Development and analysis of multiplex microsatellite markers sets in common bean ( Phaseolus vulgaris L.)

Abstract

Multiplexing involves the simultaneous amplification of several loci in a single PCR reaction, and subsequent analysis of multiple molecular markers in a single gel lane. This study focuses on the usefulness of SSR (simple sequence repeat) multiplex-PCR, and how this method can be both highly informative and amenable to automation using a fluorescence-based, semi-automated DNA sequencing platform. We also discuss the relevance of this approach to the issue of the multiplex ratio of SSR markers and show that it is effective as an economic procedure for germplasm evaluation and evolutionary studies of plants. Various conditions of multiplex-PCR were examined and a method is proposed for developing multiplex sets of SSR markers in common bean (Phaseolus vulgaris L.). Seven multiplex sets, based on 30 SSR markers selected from GenBank sequences, were developed. The method was tested using a large number of samples from three common bean landraces. Technical aspects of the application and a description of the allelic constitution at various loci of all genotypes analysed are described. From a sample of 264 genotypes, selected from three landraces, we detected a total of 135 alleles, equivalent to 4.3 alleles per SSR. Null alleles were observed in each of the three landraces analysed. The procedures used in this study are applicable for the study of genetic diversity in common bean germplasm collections consisting of a significant number of accessions, and should be transferable to similar analyses of any species.