Genome-Environment Associations, an Innovative Tool for Studying Heritable Evolutionary Adaptation in Orphan Crops and Wild Relatives.

Leveraging innovative tools to speed up prebreeding and discovery of genotypic sources of adaptation from landraces, crop wild relatives, and orphan crops is a key prerequisite to accelerate genetic gain of abiotic stress tolerance in annual crops such as legumes and cereals, many of which are still orphan species despite advances in major row crops. Here, we review a novel, interdisciplinary approach to combine ecological climate data with evolutionary genomics under the paradigm of a new field of study: genome-environment associations (GEAs). We first exemplify how GEA utilizes in situ georeferencing from genotypically characterized, gene bank accessions to pinpoint genomic signatures of natural selection. We later discuss the necessity to update the current GEA models to predict both regional- and local- or micro-habitat-based adaptation with mechanistic ecophysiological climate indices and cutting-edge GWAS-type genetic association models. Furthermore, to account for polygenic evolutionary adaptation, we encourage the community to start gathering genomic estimated adaptive values (GEAVs) for genomic prediction (GP) and multi-dimensional machine learning (ML) models. The latter two should ideally be weighted by de novo GWAS-based GEA estimates and optimized for a scalable marker subset. We end the review by envisioning avenues to make adaptation inferences more robust through the merging of high-resolution data sources, such as environmental remote sensing and summary statistics of the genomic site frequency spectrum, with the epigenetic molecular functionality responsible for plastic inheritance in the wild. Ultimately, we believe that coupling evolutionary adaptive predictions with innovations in ecological genomics such as GEA will help capture hidden genetic adaptations to abiotic stresses based on crop germplasm resources to assist responses to climate change. "I shall endeavor to find out how nature's forces act upon one another, and in what manner the geographic environment exerts its influence on animals and plants. In short, I must find out about the harmony in nature" Alexander von Humboldt-Letter to Karl Freiesleben, June 1799.

Allelic Diversity at Abiotic Stress Responsive Genes in Relationship to Ecological Drought Indices for Cultivated Tepary Bean, Phaseolus acutifolius A. Gray, and Its Wild Relatives.

Some of the major impacts of climate change are expected in regions where drought stress is already an issue. Grain legumes are generally drought susceptible. However, tepary bean and its wild relatives within Phaseolus acutifolius or P. parvifolius are from arid areas between Mexico and the United States. Therefore, we hypothesize that these bean accessions have diversity signals indicative of adaptation to drought at key candidate genes such as: Asr2, Dreb2B, and ERECTA. By sequencing alleles of these genes and comparing to estimates of drought tolerance indices from climate data for the collection site of geo-referenced, tepary bean accessions, we determined the genotype x environmental association (GEA) of each gene. Diversity analysis found that cultivated and wild P. acutifolius were intermingled with var. tenuifolius and P. parvifolius, signifying that allele diversity was ample in the wild and cultivated clade over a broad sense (sensu lato) evaluation. Genes Dreb2B and ERECTA harbored signatures of directional selection, represented by six SNPs correlated with the environmental drought indices. This suggests that wild tepary bean is a reservoir of novel alleles at genes for drought tolerance, as expected for a species that originated in arid environments. Our study corroborated that candidate gene approach was effective for marker validation across a broad genetic base of wild tepary accessions.

Source-to-Tap Assessment of Microbiological Water Quality in Small Rural Drinking Water Systems in Puerto Rico Six Months After Hurricane Maria.

Maria made a landfall in Puerto Rico on September 20, 2017 as a category 4 hurricane, causing severe flooding, widespread electricity outages, damage to infrastructure, and interruptions in water and wastewater treatment. Small rural community water systems face unique challenges in providing drinking water, which intensify after natural disasters. The purpose of this study was to evaluate the functionality of six very small rural public water systems and one large regulated system in Puerto Rico six months after Maria and survey a broad sweep of fecal, zoonotic, and opportunistic pathogens from the source to tap. Samples were collected from surface and groundwater sources, after water treatment and after distribution to households. Genes indicative of pathogenic Leptospira spp. were detected by polymerase chain reaction (PCR) in all systems reliant on surface water sources. Salmonella spp. was detected in surface and groundwater sources and some distribution system water both by culture and PCR. Legionella spp. and Mycobacteria spp. gene numbers measured by quantitative PCR were similar to nonoutbreak conditions in the continental U.S. Amplicon sequencing provided a nontarget screen for other potential pathogens of concern. This study aids in improving future preparedness, assessment, and recovery operations for small rural water systems after natural disasters.

Seed Structural Variability and Germination Capacity in Passiflora edulis Sims f. edulis.

Purple passion fruit, Passiflora edulis Sims f. edulis, is an important semi-perennial, fruit bearing vine originating in South America that produces a commercial tropical juice pulp for international and national consumption. Within the round purple passion fruit are a large number of membranous seed sacs each containing individual seeds. Little is known about the seed anatomy of the commercial passion fruit, differences between wild collected and commercial types, and its effect on seedling germination. Therefore, our main objective for this study was to analyze the seed anatomy variability of different germplasm as well as the effect on viability and germination of the seeds of this species. Germplasm was evaluated from three sources: (1) commercial cultivars grown in current production areas, (2) genebank accession from the national seed bank, and (3) landraces collected across different high and mid-elevation sites of the Andean region. A total of 12 morphometric descriptors related to seed anatomy were evaluated on the 56 genotypes, of which three were most informative: Angle to the vertex which is related to the shape of the seed, the thickness of the tegument and the horizontal length; separating the seed according to its source of origin. Germination was found to be positively correlated with the number (r = 0.789) and depth (r = 0.854) of seed pitting. Seeds of the commercial cultivars had more seed pits and higher germination compared to seeds of landraces or genebank accessions showing a possible effect of domestication on the crop. Interestingly, passion fruits often germinate during the rainy season as escaped or wild seedlings especially in the disturbed landscapes of coffee plantations, so some dormancy is needed but faster germination is needed for intensive cultivation. Harnessing passion fruit diversity would be useful as the semi-domesticated landraces have valuable adaptation characteristics to combine with rapid germination selected in the commercial cultivars. The variability of seed pitting with cultivars more pitted than landraces possibly resulting in faster germination may indicate that purple passion fruit is still undergoing a process of selection and domestication for this trait.

Does the Genomic Landscape of Species Divergence in Phaseolus Beans Coerce Parallel Signatures of Adaptation and Domestication?

Exploring the genomic architecture of species and populations divergence aids understanding how lineages evolve and adapt, and ultimately can show the repeatability of evolutionary processes. Yet, the genomic signatures associated with divergence are still relatively unexplored, leading to a knowledge gap on whether species divergence ultimately differs in its genetic architecture from divergence at other spatial scales (i.e., populations, ecotypes). Our goal in this research was to determine whether genomic islands of speciation are more prone to harbor within-species differentiation due to genomic features, suppressed recombination, smaller effective population size or increased drift, across repeated hierarchically nested levels of divergence. We used two species of Phaseolus beans with strong genepool and population sub-structure produced by multiple independent domestications each especially in Andean and Mesoamerican / Middle American geographies. We genotyped 22,531 GBS-derived SNP markers in 209 individuals of wild and cultivated Phaseolus vulgaris and Phaseolus lunatus. We identified six regions for species-associated divergence. Out of these divergence peaks, 21% were recovered in the four within-species between-genepool comparisons and in the five within-genepool wild-cultivated comparisons (some of the latter did retrieve genuine signatures of the well described multiple domestication syndromes). However, genomic regions with overall high relative differentiation (measured by FST) coincided with regions of low SNP density and regions of elevated delta divergence between-genepools (ΔDiv), independent of the scale of divergence. The divergence in chromosome Pv10 further coincided with a between-species pericentric inversion. These convergences suggest that shared variants are being recurrently fixed at replicated regions of the genome, and in a similar manner across different hierarchically nested levels of divergence, likely as result of genomic features that make certain regions more prone to accumulate islands of speciation and within-species divergence. In summary, neighboring signatures of speciation, adaptation and domestication in Phaseolus beans are influenced by ubiquitous genomic constrains, which may continue to fortuitously shape genomic differentiation at various others scales of divergence.

Mapping QTLs for drought tolerance in a SEA 5 x AND 277 common bean cross with SSRs and SNP markers

Abstract The common bean is characterized by high sensitivity to drought and low productivity. Breeding for drought resistance in this species involves genes of different genetic groups. In this work, we used a SEA 5 x AND 277 cross to map quantitative trait loci associated with drought tolerance in order to assess the factors that determine the magnitude of drought response in common beans. A total of 438 polymorphic markers were used to genotype the F8 mapping population. Phenotyping was done in two greenhouses, one used to simulate drought and the other to simulate irrigated conditions. Fourteen traits associated with drought tolerance were measured to identify the quantitative trait loci (QTLs). The map was constructed with 331 markers that covered all 11 chromosomes and had a total length of 1515 cM. Twenty-two QTLs were discovered for chlorophyll, leaf and stem fresh biomass, leaf biomass dry weight, leaf temperature, number of pods per plant, number of seeds per plant, seed weight, days to flowering, dry pod weight and total yield under well-watered and drought (stress) conditions. All the QTLs detected under drought conditions showed positive effects of the SEA 5 allele. This study provides a better understanding of the genetic inheritance of drought tolerance in common bean.

Mapping QTLs for drought tolerance in a SEA 5 x AND 277 common bean cross with SSRs and SNP markers.

The common bean is characterized by high sensitivity to drought and low productivity. Breeding for drought resistance in this species involves genes of different genetic groups. In this work, we used a SEA 5 x AND 277 cross to map quantitative trait loci associated with drought tolerance in order to assess the factors that determine the magnitude of drought response in common beans. A total of 438 polymorphic markers were used to genotype the F8 mapping population. Phenotyping was done in two greenhouses, one used to simulate drought and the other to simulate irrigated conditions. Fourteen traits associated with drought tolerance were measured to identify the quantitative trait loci (QTLs). The map was constructed with 331 markers that covered all 11 chromosomes and had a total length of 1515 cM. Twenty-two QTLs were discovered for chlorophyll, leaf and stem fresh biomass, leaf biomass dry weight, leaf temperature, number of pods per plant, number of seeds per plant, seed weight, days to flowering, dry pod weight and total yield under well-watered and drought (stress) conditions. All the QTLs detected under drought conditions showed positive effects of the SEA 5 allele. This study provides a better understanding of the genetic inheritance of drought tolerance in common bean.

Linkage and mapping of quantitative trait loci associated with angular leaf spot and powdery mildew resistance in common beans

Abstract Angular leaf spot (ALS) and powdery mildew (PWM) are two important fungi diseases causing significant yield losses in common beans. In this study, a new genetic linkage map was constructed using single sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in a segregating population derived from the AND 277 x SEA 5 cross, with 105 recombinant inbred lines. Phenotypic evaluations were performed in the greenhouse to identify quantitative trait loci (QTLs) associated with resistance by means of the composite interval mapping analysis. Four QTLs were identified for ALS resistance. The QTL ALS11AS, linked on the SNP BAR 5054, mapped on chromosome Pv11, showed the greatest effect (R2 = 26.5%) on ALS phenotypic variance. For PWM resistance, two QTLs were detected, PWM2AS and PWM11AS, on Pv2 and Pv11, explaining 7% and 66% of the phenotypic variation, respectively. Both QTLs on Pv11 were mapped on the same genomic region, suggesting that it is a pleiotropic region. The present study resulted in the identification of new markers closely linked to ALS and PWM QTLs, which can be used for marker-assisted selection, fine mapping and positional cloning.

Linkage and mapping of quantitative trait loci associated with angular leaf spot and powdery mildew resistance in common beans.

Angular leaf spot (ALS) and powdery mildew (PWM) are two important fungi diseases causing significant yield losses in common beans. In this study, a new genetic linkage map was constructed using single sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in a segregating population derived from the AND 277 x SEA 5 cross, with 105 recombinant inbred lines. Phenotypic evaluations were performed in the greenhouse to identify quantitative trait loci (QTLs) associated with resistance by means of the composite interval mapping analysis. Four QTLs were identified for ALS resistance. The QTL ALS11AS, linked on the SNP BAR 5054, mapped on chromosome Pv11, showed the greatest effect (R2 = 26.5%) on ALS phenotypic variance. For PWM resistance, two QTLs were detected, PWM2AS and PWM11AS, on Pv2 and Pv11, explaining 7% and 66% of the phenotypic variation, respectively. Both QTLs on Pv11 were mapped on the same genomic region, suggesting that it is a pleiotropic region. The present study resulted in the identification of new markers closely linked to ALS and PWM QTLs, which can be used for marker-assisted selection, fine mapping and positional cloning.

Identification of an ERECTA gene and its drought adaptation associations with wild and cultivated common bean.

In this research, we cloned and accessed nucleotide diversity in the common bean ERECTA gene which has been implicated in drought tolerance and stomatal patterning. The homologous gene segment was isolated with degenerate primer and was found to be located on Chromosome 1. The gene had at least one paralog on Chromosome 9 and duplicate copies in soybean for each homolog. ERECTA-like genes were also discovered but the function of these was of less interest due to low similarity with the ERECTA gene from Arabidopsis. The diversity of the 5' end of the large Chr. 1 PvERECTA gene was evaluated in a collection of 145 wild and cultivated common beans that were also characterized by geographic source and drought tolerance, respectively. Our wild population sampled a range of wet to dry habitats, while our cultivated samples were representative of landrace diversity and the patterns of nucleotide variation differed between groups. The 5' region exhibited lower levels of diversity in the cultivated collection, which was indicative of population bottlenecks associated with the domestication process, compared to the wild collection where diversity was associated with ecological differences. We discuss associations of nucleotide diversity at PvERECTA with drought tolerance prediction for the genotypes.

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.

EST-SSR markers from five sequenced cDNA libraries of common bean (Phaseolus vulgaris L.) comparing three bioinformatic algorithms.

Expressed sequence tags (ESTs) are a rich source of SSR sequences, but the proportion of long Class I microsatellites with many repeats vs. short Class II microsatellites with few repeats is an important factor to consider. Class I microsatellites, with more than 20 bp of repeats, tend to make better markers with higher polymorphism. The goal of this study was to determine the frequency of Class I and Class II microsatellites in a collection of over 21,000 ESTs from a single study of five different tissues of common bean: two types of leaves, nodules, pods and roots. For this objective, we used three different bioinformatics pipelines: Automated Microsatellite Marker Development (AMMD), Batchprimer3 and SSRLocator. In addition, we determined the frequency of single or multiple SSRs in the assembled ESTs, the frequency of perfect and compound repeats and whether Class I microsatellites were mainly di-nucleotide or tri-nucleotide motifs with each of the search engines. Primers were designed for a total of 175 microsatellites concentrating on class I microsatellites identified with SSR locator. A few other microsatellites were included from the other search engines, AMMD and Batchprimer3 programs so as to have a representative set of class II markers for comparison sake. The comparison of 95 class I vs. 80 class II markers confirmed that the Class I were more polymorphic and therefore more useful.

Phenotyping common beans for adaptation to drought.

Common beans (Phaseolus vulgaris L.) originated in the New World and are the grain legume of greatest production for direct human consumption. Common bean production is subject to frequent droughts in highland Mexico, in the Pacific coast of Central America, in northeast Brazil, and in eastern and southern Africa from Ethiopia to South Africa. This article reviews efforts to improve common bean for drought tolerance, referring to genetic diversity for drought response, the physiology of drought tolerance mechanisms, and breeding strategies. Different races of common bean respond differently to drought, with race Durango of highland Mexico being a major source of genes. Sister species of P. vulgaris likewise have unique traits, especially P. acutifolius which is well adapted to dryland conditions. Diverse sources of tolerance may have different mechanisms of plant response, implying the need for different methods of phenotyping to recognize the relevant traits. Practical considerations of field management are discussed including: trial planning; water management; and field preparation.

Diversification and population structure in common beans (Phaseolus vulgaris L.).

Wild accessions of crops and landraces are valuable genetic resources for plant breeding and for conserving alleles and gene combinations in planta. The primary genepool of cultivated common beans includes wild accessions of Phaseolus vulgaris. These are of the same species as the domesticates and therefore are easily crossable with cultivated accessions. Molecular marker assessment of wild beans and landraces is important for the proper utilization and conservation of these important genetic resources. The goal of this research was to evaluate a collection of wild beans with fluorescent microsatellite or simple sequence repeat markers and to determine the population structure in combination with cultivated beans of all known races. Marker diversity in terms of average number of alleles per marker was high (13) for the combination of 36 markers and 104 wild genotypes that was similar to the average of 14 alleles per marker found for the 606 cultivated genotypes. Diversity in wild beans appears to be somewhat higher than in cultivated beans on a per genotype basis. Five populations or genepools were identified in structure analysis of the wild beans corresponding to segments of the geographical range, including Mesoamerican (Mexican), Guatemalan, Colombian, Ecuadorian-northern Peruvian and Andean (Argentina, Bolivia and Southern Peru). The combined analysis of wild and cultivated accessions showed that the first and last of these genepools were related to the cultivated genepools of the same names and the penultimate was found to be distinct but not ancestral to the others. The Guatemalan genepool was very novel and perhaps related to cultivars of race Guatemala, while the Colombian population was also distinct. Results suggest geographic isolation, founder effects or natural selection could have created the different semi-discrete populations of wild beans and that multiple domestications and introgression were involved in creating the diversity of cultivated beans.

Molecular ecology and selection in the drought-related Asr gene polymorphisms in wild and cultivated common bean (Phaseolus vulgaris L.).

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.

Use of the advanced backcross-QTL method to transfer seed mineral accumulation nutrition traits from wild to Andean cultivated common beans.

Iron deficiency anemia and zinc deficiency are major health concerns across the world and can be addressed by biofortification breeding of higher mineral concentration in staple crops, such as common bean. Wild common beans have for the most part had higher average seed mineral concentration than cultivars of this species but have small un-commercial seeds. A logical approach for the transfer of the seed mineral trait from wild beans to cultivated beans is through the advanced backcross breeding approach. The goal of this study was to analyze a population of 138 BC(2)F(3:5) introgression lines derived from the very high iron wild genotype G10022 backcrossed into the genetic background of the commercial-type variety 'Cerinza', a large-red seeded bush bean cultivar of the Andean genepool. In addition to measuring seed mineral accumulation traits and the quantitative trait loci (QTL) controlling these traits we were interested in simultaneously testing the adaptation of the introgression lines in two replicated yield trials. We found the cross to have high polymorphism and constructed an anchored microsatellite map for the population that was 1,554-cM long and covered all 11 linkage groups of the common bean genome. Through composite interval mapping (CIM) and single point analysis (SPA), we identified associations of markers and mineral traits on b01, b06, b07, b08, b10 and b11 for seed iron concentration, and markers on b01, b04 and b10 for seed zinc concentration. The b07 and b08 QTL aligned with previous QTL for iron concentration. A large number of QTL were found for seed weight (9 with CIM and 36 with SPA analysis) and correlations between seed size and mineral content affected the identification of iron and zinc contents' QTL on many linkage groups. Segregation distortion around domestication genes made some areas difficult to introgress. However, in conclusion, the advanced backcross program produced some introgression lines with high mineral accumulation traits using a wild donor parent.

First use of microsatellite markers in a large collection of cultivated and wild accessions of tepary bean (Phaseolus acutifolius A. Gray).

Tepary bean (Phaseolus acutifolius A. Gray) is a dry-land crop species that originated in the deserts of Mexico and the south-western United States and therefore is proposed as a source of drought and salt tolerance for related species and for production in marginal rainfall areas. Few genetic tools have been developed or tested for tepary bean but microsatellites from common bean are an obvious choice for diversity analysis in the crop. The first goal of this study was to validate a set of gene-derived and non-gene simple sequence repeat or microsatellite markers from common bean in tepary bean cultivars and wild relative accessions. The second and more extensive objective of this study was to evaluate the genetic diversity and population structure of the tepary bean accessions to determine if leaf-morphology variants are valid as separate sub-groups of wild tepary beans; if P. parvifolius exist as a separate variants or species; and if cultivated tepary beans originated from one domestication event or several events. Our analysis of 140 tepary bean genotypes showed that a single domestication was likely as the cultivars were most closely related to accessions from Sinaloa and northern Mexico and that diversity was much higher in the wild genotypes compared to the cultivated ones. Other results were that P. parvifolius was classified as a separate species by population structure analysis while the variants P. acutifolius var. acutifolius and var. tenuifolius were admixed and inter-crossed. P. latifolius is not a valid species or variant of P. acutifolius but represents a group of cultivars within tepary bean. This is the first analysis of microsatellite diversity in tepary beans and has implications for breeding and conservation of this crop and its wild relatives.

New gene-derived simple sequence repeat markers for common bean (Phaseolus vulgaris L.).

Common bean is an important and diverse crop legume with several wild relatives that are all part of the Phaseoleae tribe of tropical crop legumes. Sequence databases have been a good source of sequences to mine for simple sequence repeats (SSRs). The objective of this research was to evaluate 14 sequence collections from common bean for SSRs and to evaluate the diversity of the polymorphic microsatellites derived from these collections. SSRs were found in 10 of the GenBank sequence collections with an average of 11.3% of sequences containing microsatellite motifs. The most common motifs were based on tri- and dinucleotides. In a marker development programme, primers were designed for 125 microsatellites which were tested on a panel of 18 common bean genotypes. The markers were named as part of the bean microsatellite-database (BMd) series, and the average polymorphism information content was 0.404 for polymorphic markers and predicted well the genepool structure of common beans and the status of the wild and cultivated accessions that were included in the study. Therefore, the BMd series of microsatellites is useful for multiple studies of genetic relatedness and as anchor markers in future mapping of wide crosses in the species.

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.).

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.

SNP marker diversity in common bean (Phaseolus vulgaris L.).

Single nucleotide polymorphism (SNP) markers have become a genetic technology of choice because of their automation and high precision of allele calls. In this study, our goal was to develop 94 SNPs and test them across well-chosen common bean (Phaseolus vulgaris L.) germplasm. We validated and accessed SNP diversity at 84 gene-based and 10 non-genic loci using KASPar technology in a panel of 70 genotypes that have been used as parents of mapping populations and have been previously evaluated for SSRs. SNPs exhibited high levels of genetic diversity, an excess of middle frequency polymorphism, and a within-genepool mismatch distribution as expected for populations affected by sudden demographic expansions after domestication bottlenecks. This set of markers was useful for distinguishing Andean and Mesoamerican genotypes but less useful for distinguishing within each gene pool. In summary, slightly greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool but polymorphism rate between genotypes was consistent with genepool and race identity. Our survey results represent a baseline for the choice of SNP markers for future applications because gene-associated SNPs could themselves be causative SNPs for traits. Finally, we discuss that the ideal genetic marker combination with which to carry out diversity, mapping and association studies in common bean should consider a mix of both SNP and SSR markers.