Adaptation of fluorescent technique for genotyping with new microsatellite markers in common bean

Abstract

-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...

Construction of High-Density Genetic Linkage Maps and Mapping of Growth-Related Quantitative Trail Loci in the Japanese Flounder (Paralichthys olivaceus)

“…Microsatellites comprise an excellent opportunity for genomic mapping due to their abundance in most vertebrate genomes, as well as their genomic distribution pattern, high polymorphism rate and ease of typing, all of which are determinable via PCR. Meanwhile, the simple sequence repeat (SSR) alleles are typically co-dominant, and their polymorphisms can be scored in either a simple polyacrylamide gel separation format or with high-throughput capillary arrays [1]. Genetic linkage maps based on microsatellite markers have been produced for economically important fish species, including salmon [2], tilapia [3], European sea bass [4], rainbow trout [5], sea bream [6], Barramundi [7], catfish [8], grass carp [9], Japanese flounder [10] and Asian sea bass [11].…”

Genetic diversity in cultivated carioca common beans based on molecular marker analysis

“…A total of 85 SSRs ( Table 2 ) were developed from two microsatellite-enriched libraries, one for the ‘CAL-143’ line and another for the ‘IAC-UNA’ variety ( Benchimol et al , 2007 ; Campos et al , 2007 ; Cardoso et al , 2008 ; Oblessuc et al , 2009 ). Sixty-five of the 85 SSRs were genotyped using 6% polyacrylamide silver stained gels ( Creste et al , 2001 ) whereas the remaining 20 SSRs were genotyped using a fluorescent labeling method that allowed high-throughput genotyping ( Schuelke, 2000 ).…”

Genetic diversity of Colombian landraces of common bean as detected through the use of silver-stained and fluorescently labelled microsatellites

“…SSR markers can determine genetic structure more precisely than other types of markers (Liu et al , 2003). While some authors have used non-specific fluorescent dyes based on M13 labelling with common bean microsatellites (Kwak and Gepts, 2009; Oblessuc et al , 2009), we have developed a group of specific, fluorescent dye-labelled microsatellites that have proved to be a highly efficient tool for analyzing allelic diversity in beans (Blair et al , 2009), given that the estimate of allele sizes is not biased by the effect of the M13 label.…”