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More than 60% of common bean (Phaseolus vulgaris L.) production worldwide is impacted by the risk of drought. In this study, the goal was to evaluate 64 bush bean genotypes from the CIAT reference collection to identify possible sources of drought resistance in the Andean gene pool. Phenotypic traits such as yield, 100-seed weight (P100) and days to physiological maturity (Dpm) were evaluated on selected accessions of this collection which was grown in an 8x8 lattice with two repetitions under three environments: intermittent drought (SI) and irrigation (R) in Palmira as well as early drought (ST) in Darién, Colombia. The genotypes included 20 from the Nueva Granada 1 (NG1) sub-race, 19 from the Nueva Granada 2 (NG2) sub-race, 10 from race Peru (P), 14 Andean control genotypes and one Mesoamerican check. The variables were analyzed through a combined ANOVA across environments, while simple correlations between yield and others variables were determinate. The genotypes with better adaptation to drought showed higher yields, 100-seed weight and fewer days to physiological maturity. The coefficients of correlations among yield and 100-seed weight were significant and positive, while Dpm showed negative correlation. Fourteen genotypes were identified as drought tolerant: G4001, G5625, G6639, G16115, G17070, G18255, G21210 and G22247 from the NG1 sub-race; G5708, G14253, G18264 and LRK31 from the NG2 sub-race; and DRK47 and G22147 from race Peru.More than 60% of common bean (Phaseolus vulgaris L.) production worldwide is impacted by the risk of drought. In this study, the goal was to evaluate 64 bush bean genotypes from the CIAT reference collection to identify possible sources of drought resistance in the Andean gene pool. Phenotypic traits such as yield, 100-seed weight (P100) and days to physiological maturity (Dpm) were evaluated on selected accessions of this collection which was grown in an 8x8 lattice with two repetitions under three environments: intermittent drought (SI) and irrigation (R) in Palmira as well as early drought (ST) in Darién, Colombia. The genotypes included 20 from the Nueva Granada 1 (NG1) sub-race, 19 from the Nueva Granada 2 (NG2) sub-race, 10 from race Peru (P), 14 Andean control genotypes and one Mesoamerican check. The variables were analyzed through a combined ANOVA across environments, while simple correlations between yield and others variables were determinate. The genotypes with better adaptation to drought showed higher yields, 100-seed weight and fewer days to physiological maturity. The coefficients of correlations among yield and 100-seed weight were significant and positive, while Dpm showed negative correlation. Fourteen genotypes were identified as drought tolerant: G4001, G5625, G6639, G16115, G17070, G18255, G21210 and G22247 from the NG1 sub-race; G5708, G14253, G18264 and LRK31 from the NG2 sub-race; and DRK47 and G22147 from race Peru.

El mejoramiento del fríjol en Colombia ha producido un amplio número de variedades para diferentes zonas agroecológicas y con diferentes rangos de adaptación. Los objetivos de esta investigación fueron determinar la calidad nutricional del fríjol en Colombia en cuanto al contenido de hierro y cinc en el grano y entender cómo los niveles de fertilización del suelo alta y baja en fósforo afectan tanto el rendimiento como el nivel de estos minerales. Se evaluaron 40 genotipos arbustivos dentro de los que se encuentran variedades liberadas por el ICA o Corpoica (series Diacol e ICA), cultivares tradicionales de los departamentos de Valle, Nariño, Norte de Santander, y líneas CIAT como testigos. Estos se sembraron en la localidad de Darién en el primer semestre de 2004, en los dos tratamientos de fertilización, con diseño de bloques completos aleatorizados con tres repeticiones. El contenido de hierro y cinc en la semilla cosechada fue determinado por espectroscopia de absorción atómica. El análisis de varianza mostró diferencias significativas para la concentración de los minerales entre las variedades y entre los niveles de alta y baja fertilización con fósforo con promedios de 61,5 y 53,2 ppm, respectivamente, para hierro, y 26,3 y 23,1 ppm, respectivamente, para cinc. Teniendo en cuenta el análisis combinado para hierro y cinc a través de los dos tratamientos de fósforo, se sugiere que la aplicación de fósforo puede incrementar la concentración de hierro en la semilla, pero no la de cinc.

Abstract Background The abscisic acid (ABA) pathway plays an important role in the plants’ reaction to drought stress and ABA-stress response (Asr) genes are important in controlling this process. In this sense, we accessed nucleotide diversity at two candidate genes for drought tolerance (Asr1 and Asr2), involved in an ABA signaling pathway, in the reference collection of cultivated common bean (Phaseolus vulgaris L.) and a core collection of wild common bean accessions. Results Our wild population samples covered a range of mesic (semi-arid) to very dry (desert) habitats, while our cultivated samples presented a wide spectrum of drought tolerance. Both genes showed very different patterns of nucleotide variation. Asr1 exhibited very low nucleotide diversity relative to the neutral reference loci that were previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Asr2 exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection. These patterns were more notable in wild beans than in cultivated common beans indicting that natural selection has played a role over long time periods compared to farmer selection since domestication. Conclusions Together these results suggested the importance of Asr1 in the context of drought tolerance, and constitute the first steps towards an association study between genetic polymorphism of this gene family and variation in drought tolerance traits. Furthermore, one of our major successes was to find that wild common bean is a reservoir of genetic variation and selection signatures at Asr genes, which may be useful for breeding drought tolerance in cultivated common bean.

Abstract Background In common bean, expressed sequence tags (ESTs) are an underestimated source of gene-based markers such as insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). However, due to the nature of these conserved sequences, detection of markers is difficult and portrays low levels of polymorphism. Therefore, development of intron-spanning EST-SNP markers can be a valuable resource for genetic experiments such as genetic mapping and association studies. Results In this study, a total of 313 new gene-based markers were developed at target genes. Intronic variation was deeply explored in order to capture more polymorphism. Introns were putatively identified after comparing the common bean ESTs with the soybean genome, and the primers were designed over intron-flanking regions. The intronic regions were evaluated for parental polymorphisms using the single strand conformational polymorphism (SSCP) technique and Sequenom MassARRAY system. A total of 53 new marker loci were placed on an integrated molecular map in the DOR364 × G19833 recombinant inbred line (RIL) population. The new linkage map was used to build a consensus map, merging the linkage maps of the BAT93 × JALO EEP558 and DOR364 × BAT477 populations. A total of 1,060 markers were mapped, with a total map length of 2,041 cM across 11 linkage groups. As a second application of the generated resource, a diversity panel with 93 genotypes was evaluated with 173 SNP markers using the MassARRAY-platform and KASPar technology. These results were coupled with previous SSR evaluations and drought tolerance assays carried out on the same individuals. This agglomerative dataset was examined, in order to discover marker-trait associations, using general linear model (GLM) and mixed linear model (MLM). Some significant associations with yield components were identified, and were consistent with previous findings. Conclusions In short, this study illustrates the power of intron-based markers for linkage and association mapping in common bean. The utility of these markers is discussed in relation with the usefulness of microsatellites, the molecular markers by excellence in this crop.

Abstract Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.

Abstract Background Our objective was to compare the capacities of biofortified and standard colored beans to deliver iron (Fe) for hemoglobin synthesis. Two isolines of large-seeded, red mottled Andean beans (Phaseolus vulgaris L.), one standard ("Low Fe") and the other biofortified ("High Fe") in Fe (49 and 71 μg Fe/g, respectively) were used. This commercial class of red mottled beans is the preferred varietal type for most of the Caribbean and Eastern and Southern Africa where almost three quarters of a million hectares are grown. Therefore it is important to know the affect of biofortification of these beans on diets that simulate human feeding studies. Methods Maize-based diets containing the beans were formulated to meet the nutrient requirements for broiler except for Fe (Fe concentrations in the 2 diets were 42.9 ± 1.2 and 54.6 ± 0.9 mg/kg). One day old chicks (Gallus gallus) were allocated to the experimental diets (n = 12). For 4 wk, hemoglobin, feed-consumption and body-weights were measured. Results Hemoglobin maintenance efficiencies (HME) (means ± SEM) were different between groups on days 14 and 21 of the experiment (P < 0.05). Final total body hemoglobin Fe contents were different between the standard (12.58 ± 1.0 mg {0.228 ± 0.01 μmol}) and high Fe (15.04 ± 0.65 mg {0.273 ± 0.01 μmol}) bean groups (P < 0.05). At the end of the experiment, tissue samples were collected from the intestinal duodenum and liver for further analyses. Divalent-metal-transporter-1, duodenal-cytochrome-B, and ferroportin expressions were higher and liver ferritin was lower (P < 0.05) in the standard group vs. the biofortified group. In-vitro analysis showed lower iron bioavailability in cells exposed to standard ("Low Fe") bean based diet. Conclusions We conclude that the in-vivo results support the in-vitro observations; biofortified colored beans contain more bioavailable-iron than standard colored beans. In addition, biofortified beans seems to be a promising vehicle for increasing intakes of bioavailable Fe in human populations that consume these beans as a dietary staple. This justifies further work on the large-seeded Andean beans which are the staple of a large-region of Africa where iron-deficiency anemia is a primary cause of infant death and poor health status.

Abstract 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.

Abstract Background Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L.) take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 × G19833), to identify quantitative trait loci (QTL) for this trait, and to assess possible associations with seed iron levels. Results The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III)-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient) were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe) on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe) on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Conclusions Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO) homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity under iron limited conditions may be useful in environments where beans are grown in alkaline soils, while the QTL for iron reductase under sufficiency conditions may be useful for selecting for enhanced seed nutritional quality.

Abstract Background Common bean (Phaseolus vulgaris L.) is the most important legume for direct human consumption and the goal of this study was to integrate a recently constructed physical map for the species with a microsatellite based genetic map using a BAC library from the genotype G19833 and the recombinant inbred line population DOR364 × G19833. Results We searched for simple sequence repeats (SSRs) in the 89,017 BAC-end sequences (BES) from the physical map and genetically mapped any polymorphic BES-SSRs onto the genetic map. Among the BES it was possible to identify 623 contig-linked SSRs, most of which were highly AT-rich. A subgroup of 230 di-nucleotide and tri-nucleotide based SSR primer pairs from these BACs was tested on the mapping parents with 176 single copy loci and 114 found to be polymorphic markers. Of these, 99 were successfully integrated into the genetic map. The 99 linkages between the genetic and physical maps corresponded to an equal number of contigs containing a total of 5,055 BAC clones. Conclusions Class II microsatellites were more common in the BES than longer class I microsatellites. Both types of markers proved to be valuable for linking BAC clones to the genetic map and were successfully placed across all 11 linkage groups. The integration of common bean physical and genetic maps is an important part of comparative genome analysis and a prelude to positional cloning of agronomically important genes for this crop.

Abstract Background An interesting seed protein family with a role in preventing insect herbivory is the multi-gene, APA family encoding the α-amylase inhibitor, phytohemagglutinin and arcelin proteins of common bean (Phaseolus vulgaris). Variability for this gene family exists and has been exploited to breed for insect resistance. For example, the arcelin locus has been successfully transferred from wild to cultivated common bean genotypes to provide resistance against the bruchid species Zabrotes subfasciatus although the process has been hampered by a lack of genetic tools for and understanding about the locus. In this study, we analyzed linkage disequilibrium (LD) between microsatellite markers at the APA locus and bruchid resistance in a germplasm survey of 105 resistant and susceptible genotypes and compared this with LD in other parts of the genome. Results Microsatellite allele diversity was found to vary with each of the eight APA-linked markers analyzed, and two markers within the APA locus were found to be diagnostic for bruchid resistance or susceptibility and for the different arcelin alleles inherited from the wild accessions. Arc1 was found to provide higher levels of resistance than Arc5 and the markers in the APA locus were highly associated with resistance showing that introgression of this gene-family from wild beans provides resistance in cultivated beans. LD around the APA locus was found to be intermediate compared to other regions of the genome and the highest LD was found within the APA locus itself for example between the markers PV-atct001 and PV-ag004. Conclusions We found the APA locus to be an important genetic determinant of bruchid resistance and also found that LD existed mostly within the APA locus but not beyond it. Moderate LD was also found for some other regions of the genome perhaps related to domestication genes. The LD pattern may reflect the introgression of arcelin from the wild into the cultivated background through breeding. LD and association studies for the arcelin gene, linked genes and other members of the APA family are essential for breaking linkage drag while maintaining high levels of bruchid resistance in common bean.

Abstract Background Iron is an important micronutrient for all living organisms. Almost 25% of the world population is affected by iron deficiency, a leading cause of anemia. In plants, iron deficiency leads to chlorosis and reduced yield. Both animals and plants may suffer from iron deficiency when their diet or environment lacks bioavailable iron. A sustainable way to reduce iron malnutrition in humans is to develop staple crops with increased content of bioavailable iron. Knowledge of where and how iron accumulates in seeds of crop plants will increase the understanding of plant iron metabolism and will assist in the production of staples with increased bioavailable iron. Results Here we reveal the distribution of iron in seeds of three Phaseolus species including thirteen genotypes of P. vulgaris, P. coccineus, and P. lunatus. We showed that high concentrations of iron accumulate in cells surrounding the provascular tissue of P. vulgaris and P. coccineus seeds. Using the Perls' Prussian blue method, we were able to detect iron in the cytoplasm of epidermal cells, cells near the epidermis, and cells surrounding the provascular tissue. In contrast, the protein ferritin that has been suggested as the major iron storage protein in legumes was only detected in the amyloplasts of the seed embryo. Using the non-destructive micro-PIXE (Particle Induced X-ray Emission) technique we show that the tissue in the proximity of the provascular bundles holds up to 500 μg g-1 of iron, depending on the genotype. In contrast to P. vulgaris and P. coccineus, we did not observe iron accumulation in the cells surrounding the provascular tissues of P. lunatus cotyledons. A novel iron-rich genotype, NUA35, with a high concentration of iron both in the seed coat and cotyledons was bred from a cross between an Andean and a Mesoamerican genotype. Conclusions The presented results emphasize the importance of complementing research in model organisms with analysis in crop plants and they suggest that iron distribution criteria should be integrated into selection strategies for bean biofortification.

Abstract Background Expressed sequence tags (ESTs) are an important source of gene-based markers such as those based on insertion-deletions (Indels) or single-nucleotide polymorphisms (SNPs). Several gel based methods have been reported for the detection of sequence variants, however they have not been widely exploited in common bean, an important legume crop of the developing world. The objectives of this project were to develop and map EST based markers using analysis of single strand conformation polymorphisms (SSCPs), to create a transcript map for common bean and to compare synteny of the common bean map with sequenced chromosomes of other legumes. Results A set of 418 EST based amplicons were evaluated for parental polymorphisms using the SSCP technique and 26% of these presented a clear conformational or size polymorphism between Andean and Mesoamerican genotypes. The amplicon based markers were then used for genetic mapping with segregation analysis performed in the DOR364 × G19833 recombinant inbred line (RIL) population. A total of 118 new marker loci were placed into an integrated molecular map for common bean consisting of 288 markers. Of these, 218 were used for synteny analysis and 186 presented homology with segments of the soybean genome with an e-value lower than 7 × 10-12. The synteny analysis with soybean showed a mosaic pattern of syntenic blocks with most segments of any one common bean linkage group associated with two soybean chromosomes. The analysis with Medicago truncatula and Lotus japonicus presented fewer syntenic regions consistent with the more distant phylogenetic relationship between the galegoid and phaseoloid legumes. Conclusion The SSCP technique is a useful and inexpensive alternative to other SNP or Indel detection techniques for saturating the common bean genetic map with functional markers that may be useful in marker assisted selection. In addition, the genetic markers based on ESTs allowed the construction of a transcript map and given their high conservation between species allowed synteny comparisons to be made to sequenced genomes. This synteny analysis may support positional cloning of target genes in common bean through the use of genomic information from these other legumes.

Abstract 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 genepools and therefore will be useful for diversity analyses and for comparative and transcript mapping in common bean.

Cowpea is one of the most popular dry-land legumes cultivated for food and forage in arid and semi-arid areas. Genetic diversity for global germplasm can be organized into core collections providing optimum resources to serve breeding requirements. Here, we present diversity analysis and genome-wide association study (GWAS) results for part of the cowpea core collection of the United States Department of Agriculture (USDA) along with breeding line controls. Included in the analysis were a total of 373 accessions analyzed with 6880 Single Nucleotide Polymorphism (SNP) markers from Genotyping by Sequencing (GBS). Population structure differentiated accessions into two groups irrespective of geographical origin and formed three clusters based on taxa upon phylogenetic analysis. A total of 56 SNPs were significantly associated to nine traits including pod length (25 Quantitative Trait Nucleotides, QTNs), seed anti-oxidant content (7 QTNs), dry pod color (7 QTNs), plant maturity (5 QTNs), flower color (5 QTNs), seed weight (4 QTNs), tolerance to low phosphate (1 QTN), growth habit (1 QTN), and response to rock phosphate (1 QTN) using Bayesian-information, Linkage-disequilibrium Iteratively Nested Keyway (BLINK), and Fixed and random model Circulating Probability Unification (FarmCPU) association models. Key genes related to all significant SNPs were identified based on annotations of the cowpea reference genome, including a flavonoid gene controlling flower color (Vigun08g040200.1), a root nodulation regulator for tolerance to low phosphate (Vigun11g168000.1), and numerous genes involved in signaling, biosynthesis, metabolite transport, and abiotic stress. Our results highlight the importance of maintaining public phenotyping databases at USDA and strengthening collaborations for data collection in cowpea to maximize research impacts. [ABSTRACT FROM AUTHOR]

Micronutrient deficiencies have a negative impact in the lives of millions of people worldwide. Most affected are children and pregnant women in developing regions. Biofortification is a sustainable way to alleviate micronutrient deficiencies in at-risk populations. To optimize the biofortification approach, it is important to consider both the quantities and bioavailability of the target micronutrients. Both the speciation and the localization of the micronutrients within the seed can have an impact on bioavailability. In this study we use the sensitive and non-destructive micro-PIXE technique to reveal the distribution of iron, zinc, manganese and phosphorus within soybean seeds. We show that high concentrations of iron accumulate in the seed coats of mature soybean seeds. This iron accounted for 20 to 40% of the total seed iron. Furthermore, manganese and iron accumulated in close proximity to each other in the provascular tissue of the soybean radicle. No regions with increased accumulation of iron, zinc, or manganese were observed in the cotyledons. The concentrations of both phosphorus and zinc were higher in the radicle compared to the cotyledons, and zinc accumulated primarily near the radicle tip.Our study provides a thorough description of the distribution of important micronutrients within the mature soybean seed.