5 × TBE buffer (pH 8.0), and silver stained to visualize the PCR products. The polymorphic BMr markers were evaluated in the DOR364 × G19833 population of F9:11 recombinant Bleomycin research buy inbred lines (RIL) as described in Blair et al. [16]. DOR364 is a small red-seeded variety of the Mesoamerican genepool, grown

in several countries of Central America. G19833 is a landrace originally collected in Peru of the Andean genepool with large, yellow and red-mottled seed and has been selected for genomic sequencing. An anchor genetic map for this population was built with single-copy RFLP and SSR markers (of the series BM, BMa and BMd) using the software Mapmaker 3.0 for Windows [31]. Genotyping results of the present work were recorded in a Microsoft Excel worksheet with female alleles as “A” and

male alleles as “B”. Heterozygotes or missing data was not considered. The BMr markers were added to the genetic map using the software program MapDisto v. 1.7 (http://mapdisto.free.fr/) with “find groups” at a minimum of LOD > 3.0. The “order sequence” and “compare all orders” commands were then used to identify the best marker order for each linkage group. The location of anchor markers was cross-checked with the map of Blair et al. [16]. Linkage groups were drawn according to the cytogenetic orientation of their corresponding chromosomes based on Fonsêca et al. [32]. R-genes or QTL were added Protein Tyrosine Kinase inhibitor to the map based on the estimated positions in Miklas et al. [9]. Genetic distances between markers in centiMorgans (cM) were obtained using the Kosambi function, which assumes crossover interference. The first step in the detection of RGH-SSRs in common bean was probe design, which was based on singleton and assembled RGH gene and pseudogene sequences (Table 1). A total of 86 probes were amplified for screening of the G19833 BAC library. Based on the phylogenetic analysis of Garzón et al. [26], 38 of these represented TIR clades and 48 non-TIR clades. Some sequences

with premature Thiamine-diphosphate kinase stop codon or with no evident open reading frame (ORF) were considered pseudogenes (22 out of 86) but were also used for probe design. If a probe was designed from two or more sequences, it was classified as from assembled sequences. Singleton probes were those designed from only one sequence sharing no more than 90% identity with any other sequence according to Garzón et al. [26]. Almost all the TIR probes were designed from assembled RGH gene sequences. Most of the non-TIR probes were designed from single RGH. Pseudogenes, even those with a low identity value with other sequences, were used for probe design because we were interested in identifying the maximum number of putative common bean RGHs. The next step was confirming probe amplification and deciding which probes to hybridize to the G19833 BAC library. This was done by sequencing the PCR products of the probe amplification described above.