Variation in cytonuclear expression accommodation among allopolyploid plants.

Cytonuclear coevolution is a common feature among plants, which coordinates gene expression and protein products between the nucleus and organelles. Consequently, lineage-specific differences may result in incompatibilities between the nucleus and cytoplasm in hybrid taxa. Allopolyploidy is also a common phenomenon in plant evolution. The hybrid nature of allopolyploids may result in cytonuclear incompatibilities, but the massive nuclear redundancy created during polyploidy affords additional avenues for resolving cytonuclear conflict (i.e. cytonuclear accommodation). Here we evaluate expression changes in organelle-targeted nuclear genes for 6 allopolyploid lineages that represent 4 genera (i.e. Arabidopsis, Arachis, Chenopodium, and Gossypium) and encompass a range in polyploid ages. Because incompatibilities between the nucleus and cytoplasm could potentially result in biases toward the maternal homoeolog and/or maternal expression level, we evaluate patterns of homoeolog usage, expression bias, and expression-level dominance in cytonuclear genes relative to the background of noncytonuclear expression changes and to the diploid parents. Although we find subsets of cytonuclear genes in most lineages that match our expectations of maternal preference, these observations are not consistent among either allopolyploids or categories of organelle-targeted genes. Our results indicate that cytonuclear expression evolution may be subtle and variable among genera and genes, likely reflecting a diversity of mechanisms to resolve nuclear-cytoplasmic incompatibilities in allopolyploid species.

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange.

The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii-enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.

Brazilian Kayabi Indian accessions of peanut, Arachis hypogaea (Fabales, Fabaceae): origin, diversity and evolution.

Peanut is a crop of the Kayabi tribe, inhabiting the Xingu Indigenous Park, Brazil. Morphological analysis of Xingu accessions showed variation exceeding that described for cultivated peanuts. This raised questions as to the origin of the Xingu accessions: are they derived from different species, or is their diversity a result of different evolutionary and selection processes? To answer these questions, cytogenetic and genotyping analyses were conducted. The karyotypes of Xingu accessions analyzed are very similar to each other, to an A. hypogaea subsp. fastigiata accession and to the wild allotetraploid A. monticola. The accessions share the number and general morphology of the chromosomes; DAPI+ bands; 5S and 45S rDNA loci distribution and a high genomic affinity with A. duranensis and A. ipaënsis genomic probes. However, the number of CMA3+ bands differs from those determined for A. hypogaea and A. monticola, which are also different from each other. SNP genotyping grouped all Arachis allotetraploids into four taxonomic groups: Xingu accessions were closer to A. monticola and A. hypogaea subsp. hypogaea. Our data suggests that the morphological diversity within these accessions is not associated with a different origin and can be attributed to morphological plasticity and different selection by the Indian tribes.

Pod and Seed Trait QTL Identification To Assist Breeding for Peanut Market Preferences.

Although seed and pod traits are important for peanut breeding, little is known about the inheritance of these traits. A recombinant inbred line (RIL) population of 156 lines from a cross of Tifrunner x NC 3033 was genotyped with the Axiom_Arachis1 SNP array and SSRs to generate a genetic map composed of 1524 markers in 29 linkage groups (LG). The genetic positions of markers were compared with their physical positions on the peanut genome to confirm the validity of the linkage map and explore the distribution of recombination and potential chromosomal rearrangements. This linkage map was then used to identify Quantitative Trait Loci (QTL) for seed and pod traits that were phenotyped over three consecutive years for the purpose of developing trait-associated markers for breeding. Forty-nine QTL were identified in 14 LG for seed size index, kernel percentage, seed weight, pod weight, single-kernel, double-kernel, pod area and pod density. Twenty QTL demonstrated phenotypic variance explained (PVE) greater than 10% and eight more than 20%. Of note, seven of the eight major QTL for pod area, pod weight and seed weight (PVE >20% variance) were attributed to NC 3033 and located in a single linkage group, LG B06_1. In contrast, the most consistent QTL for kernel percentage were located on A07/B07 and derived from Tifrunner.

Mapping quantitative trait loci (QTLs) and estimating the epistasis controlling stem rot resistance in cultivated peanut (Arachis hypogaea).

KEY MESSAGE: A total of 33 additive stem rot QTLs were identified in peanut genome with nine of them consistently detected in multiple years or locations. And 12 pairs of epistatic QTLs were firstly reported for peanut stem rot disease. Stem rot in peanut (Arachis hypogaea) is caused by the Sclerotium rolfsii and can result in great economic loss during production. In this study, a recombinant inbred line population from the cross between NC 3033 (stem rot resistant) and Tifrunner (stem rot susceptible) that consists of 156 lines was genotyped by using 58 K peanut single nucleotide polymorphism (SNP) array and phenotyped for stem rot resistance at multiple locations and in multiple years. A linkage map consisting of 1451 SNPs and 73 simple sequence repeat (SSR) markers was constructed. A total of 33 additive quantitative trait loci (QTLs) for stem rot resistance were detected, and six of them with phenotypic variance explained of over 10% (qSR.A01-2, qSR.A01-5, qSR.A05/B05-1, qSR.A05/B05-2, qSR.A07/B07-1 and qSR.B05-1) can be consistently detected in multiple years or locations. Besides, 12 pairs of QTLs with epistatic (additive × additive) interaction were identified. An additive QTL qSR.A01-2 also with an epistatic effect interacted with a novel locus qSR.B07_1-1 to affect the percentage of asymptomatic plants in a row. A total of 193 candidate genes within 38 stem rot QTLs intervals were annotated with functions of biotic stress resistance such as chitinase, ethylene-responsive transcription factors and pathogenesis-related proteins. The identified stem rot resistance QTLs, candidate genes, along with the associated SNP markers in this study, will benefit peanut molecular breeding programs for improving stem rot resistance.

The genome sequence of segmental allotetraploid peanut Arachis hypogaea.

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.

Heterochromatin evolution in Arachis investigated through genome-wide analysis of repetitive DNA.

MAIN CONCLUSION: The most conspicuous difference among chromosomes and genomes in Arachis species, the patterns of heterochromatin, was mainly modeled by differential amplification of different members of one superfamily of satellite DNAs. Divergence in repetitive DNA is a primary driving force for genome and chromosome evolution. Section Arachis is karyotypically diverse and has six different genomes. Arachis glandulifera (D genome) has the most asymmetric karyotype and the highest reproductive isolation compared to the well-known A and B genome species. These features make A. glandulifera an interesting model species for studying the main repetitive components that accompanied the genome and chromosome diversification in the section. Here, we performed a genome-wide analysis of repetitive sequences in A. glandulifera and investigated the chromosome distribution of the identified satellite DNA sequences (satDNAs). LTR retroelements, mainly the Ty3-gypsy families "Fidel/Feral" and "Pipoka/Pipa", were the most represented. Comparative analyses with the A and B genomes showed that many of the previously described transposable elements (TEs) were differently represented in the D genome, and that this variation accompanied changes in DNA content. In addition, four major satDNAs were characterized. Agla_CL8sat was the major component of pericentromeric heterochromatin, while Agla_CL39sat, Agla_CL69sat, and Agla_CL122sat were found in heterochromatic and/or euchromatic regions. Even though Agla_CL8sat belong to a different family than that of the major satDNA (ATR-2) found in the heterochromatin of the A, K, and F genomes, both satDNAs are members of the same superfamily. This finding suggests that closely related satDNAs of an ancestral library were differentially amplified leading to the major changes in the heterochromatin patterns that accompanied the karyotype and genome differentiation in Arachis.

Mapping Late Leaf Spot Resistance in Peanut (Arachis hypogaea) Using QTL-seq Reveals Markers for Marker-Assisted Selection.

Late leaf spot (LLS; Cercosporidium personatum) is a major fungal disease of cultivated peanut (Arachis hypogaea). A recombinant inbred line population segregating for quantitative field resistance was used to identify quantitative trait loci (QTL) using QTL-seq. High rates of false positive SNP calls using established methods in this allotetraploid crop obscured significant QTLs. To resolve this problem, robust parental SNPs were first identified using polyploid-specific SNP identification pipelines, leading to discovery of significant QTLs for LLS resistance. These QTLs were confirmed over 4 years of field data. Selection with markers linked to these QTLs resulted in a significant increase in resistance, showing that these markers can be immediately applied in breeding programs. This study demonstrates that QTL-seq can be used to rapidly identify QTLs controlling highly quantitative traits in polyploid crops with complex genomes. Markers identified can then be deployed in breeding programs, increasing the efficiency of selection using molecular tools. Key Message: Field resistance to late leaf spot is a quantitative trait controlled by many QTLs. Using polyploid-specific methods, QTL-seq is faster and more cost effective than QTL mapping.

Development and Evaluation of a High Density Genotyping 'Axiom_Arachis' Array with 58 K SNPs for Accelerating Genetics and Breeding in Groundnut.

Single nucleotide polymorphisms (SNPs) are the most abundant DNA sequence variation in the genomes which can be used to associate genotypic variation to the phenotype. Therefore, availability of a high-density SNP array with uniform genome coverage can advance genetic studies and breeding applications. Here we report the development of a high-density SNP array 'Axiom_Arachis' with 58 K SNPs and its utility in groundnut genetic diversity study. In this context, from a total of 163,782 SNPs derived from DNA resequencing and RNA-sequencing of 41 groundnut accessions and wild diploid ancestors, a total of 58,233 unique and informative SNPs were selected for developing the array. In addition to cultivated groundnuts (Arachis hypogaea), fair representation was kept for other diploids (A. duranensis, A. stenosperma, A. cardenasii, A. magna and A. batizocoi). Genotyping of the groundnut 'Reference Set' containing 300 genotypes identified 44,424 polymorphic SNPs and genetic diversity analysis provided in-depth insights into the genetic architecture of this material. The availability of the high-density SNP array 'Axiom_Arachis' with 58 K SNPs will accelerate the process of high resolution trait genetics and molecular breeding in cultivated groundnut.

Genome-wide SNP Genotyping Resolves Signatures of Selection and Tetrasomic Recombination in Peanut.

Peanut (Arachis hypogaea; 2n = 4x = 40) is a nutritious food and a good source of vitamins, minerals, and healthy fats. Expansion of genetic and genomic resources for genetic enhancement of cultivated peanut has gained momentum from the sequenced genomes of the diploid ancestors of cultivated peanut. To facilitate high-throughput genotyping of Arachis species, 20 genotypes were re-sequenced and genome-wide single nucleotide polymorphisms (SNPs) were selected to develop a large-scale SNP genotyping array. For flexibility in genotyping applications, SNPs polymorphic between tetraploid and diploid species were included for use in cultivated and interspecific populations. A set of 384 accessions was used to test the array resulting in 54 564 markers that produced high-quality polymorphic clusters between diploid species, 47 116 polymorphic markers between cultivated and interspecific hybrids, and 15 897 polymorphic markers within A. hypogaea germplasm. An additional 1193 markers were identified that illuminated genomic regions exhibiting tetrasomic recombination. Furthermore, a set of elite cultivars that make up the pedigree of US runner germplasm were genotyped and used to identify genomic regions that have undergone positive selection. These observations provide key insights on the inclusion of new genetic diversity in cultivated peanut and will inform the development of high-resolution mapping populations. Due to its efficiency, scope, and flexibility, the newly developed SNP array will be very useful for further genetic and breeding applications in Arachis.

A Comparative Epigenomic Analysis of Polyploidy-Derived Genes in Soybean and Common Bean.

Soybean (Glycine max) and common bean (Phaseolus vulgaris) share a paleopolyploidy (whole-genome duplication [WGD]) event, approximately 56.5 million years ago, followed by a genus Glycine-specific polyploidy, approximately 10 million years ago. Cytosine methylation is an epigenetic mark that plays an important role in the regulation of genes and transposable elements (TEs); however, the role of DNA methylation in the fate/evolution of genes following polyploidy and speciation has not been fully explored. Whole-genome bisulfite sequencing was used to produce nucleotide resolution methylomes for soybean and common bean. We found that, in soybean, CG body-methylated genes were abundant in WGD genes, which were, on average, more highly expressed than single-copy genes and had slower evolutionary rates than unmethylated genes, suggesting that WGD genes evolve more slowly than single-copy genes. CG body-methylated genes were also enriched in shared single-copy genes (single copy in both species) that may be responsible for the broad and high expression patterns of this class of genes. In addition, diverged methylation patterns in non-CG contexts between paralogs were due mostly to TEs in or near genes, suggesting a role for TEs and non-CG methylation in regulating gene expression post polyploidy. Reference methylomes for both soybean and common bean were constructed, providing resources for investigating epigenetic variation in legume crops. Also, the analysis of methylation patterns of duplicated and single-copy genes has provided insights into the functional consequences of polyploidy and epigenetic regulation in plant genomes.

Genome-wide identification of evolutionarily conserved alternative splicing events in flowering plants.

Alternative splicing (AS) plays important roles in many plant functions, but its conservation across the plant kingdom is not known. We describe a methodology to identify AS events and identify conserved AS events across large phylogenetic distances using RNA-Seq datasets. We applied this methodology to transcriptome data from nine angiosperms including Amborella, the single sister species to all other extant flowering plants. AS events within 40-70% of the expressed multi-exonic genes per species were found, 27,120 of which are conserved among two or more of the taxa studied. While many events are species specific, many others are shared across long evolutionary distances suggesting they have functional significance. Conservation of AS event data provides an estimate of the number of ancestral AS events present at each node of the tree representing the nine species studied. Furthermore, the presence or absence of AS isoforms between species with different whole genome duplication (WGD) histories provides the opportunity to examine the impact of WDG on AS potential. Examining AS in gene families identifies those with high rates of AS, and conservation can distinguish ancient events vs. recent or species specific adaptations. The MADS-box and SR protein families are found to represent families with low and high occurrences of AS, respectively, yet their AS events were likely present in the MRCA of angiosperms.

Transcriptomic changes due to water deficit define a general soybean response and accession-specific pathways for drought avoidance.

BACKGROUND: Among abiotic stresses, drought is the most common reducer of crop yields. The slow-wilting soybean genotype PI 416937 is somewhat robust to water deficit and has been used previously to map the trait in a bi-parental population. Since drought stress response is a complex biological process, whole genome transcriptome analysis was performed to obtain a deeper understanding of the drought response in soybean. RESULTS: Contrasting data from PI 416937 and the cultivar 'Benning', we developed a classification system to identify genes that were either responding to water-deficit in both genotypes or that had a genotype x environment (GxE) response. In spite of very different wilting phenotypes, 90% of classifiable genes had either constant expression in both genotypes (33%) or very similar response profiles (E genes, 57%). By further classifying E genes based on expression profiles, we were able to discern the functional specificity of transcriptional responses at particular stages of water-deficit, noting both the well-known reduction in photosynthesis genes as well as the less understood up-regulation of the protein transport pathway. Two percent of classifiable genes had a well-defined GxE response, many of which are located within slow-wilting QTLs. We consider these strong candidates for possible causal genes underlying PI 416937's unique drought avoidance strategy. CONCLUSIONS: There is a general and functionally significant transcriptional response to water deficit that involves not only known pathways, such as down-regulation of photosynthesis, but also up-regulation of protein transport and chromatin remodeling. Genes that show a genotypic difference are more likely to show an environmental response than genes that are constant between genotypes. In this study, at least five genes that clearly exhibited a genotype x environment response fell within known QTL and are very good candidates for further research into slow-wilting.

Tetrasomic recombination is surprisingly frequent in allotetraploid Arachis.

Arachis hypogaea L. (cultivated peanut) is an allotetraploid (2n = 4x = 40) with an AABB genome type. Based on cytogenetic studies it has been assumed that peanut and wild-derived induced AABB allotetraploids have classic allotetraploid genetic behavior with diploid-like disomic recombination only between homologous chromosomes, at the exclusion of recombination between homeologous chromosomes. Using this assumption, numerous linkage map and quantitative trait loci studies have been carried out. Here, with a systematic analysis of genotyping and gene expression data, we show that this assumption is not entirely valid. In fact, autotetraploid-like tetrasomic recombination is surprisingly frequent in recombinant inbred lines generated from a cross of cultivated peanut and an induced allotetraploid derived from peanut's most probable ancestral species. We suggest that a better, more predictive genetic model for peanut is that of a "segmental allotetraploid" with partly disomic, partly tetrasomic genetic behavior. This intermediate genetic behavior has probably had a previously overseen, but significant, impact on the genome and genetics of cultivated peanut.

Single Nucleotide Polymorphism Identification in Polyploids: A Review, Example, and Recommendations.

Understanding the relationship between genotype and phenotype is a major biological question and being able to predict phenotypes based on molecular genotypes is integral to molecular breeding. Whole-genome duplications have shaped the history of all flowering plants and present challenges to elucidating the relationship between genotype and phenotype, especially in neopolyploid species. Although single nucleotide polymorphisms (SNPs) have become popular tools for genetic mapping, discovery and application of SNPs in polyploids has been difficult. Here, we summarize common experimental approaches to SNP calling, highlighting recent polyploid successes. To examine the impact of software choice on these analyses, we called SNPs among five peanut genotypes using different alignment programs (BWA-mem and Bowtie 2) and variant callers (SAMtools, GATK, and Freebayes). Alignments produced by Bowtie 2 and BWA-mem and analyzed in SAMtools shared 24.5% concordant SNPs, and SAMtools, GATK, and Freebayes shared 1.4% concordant SNPs. A subsequent analysis of simulated Brassica napus chromosome 1A and 1C genotypes demonstrated that, of the three software programs, SAMtools performed with the highest sensitivity and specificity on Bowtie 2 alignments. These results, however, are likely to vary among species, and we therefore propose a series of best practices for SNP calling in polyploids.

A reference genome for common bean and genome-wide analysis of dual domestications.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption and has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. We assembled 473 Mb of the 587-Mb genome and genetically anchored 98% of this sequence in 11 chromosome-scale pseudomolecules. We compared the genome for the common bean against the soybean genome to find changes in soybean resulting from polyploidy. Using resequencing of 60 wild individuals and 100 landraces from the genetically differentiated Mesoamerican and Andean gene pools, we confirmed 2 independent domestications from genetic pools that diverged before human colonization. Less than 10% of the 74 Mb of sequence putatively involved in domestication was shared by the two domestication events. We identified a set of genes linked with increased leaf and seed size and combined these results with quantitative trait locus data from Mesoamerican cultivars. Genes affected by domestication may be useful for genomics-enabled crop improvement.

The Subtelomeric khipu Satellite Repeat from Phaseolus vulgaris: Lessons Learned from the Genome Analysis of the Andean Genotype G19833.

Subtelomeric regions in eukaryotic organisms are known for harboring species-specific tandemly repeated satellite sequences. However, studies on the molecular organization and evolution of subtelomeric repeats are scarce, especially in plants. Khipu is a satellite DNA of 528-bp repeat unit, specific of the Phaseolus genus, with a subtelomeric distribution in common bean, P. vulgaris. To investigate the genomic organization and the evolution of khipu, we performed genome-wide analysis on the complete genome sequence of the common bean genotype G19833. We identified 2,460 khipu units located at most distal ends of the sequenced regions. Khipu units are arranged in discrete blocks of 2-55 copies and are heterogeneously distributed among the different chromosome ends of G19833 (from 0 to 555 khipus units per chromosome arm). Phylogenetically related khipu units are spread between numerous chromosome ends, suggesting frequent exchanges between non-homologous subtelomeres. However, most subclades contain numerous khipu units from only one or few chromosome ends indicating that local duplication is also driving khipu expansion. Unexpectedly, we also identified 81 khipu units located at centromeres. All the centromeric khipu units belong to a single divergent clade also comprised of a few units from several subtelomeres, suggesting that a few sequence exchanges between centromeres and subtelomeres took place in the common bean genome. The divergence and low copy number of these centromeric units from the subtelomeric units could explain why they were not detected by FISH (Fluorescence in situ Hybridization) although it can not be excluded that these centromeric units may have resulted from errors in the pseudomolecule assembly. Altogether our data highlight extensive sequence exchanges in subtelomeres between non-homologous chromosomes in common bean and confirm that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes.

Nucleotide diversity patterns at the drought-related DREB2 encoding genes in wild and cultivated common bean (Phaseolus vulgaris L.).

Common beans are an important food legume faced with a series of abiotic stresses the most severe of which is drought. The crop is interesting as a model for the analysis of gene phylogenies due to its domestication process, race structure, and origins in a group of wild common beans found along the South American Andes and the region of Mesoamerica. Meanwhile, the DREB2 transcription factors have been implicated in controlling non-ABA dependent responses to drought stress. With this in mind our objective was to study in depth the genetic diversity for two DREB2 genes as possible candidates for association with drought tolerance through a gene phylogenetic analysis. In this genetic diversity assessment, we analyzed nucleotide diversity at the two candidate genes Dreb2A and Dreb2B, in partial core collections of 104 wild and 297 cultivated common beans with a total of 401 common bean genotypes from world-wide germplasm analyzed. Our wild population sample covered a range of semi-mesic to very dry habitats, while our cultivated samples presented a wide spectrum of low to high drought tolerance. Both genes showed very different patterns of nucleotide variation. Dreb2B exhibited very low nucleotide diversity relative to neutral reference loci previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Dreb2A exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection and population expansion. These patterns were more distinct in wild compared to cultivated common beans. These approximations suggested the importance of Dreb2 genes 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. We discuss the utility of allele mining in the DREB gene family for the discovery of new drought tolerance traits from wild common bean.

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.

Integration of physical and genetic maps of common bean through BAC-derived microsatellite markers.

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 x 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.