Abstract
BackgroundSequencing of cDNA libraries for the development of expressed sequence tags (ESTs) as well as for the discovery of simple sequence repeats (SSRs) has been a common method of developing microsatellites or SSR-based markers. In this research, our objective was to further sequence and develop common bean microsatellites from leaf and root cDNA libraries derived from the Andean gene pool accession G19833 and the Mesoamerican gene pool accession DOR364, mapping parents of a commonly used reference map. The root libraries were made from high and low phosphorus treated plants.ResultsA total of 3,123 EST sequences from leaf and root cDNA libraries were screened and used for direct simple sequence repeat discovery. From these EST sequences we found 184 microsatellites; the majority containing tri-nucleotide motifs, many of which were GC rich (ACC, AGC and AGG in particular). Di-nucleotide motif microsatellites were about half as common as the tri-nucleotide motif microsatellites but most of these were AGn microsatellites with a moderate number of ATn microsatellites in root ESTs followed by few ACn and no GCn microsatellites. Out of the 184 new SSR loci, 120 new microsatellite markers were developed in the BMc (Bean Microsatellites from cDNAs) series and these were evaluated for their capacity to distinguish bean diversity in a germplasm panel of 18 genotypes. We developed a database with images of the microsatellites and their polymorphism information content (PIC), which averaged 0.310 for polymorphic markers.ConclusionsThe present study produced information about microsatellite frequency in root and leaf tissues of two important genotypes for common bean genomics: namely G19833, the Andean genotype selected for whole genome shotgun sequencing from race Peru, and DOR364 a race Mesoamerica subgroup 2 genotype that is a small-red seeded, released variety in Central America. Both race Peru and Mesoamerica subgroup 2 (small red beans) have been understudied in comparison to race Nueva Granada and Mesoamerica subgroup 1 (black beans) both with regards to gene expression and as sources of markers. However, we found few differences between SSR type and frequency between the G19833 leaf and DOR364 root tissue-derived ESTs. Overall, our work adds to the analysis of microsatellite frequency evaluation for common bean and provides a new set of 120 BMc markers which combined with the 248 previously developed BMc markers brings the total in this series to 368 markers. Once we include BMd markers, which are derived from GenBank sequences, the current total of gene-based markers from our laboratory surpasses 500 markers. These markers are basic for studies of the transcriptome of common bean and can form anchor points for genetic mapping studies in the future.
Highlights
Sequencing of cDNA libraries for the development of expressed sequence tags (ESTs) as well as for the discovery of simple sequence repeats (SSRs) has been a common method of developing microsatellites or SSRbased markers
These libraries were based on 1) mRNA from leaf and stem tissues as described in Blair et al [12] and Ramirez et al [17] for the genotype G19833; 2) a library that was made in the pBS-SKII vector from mRNAs of hydroponically grown DOR364 roots which were produced under low phosphorus (LP) conditions and 3) a final library made in the pBS-SKII vector from mRNAs of hydroponically grown DOR364 roots but which were from a high phosphorus (HP) treatment
Across all the EST sequencing sets the percentage of ESTs containing SSRs varied from 3.5 to 11.9% with the highest number found in the first sequencing of the leaf library and the least in the second sequencing of the leaf library which may have been due to sampling differences
Summary
Sequencing of cDNA libraries for the development of expressed sequence tags (ESTs) as well as for the discovery of simple sequence repeats (SSRs) has been a common method of developing microsatellites or SSRbased markers. The root libraries were made from high and low phosphorus treated plants Genic microsatellites are those microsatellites based on simple sequence repeats (SSRs) found within, or closely associated with, gene sequences from a given genome [1]. Genic microsatellites have been limited in number for this crop This is perhaps due to two main reasons: 1) a lack of funding has precluded large scale expressed sequence tag (EST) sequencing or even the sufficient construction of many cDNA libraries for the crop and 2) those ESTs and cDNA libraries that exist have not been extensively screened for gene-based SSRs with the exception of the work of Blair et al [6] and Hanai et al [7,8]. A more complete toolbox of molecular tools for this crop is needed especially in the case of gene-based markers which can be based on SSRs polymorphisms as will be discussed here
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