Gene-based SSR markers for common bean (Phaseolus vulgaris L.) derived from root and leaf tissue ESTs: an integration of the BMc series

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.

Cross Species Amplification of Cowpea Derived Unigene-SSR Markers and Diversity Analysis in Black Gram [Vigna mungo (L.) Hepper]

“…Moreover, the unigene-SSRs based on longer motifs such as tetra-and penta-nucleotide repeat motifs were found to be polymorphic, suggesting that long-motif SSRs are also useful for generating informative markers. The polymorphism level of the SSR markers in this study was comparable to other studies (Blair et al 2011;Mishra et al 2012). Moreover, 39 SSR loci matched known genes, which were involved in different functional types of proteins and would assist in identifying marker trait association.…”

Molecular characterization of high performance inbred lines of Brazilian common beans

“…Despite the inherent limitations of analysis based on di-nucleotides, such as difficulties in interpretation due to stuttering and low detection power for alleles that differ by a few base pairs even when using high-resolution electrophoresis, genotyping based on di-nucleotide repeats still predominates among plants due to their high abundance and ease of identification in the genomes. More recently, because of efforts in genomic sequencing and the availability of expressed sequence tag databanks, a greater number of tri-nucleotide markers are being developed (Blair et al, 2011).…”

Development of cDNA-derived SSR markers and their efficiency in diversity assessment of Cymbidium accessions

“…There are three possible reasons for these differences: (1) the use of different number of Cymbidium accessions during polymorphic screening, (2) only 39.3% of designed primers (not all primers) can be screened in the EST-SSRs, and (3) polymorphic primer pairs may be selected by chance while primer selection, although they were selected proportionally according to their repeat motifs types. In comparison, amplification with non-gene based microsatellites is prone to some pitfalls for AT-rich hybridization-derived genomic microsatellites (Blair et al 2011). Differences between genic and other types of genomic microsatellites have been observed in marker sets of other crops Hanai et al 2007).…”