-The objectives of this work were to adapt the fluorescent labeling polymerase chain reaction (PCR) technique using M13 universal primer for genotyping purposes, and to present a new set of microsatellite markers for common bean (Phaseolus vulgaris L.). A large population (380 common bean lines) was used for microsatellite genotyping. PCR fluorescent labeling method showed to be very efficient for multiplex analysis, providing lower costs and saving time, thus increasing the quality of genotyping analysis. A new set of 50 microsatellites developed from an enriched library derived from cultivar IAC-UNA was presented. This study provides better tools for assisting common bean breeding programs.Index terms: Phaseolus vulgaris, fluorescence, molecular marker, multiplex analysis.
Adaptação da técnica de fluorescência para fins de genotipagem com novos marcadores microssatélite em feijoeiroResumo -Os objetivos deste trabalho foram adaptar a técnica de marcação fluorescente de produtos da reação em cadeia da polimerase (PCR) com uso do iniciador universal M13, para aplicação em genotipagem, e apresentar novos marcadores microssatélite para o feijoeiro (Phaseolus vulgaris L.). Uma população de grande tamanho amostral (380 linhagens) foi utilizada para genotipagem dos microssatélites. O método de PCR marcado por fluorescência demonstrou ser muito eficiente para a análise "multiplex" e proporcionou a redução de custos e ganho de tempo, aumentando a qualidade de análise da genotipagem. Foram apresentados 50 novos locos de microssatélites, desenvolvidos a partir de biblioteca enriquecida a partir da cultivar IAC-UNA. Este estudo fornece ferramentas melhores para assistir aos programas de melhoramento do feijoeiro.Termos para indexação: Phaseolus vulgaris, fluorescência, marcador molecular, análise multiplex.Microsatellite genotyping is widely used nowadays for different goals, such as diversity studies and genetic mapping. Microsatellites (Simple Sequence RepeatsSSRs) are molecular markers characterized as small DNA sequences of one to six base pairs tandemly repeated, spread all over the genome of plants and animals (Li et al., 2002;Varshney et al., 2005). They are multiallelic, codominant and have Mendelian inheritance. Moreover, they are easy to assay and are reproducible by polymerase chain reaction (PCR). Currently, the most used technique for SSR genotyping is the 6% polyacrylamide gel electrophoresis stained with silver nitrate (Creste et al., 2001). However, this method is time consuming and costly, especially when working with a large amount of individuals.Due to this fact, microsatellite fluorescencebased detection has been used in many crops in order to reduce general labor and costs. Oetting et al. (1995) were the first group to use the fluorescent method for genotyping purposes and used five primer pairs to perform different multiplex amplification reactions. The technique was based on a fluorescent-labeled universal M13 primer, which allowed automated DNA sequencer analysis and subsequent genotyping w...