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

BACKGROUND: 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 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. RESULTS: A 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. CONCLUSIONS: The 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.

Development of microsatellite markers for common bean (Phaseolus vulgaris L.) based on screening of non-enriched, small-insert genomic libraries.

Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries.

Development and diversity of Andean-derived, gene-based microsatellites for common bean (Phaseolus vulgaris L.).

BACKGROUND: Gene-based (genic) microsatellites are a useful tool for plant genetics and simple sequence repeat loci can often be found in coding regions of the genome. While EST sequencing can be used to discover genic microsatellites, direct screening of cDNA libraries for repeat motifs can save on overall sequencing costs. The objective of this research was to screen a large cDNA library from and Andean common bean genotype for six di-nucleotide and tri-nucleotide repeat motifs through a filter hybridization approach and to develop microsatellite markers from positive clones. RESULTS: Robotics were used for high-throughput colony picking and to create a high-density filter of 18,432 double spotted cDNA clones which was followed by hybridization with repeat motif containing probes based on GA, CA, AAT, CAG, CAA and ACG repeats. A total of 1203 positive clones were identified by their addresses and sequenced from 5' ends and if required from 3' ends to confirm repeat motif and length. Out of 886 high quality sequences, 497 had complete microsatellite loci that were not truncated by the sequencing reaction and of these tri-nucleotide repeats were more common than di-nucleotide repeats. Different motifs were found in different frequencies in the 5' and 3' ends of the cDNAs. In a microsatellite development program, primers were designed for 248 SSR loci which were tested on a panel of 18 common bean genotypes to determine their potential as genetic markers finding higher average polymorphism information content for di-nucleotide repeat markers (0.3544) than for tri-nucleotide repeat markers (0.1536). CONCLUSION: The present study provides a set of validated gene-based markers for common bean that are derived from G19833, an Andean landrace that is an important source of disease and abiotic stress tolerance which has been used for physical map development and as a mapping parent. Gene-based markers appear to be very efficient at separating divergent wild and cultivated accessions as well as Andean and Mesoamerican gene pools and therefore will be useful for diversity analyses and for comparative and transcript mapping in common bean.