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The Great Lakes region of Central Africa is a major producer of common beans in Africa. The region is known for high population density and small average farm size. The common bean represents the most important legume crop of the region, grown on over a third of the cultivated land area, and the per capita consumption is among the highest in the world for the food crop. The objective of this study was to evaluate the genetic diversity in a collection of 365 genotypes from the Great Lakes region of Central Africa, including a large group of landraces from Rwanda as well as varieties from primary centers of diversity and from neighboring countries of Central Africa, such as the Democratic Republic of Congo and Uganda, using 30 fluorescently labeled microsatellite markers and automated allele detection. In addition, the landraces were evaluated for their seed iron and zinc concentration to determine if genetic diversity influenced nutritional quality. Principal coordinate and neighbor-joining analyses allowed the separation of the landraces into 132 Andean and 195 Mesoamerican (or Middle American) genotypes with 32 landraces and 6 varieties intermediate between the gene pools and representing inter-gene pool introgression in terms of seed characteristics and alleles. Genetic diversity and the number of alleles were high for the collection, reflecting the preference for a wide range of seed types in the region and no strong commercial class preference, although red, red mottled and brown seeded beans were common. Observed heterozygosity was also high and may be explained by the common practice of maintaining seed and plant mixtures, a coping strategy practiced by Central African farmers to reduce the effects of abiotic and biotic stresses. Finally, nutritional quality differed between the gene pools with respect to seed iron and zinc concentration, while genotypes from the intermediate group were notably high in both minerals. In conclusion, this study has shown that Central African varieties of common bean are a source of wide genetic diversity with variable nutritional quality that can be used in crop improvement programs for the region.

The deployment in common beans (Phaseolus vulgaris L.) of arcelin-based bruchid resistance could help reduce post-harvest storage losses to the Mexican bean weevil [(Zabrotes subfasciatus (Boheman)]. Arcelin is a member of the arcelin-phytohemagglutinin-alpha-amylase inhibitor (APA) family of seed proteins, which has been extensively studied but not widely used in bean breeding programs. The purpose of this study was to evaluate microsatellite markers for genetic analysis of arcelin-based bruchid resistance and to determine the orientation of markers and the rate of recombination around the APA locus. A total of 10 previously developed microsatellites and 22 newly developed markers based on a sequenced BAC from the APA locus were screened for polymorphism and of these 15 were mapped with an F(2) population of 157 individuals resulting from a susceptible x resistant cross of SEQ1006 x RAZ106 that segregated for both the arcelin 1 allele and resistance to the bruchid, Z. subfasciatus. Microsatellites derived from APA gene sequences were linked within 0.8 cM of each other and were placed relative to the rest of the b04 linkage group. In a comparison of genetic to physical distance on the BAC sequence, recombination was found to be moderate with a ratio of 125 kb/cM, but repressed within the APA locus itself. Several markers were predicted to be very effective for genetic studies or marker-assisted selection, based on their significant associations with bruchid resistance and on low adult insect emergence and positions flanking the arcelin and phytohemagglutinin genes.

Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme involved in the biosynthesis of proline in plants. By the 3' rapid amplification of cDNA ends (3'-RACE) approach, a 2,246-bp cDNA sequence was obtained from common bean (Phaseolus vulgaris L.), denominated PvP5CS2 differing from another P5CS gene that we cloned previously from common bean (PvP5CS). The predicted amino acid sequence of PvP5CS2 has an overall 93.2% identity GmP5CS (Glycine max L. P5CS). However, PvP5CS2 shows only 83.7% identity in amino acid sequence to PvP5CS, suggesting PvP5CS2 represents a homolog of the soybean P5CS gene. Abundant indel (insertion and deletion events) and SNP (single nucleotide polymorphisms) were found in the cloned PvP5CS2 genome sequence when comparing 24 cultivated and 3 wild common bean accessions and these in turn reflected aspects of common bean evolution. Sequence alignment showed that genotypes from the same gene pool had similar nucleotide variation, while genotypes from different gene pools had distinctly different nucleotide variation for PvP5CS2. Furthermore, diversity along the gene sequence was not evenly distributed, being low in the glutamic-g-semialdehyde dehydrogenase catalyzing region, moderate in the Glu-5-kinase catalyzing region and high in the intervening region. Neutrality tests showed that PvP5CS2 was a conserved gene undergoing negative selection. A new marker (Pv97) was developed for genetic mapping of PvP5CS2 based on an indel between DOR364 and G19833 sequences and the gene was located between markers Bng126 and BMd045 on chromosome b01. The relationship of PvP5CS2 and a previously cloned pyrroline-5-carboxylate synthetase gene as well as the implications of this work on selecting for drought tolerance in common bean are discussed.

Common bean can be grown as a grain crop (dry beans) or as a fresh vegetable (snap beans/green beans), both items being important in nutritional terms for providing essential minerals and vitamins to the diet. Snap beans are thought to be derived predominantly from dry beans of the Andean genepool and to be of a recent European origin; however, the existence of Mesoamerican genepool characteristics especially in traditional indeterminate growth habit snap beans indicates a wider origin. The objective of this study was to evaluate genetic diversity within a set of 120 indeterminate (pole type) snap beans and 7 control genotypes representing each genepool using amplified fragment length polymorphism (AFLP) and simple sequence repeat or microsatellite (SSR) markers. The genotypes were predominantly from Asia, Europe and the United States but included some varieties from Latin America and Africa. AFLP polymorphism ranged from 53.2 to 67.7% while SSR polymorphism averaged 95.3% for the 32 fluorescent and 11 non-fluorescent markers evaluated and total expected heterozygosity was higher for SSR markers (0.521) than for AFLP markers (0.209). Both marker systems grouped the genotypes into two genepools with Andean and Mesoamerican controls, respectively, with the Mesoamerican group being predominant in terms of the number of genotypes assigned to this genepool. Phaseolin alleles were not tightly associated with genepool assignment indicating that introgression of this locus had occurred between the genepools, especially with phaseolin "S" in the Andean group (23.5%) and phaseolins "T" and "C" in the Mesoamerican group (12.2 and 8.2%, respectively). The implications of these results on the origin of pole type snap beans and on breeding strategies for this horticultural crop are discussed.

The East African highlands are a region of important common bean production and high varietal diversity for the crop. The objective of this study was to uncover the diversity and population structure of 192 landraces from Ethiopia and Kenya together with four genepool control genotypes using morphological phenotyping and microsatellite marker genotyping. The germplasm represented different common bean production ecologies and seed types common in these countries. The landraces showed considerable diversity that corresponded well to the two recognized genepools (Andean and Mesoamerican) with little introgression between these groups. Mesoamerican genotypes were predominant in Ethiopia while Andean genotypes were predominant in Kenya. Within each country, landraces from different collection sites were clustered together indicating potential gene flow between regions within Kenya or within Ethiopia. Across countries, landraces from the same country of origin tended to cluster together indicating distinct germplasm at the national level and limited gene flow between the two countries highlighting divided social networks within the regions and a weak trans-national bean seed exchange especially for landrace varieties. One exception to this may be the case of small red-seeded beans where informal cross-border grain trade occurs. We also observed that genetic divergence was slightly higher for the Ethiopian landraces compared to Kenyan landraces and that Mesoamerican genotypes were more diverse than the Andean genotypes. Common beans in eastern Africa are often cultivated in marginal, risk-prone farming systems and the observed landrace diversity should provide valuable alleles for adaptation to stressful environments in future breeding programs in the region.

Cultivated common bean germplasm is especially diverse due to the parallel domestication of two genepools in the Mesoamerican and Andean centers of diversity and introgression between these gene pools. Classification into morphological races has helped to provide a framework for utilization of this cultivated germplasm. Meanwhile, core collections along with molecular markers are useful tools for organizing and analyzing representative sets of these genotypes. In this study, we evaluated 604 accessions from the CIAT core germplasm collection representing wide genetic variability from both primary and secondary centers of diversity with a newly developed, fluorescent microsatellite marker set of 36 genomic and gene-based SSRs to determine molecular diversity and with seed protein analysis to determine phaseolin alleles. The entire collection could be divided into two genepools and five predominant races with the division between the Mesoamerica race and the Durango-Jalisco group showing strong support within the Mesoamerican genepool and the Nueva Granada and Peru races showing less diversity overall and some between-group admixture within the Andean genepool. The Chile race could not be distinguished within the Andean genepool but there was support for the Guatemala race within the Mesoamerican genepool and this race was unique in its high level of diversity and distance from other Mesoamerican races. Based on this population structure, significant associations were found between SSR loci and seed size characteristics, some on the same linkage group as the phaseolin locus, which previously had been associated with seed size, or in other regions of the genome. In conclusion, this study has shown that common bean has very significant population structure that can help guide the construction of genetic crosses that maximize diversity as well as serving as a basis for additional association studies.

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.

Condensed tannins are major flavonoid end products that affect the nutritional quality of many legume seeds. They chelate minerals and interact with proteins, thus reducing their bioavailability. Tannins also contribute to seed coat color and pigment distribution or intensity. The objective of this study was to analyze the relationship between quantitative trait loci (QTL) for seed tannin concentration in common bean and Mendelian genes for seed coat color and pattern. Three populations of recombinant inbred lines, derived from crosses between the Andean and Mesoamerican genepools were used for QTL identification and for mapping STS markers associated with seed color loci. Seed coat condensed tannins were determined with a butanol-HCl method and a total of 12 QTL were identified on separate linkage groups (LGs) in each of the populations with individual QTL explaining from 10 to 64% of the phenotypic variation for this trait. Loci on linkage groups B3 and B10 were associated with the Mendelian genes Z and Bip for partly colored seed coat pattern, while a QTL on linkage group B7 was associated with the P gene which is the primary locus for the control of color expression in beans. In conclusion, this study found that the inheritance of tannin concentration fits an oligogenic model and identifies novel putative alleles at seed coat color and pattern genes that control tannin accumulation. The results will be important for the genetic improvement of nutritionally enhanced or biofortified beans that have health promoting effects from higher polyphenolics or better iron bioavailability.

Root gravitropism determines the relative distribution of plant roots in different soil layers, and therefore, may influence the acquisition of shallow soil resources such as phosphorus (P). Growth pouch and field studies were conducted to evaluate root gravitropism of common bean (Phaseolus vulgaris L.) in response to P deficiency and to detect quantitative trait loci (QTL) associated with this trait. A deep-rooted genotype, DOR364, was crossed with a shallow-rooted genotype, G19833, to obtain 86 F 5.7 recombinant inbred lines (RILs). Root gravitropic traits were measured as basal root growth angle (BRGA), shallow basal root length (SBRL, basal root length in the top 0-3 cm of soil) and relative shallow basal root length (RSBRL, percentage of basal root length in the top 0-3 cm of soil relative to total basal root length). Large genetic variability for these traits was found in the parents and RILs, with BRGA ranging from -18.73 to 56.69º and SBRL ranging from 0.42 to 2.63 m per plant. The parents and six RILs with contrasting root gravitropism were further evaluated in the field, where root shallowness was significantly correlated with plant growth and P uptake. QTL were detected by single point analysis (SPA), interval mapping (IM) and composite interval mapping (CIM) techniques with a genetic map for the DOR364 × G19833 population consisting of 236 molecular markers. The IM/CIM QTL were detected among the 11 linkage groups of common bean, with 16 QTL controlling the above root traits and six QTL controlling P acquisition efficiency (PAE) in the field study. At least three of the root trait QTL were associated with QTL for PAE, suggesting that root gravitropic traits are associated with PAE and that QTL for these traits can be used to facilitate selection and breeding for higher P efficiency in common bean and other crops.

The extent of 5S and 45S ribosomal DNA (rDNA) variation was investigated in wild and domesticated common beans (Phaseolus vulgaris) chosen to represent the known genetic diversity of the species. 5S and 45S rDNA probes were localized on mitotic chromosomes of 37 accessions by fluorescent in situ hybridization (FISH). The two 5S rDNA loci were largely conserved within the species, whereas a high variation in the number of 45S rDNA loci and changes in position of loci and number of repeats per locus were observed. Domesticated accessions from the Mesoamerican gene pool frequently had three 45S rDNA loci per haploid genome, and rarely four. Domesticated accessions from Andean gene pool, particularly from the race Peru, showed six, seven, eight or nine loci, but seven loci were found in all three races of this gene pool. Between three and eight loci were observed in accessions resulting from crosses between Andean and Mesoamerican genotypes. The presence of two to eight 45S rDNA loci in wild common beans from different geographic locations indicates that the 45S rDNA amplification observed in the Andean lineage took place before domestication. Our data suggest that ectopic recombination between terminal chromosomal regions might be the mechanism responsible for this variation.

Four multiplex panels of fluorescent-labeled rice microsatellite markers were designed to survey allelic diversity at a total of 27 simple sequence repeat loci. Automated fragment detection, size calling and binning were used to identify the microsatellite alleles. The panels were tested on 72 accessions representing the diversity of ecotypes and isozyme groups in cultivated rice ( Oryza sativa L.). Genetic diversity was evaluated by estimating the distribution and range of allele sizes, as well as the polymorphism information content (PIC), for each microsatellite locus. The multiplex panels were shown to be useful for fingerprinting and for clustering rice varieties, and genetic associations determined in this analysis agreed well with previous isozyme- and RFLP-based studies of these genotypes. Because of the wide cross-section of germplasm represented, we expect that the allele size-ranges reported here are close to the maximum for cultivated rice. In the future, panels of microsatellites from each rice chromosome will be made to facilitate the mapping of segregating populations and the identification of genes and QTLs underlying traits of interest.