genomic libraries and from ISSR sequences. In the first case, two enrichedgenomic libraries for microsatellite sequences, that had been developed in a previous study, were used. In the present study, 207 clones were selected from these two genomic libraries. One hundred and ninety six of these clones (94.68%) were sequenced and 184 (88.89%) of them could be analyzed, 133 clones from library 1 and 51 from library 2. Forty eight redundant clones (26.09%) were detected. Clone analysis led to the identification of 66 (49.62%) microsatellite motifs in library 1 and 20 (39.22%) in library 2, and 56 primer pairs were designed. From the 56 primer pairs developed, 34 were characterized andtested in 20 Mesoamerican and Andean genotypes, including AND277 and Rudá. All the primer pairs were able to generate PCR products and six (17.65%) generated polymorphic DNA bands among the tested genotypes. In the ISSR enrichment methodology, 250 clones were seleted with sizes over 400 bp. From these 250 clones, 168 (67.2%) were sequenced and 103 (41.2%) could be analyzed. Thirty redundant clones (29.13%) were detected. Clone analyses led to the identification of 58 microsatellite motifs (56.31%) and 32 primer pairs were developed. Out of these, 10 were characterized and tested in the same genotypes used in the previous methodology. Out of the 10 primer pairs tested, six were identified as codominant markers and the other four as dominant. The codominant markers revealed no polymorphisms among the tested genotypes. Additionally, microsatellite containing sequences obtained from both methodologies were submitted to BLAST analysis against sequences deposited in public databases. Similarity was identified between the SSR sequences and transcribed and non-transcribed regions, from nuclear, mitochondrial and chloroplast genomes, and also with retrotransposon sequences. Both methodologies were effective for selecting, in common bean, sequences that contain microsatellites. The results obtained represent an initial effort to select molecular markers that will be mapped in the future RIL's consensus population, contributing for the construction of a satured genetic map for the species. In addition, these primers can be used in different types of genetic studies which are important for common bean breeding programs that use molecular markers.