Traces of Introgression from cAus into Tropical Japonica Observed in African Upland Rice Varieties.

BACKGROUND: Asian rice Oryza sativa, first domesticated in East Asia, has considerable success in African fields. When and where this introduction occurred is unclear. Rice varieties of Asian origin may have evolved locally during and after migration to Africa, resulting in unique adaptations, particularly in relation to upland cultivation as frequently practiced in Africa. METHODS: We investigated the genetic differentiation between Asian and African varieties using the 3000 Rice Genomes SNP dataset. African upland cultivars were first characterized using principal component analysis among 292 tropical Japonica accessions from Africa and Asia. The particularities of African accessions were then explored using two inference techniques, PCA-KDE for supervised classification and chromosome painting, and ELAI for individual allelic dosage monitoring. KEY RESULTS: Ambiguities of local differentiation between Japonica and other groups pointed at genomic segments that potentially resulted from genetic exchange. Those specific to West African upland accessions were concentrated on chromosome 6 and featured several cAus introgression signals, including a large one between 17.9 and 21.7 Mb. We found iHS statistics in support of positive selection in this region and we provide a list of candidate genes enriched in GO terms that have regulatory functions involved in stress responses that could have facilitated adaptation to harsh upland growing conditions.

Evolution of flowering time in a selfing annual plant: Roles of adaptation and genetic drift.

Resurrection studies are a useful tool to measure how phenotypic traits have changed in populations through time. If these trait modifications correlate with the environmental changes that occurred during the time period, it suggests that the phenotypic changes could be a response to selection. Selfing, through its reduction of effective size, could challenge the ability of a population to adapt to environmental changes. Here, we used a resurrection study to test for adaptation in a selfing population of Medicago truncatula, by comparing the genetic composition and flowering times across 22 generations. We found evidence for evolution toward earlier flowering times by about two days and a peculiar genetic structure, typical of highly selfing populations, where some multilocus genotypes (MLGs) are persistent through time. We used the change in frequency of the MLGs through time as a multilocus fitness measure and built a selection gradient that suggests evolution toward earlier flowering times. Yet, a simulation model revealed that the observed change in flowering time could be explained by drift alone, provided the effective size of the population is small enough (<150). These analyses suffer from the difficulty to estimate the effective size in a highly selfing population, where effective recombination is severely reduced.

Fitness consequences of hybridization in a predominantly selfing species: insights into the role of dominance and epistatic incompatibilities.

Studying the consequences of hybridization on plant performance is insightful to understand the adaptive potential of populations, notably at local scales. Due to reduced effective recombination, predominantly selfing species are organized in highly homozygous multi-locus-genotypes (or lines) that accumulate genetic differentiation both among- and within-populations. This high level of homozygosity facilitates the dissection of the genetic basis of hybrid performance in highly selfing species, which gives insights into the mechanisms of reproductive isolation between lines. Here, we explored the fitness consequences of hybridization events between natural inbred lines of the predominantly selfing species Medicago truncatula, at both within- and among-populations scales. We found that hybridization has opposite effects pending on studied fitness proxies, with dry mass showing heterosis, and seed production showing outbreeding depression. Although we found significant patterns of heterosis and outbreeding depression, they did not differ significantly for within- compared to among-population crosses. Family-based analyses allowed us to determine that hybrid differentiation was mostly due to dominance and epistasis. Dominance and/or dominant epistatic interactions increased dry mass, while decreasing seed production, and recessive epistatic interactions mostly had a positive effect on both fitness proxies. Our results illustrate how genetic incompatibilities can accumulate at a very local scale among multi-locus-genotypes, and how non-additive genetic effects contribute to heterosis and outbreeding depression.

How and When Does Outcrossing Occur in the Predominantly Selfing Species Medicago truncatula?

Empirical studies on natural populations of Medicago truncatula revealed selfing rates higher than 80%, but never up to 100%. Similarly, several studies of predominantly selfing species show variability in the level of residual outcrossing between populations and also between temporal samples of the same population. However, these studies measure global selfing rates at the scale of the population and we do not know whether there is intra-population variation and how outcrossing events are distributed, between genotypes, plants, flowers, or seeds. Theoretical studies predict the maintenance of residual outcrossing in highly selfing species due to environmental (e.g., pollen biology) and/or genetic determinants and decompositions of the variation in outcrossing rate using experimental data can be very informative to test these hypotheses. Here, we focus on one natural population of M. truncatula in order to describe precisely its mating system. In particular, we investigated the determinants of the selfing rate by testing for seasonal variations (environmental determinism) and variations between genotypes (genetic determinism). We measured selfing rates in maternal progenies from plants collected widely across a natural population. For each plant, we collected pods from flowers produced at the beginning and at the end of the flowering season to test for a seasonal variation in the outcrossing rate. For each collected offspring, we also estimated the likelihood that it was issued from a self-fertilization event and assessed the genetic component of variation of this mating system measure. We found a significant, albeit small, increase in outcrossing rate in progenies collected at the end [t m = 0.137 (SD = 0.025)] compared to those collected at the beginning [t m = 0.083 (0.016)] of the flowering season. A significant between genotypes variation in selfing rate was also detected, resulting in a heritability of 9% for the rate of residual outcrossing. Altogether, our work shows that despite a predominantly selfing reproductive mode, M. truncatula displays variation in residual outcrossing rate, and that this trait is likely under a complex determinism combining environmental and genetic factors. We discuss the evolutionary implications of our results for the population.

Genetic diversity of maize landraces from the South-West of France.

From the 17th century until the arrival of hybrids in 1960s, maize landraces were cultivated in the South-West of France (SWF), a traditional region for maize cultivation. A set of landraces were collected in this area between the 1950s and 1980s and were then conserved ex situ in a germplam collection. Previous studies using molecular markers on approx. twenty landraces from this region suggested that they belonged to a Pyrenees-Galicia Flint genetic group and originated from hybridizations between Caribbean and Northern Flint germplasms introduced to Europe. In this study, we assessed the structure and genetic diversity of 194 SWF maize landraces to better elucidate their origin, using a 50K SNP array and a bulk DNA approach. We identified two weakly differentiated genetic groups, one in the Western part and the other in the Eastern part of the studied region. We highlighted the existence of a longitudinal gradient along the SWF area that was probably maintained through the interplay between genetic drifts and restricted gene flows. The contact zone between the two groups observed near the Garonne valley may be the result of these evolutionnary forces. We found in landraces from the East part of the region significant cases of admixture between landraces from the Northern Flint group and landraces from either the Caribbean, Andean or Italian groups. We then assumed that SWF landraces had a multiple origin with a predonderance of Northern Flint germplasm for the two SWF groups, notably for the East part.

Hidden genetic variance contributes to increase the short-term adaptive potential of selfing populations.

Standing genetic variation is considered a major contributor to the adaptive potential of species. The low heritable genetic variation observed in self-fertilizing populations has led to the hypothesis that species with this mating system would be less likely to adapt. However, a non-negligible amount of cryptic genetic variation for polygenic traits, accumulated through negative linkage disequilibrium, could prove to be an important source of standing variation in self-fertilizing species. To test this hypothesis, we simulated populations under stabilizing selection subjected to an environmental change. We demonstrate that, when the mutation rate is high (but realistic), selfing populations are better able to store genetic variance than outcrossing populations through genetic associations, notably due to the reduced effective recombination rate associated with predominant selfing. Following an environmental shift, this diversity can be partially remobilized, which increases the additive variance and adaptive potential of predominantly (but not completely) selfing populations. In such conditions, despite initially lower observed genetic variance, selfing populations adapt as readily as outcrossing ones within a few generations. For low mutation rates, purifying selection impedes the storage of diversity through genetic associations, in which case, as previously predicted, the lower genetic variance of selfing populations results in lower adaptability compared to their outcrossing counterparts. The population size and the mutation rate are the main parameters to consider, as they are the best predictors of the amount of stored diversity in selfing populations. Our results and their impact on our knowledge of adaptation under high selfing rates are discussed.

How does selfing affect the genetic variance of quantitative traits? An updated meta-analysis on empirical results in angiosperm species.

Most theoretical works predict that selfing should reduce the level of additive genetic variance available for quantitative traits within natural populations. Despite a growing number of quantitative genetic studies undertaken during the last two decades, this prediction is still not well supported empirically. To resolve this issue and confirm or reject theoretical predictions, we reviewed quantitative trait heritability estimates from natural plant populations with different rates of self-fertilization and carried out a meta-analysis. In accordance with models of polygenic traits under stabilizing selection, we found that the fraction of additive genetic variance is negatively correlated with the selfing rate. Although the mating system explains a moderate fraction of the variance, the mean reduction of narrow-sense heritability values between strictly allogamous and predominantly selfing populations is strong, around 60%. Because some nonadditive components of genetic variance become selectable under inbreeding, we determine whether self-fertilization affects the relative contribution of these components to genetic variance by comparing narrow-sense heritability estimates from outcrossing populations with broad-sense heritability estimated in autogamous populations. Results suggest that these nonadditive components of variance may restore some genetic variance in predominantly selfing populations; it remains, however, uncertain how these nonadditive components will contribute to adaptation.

Structure of multilocus genetic diversity in predominantly selfing populations.

Predominantly selfing populations are expected to have reduced effective population sizes due to nonrandom sampling of gametes, demographic stochasticity (bottlenecks or extinction-recolonization), and large scale hitchhiking (reduced effective recombination). Thus, they are expected to display low genetic diversity, which was confirmed by empirical studies. The structure of genetic diversity in predominantly selfing species is dramatically different from outcrossing ones, with populations often dominated by one or a few multilocus genotypes (MLGs) coexisting with several rare genotypes. Therefore, multilocus diversity indices are relevant to describe diversity in selfing populations. Here, we use simulations to provide analytical expectations for multilocus indices and examine whether selfing alone can be responsible for the high-frequency MLGs persistent through time in the absence of selection. We then examine how combining single and multilocus indices of diversity may be insightful to distinguish the effects of selfing, population size, and more complex demographic events (bottlenecks, migration, admixture, or extinction-recolonization). Finally, we examine how temporal changes in MLG frequencies can be insightful to understand the evolutionary trajectory of a given population. We show that combinations of selfing and small demographic sizes can result in high-frequency MLGs, as observed in natural populations. We also show how different demographic scenarios can be distinguished by the parallel analysis of single and multilocus indices of diversity, and we emphasize the importance of temporal data for the study of predominantly selfing populations. Finally, the comparison of our simulations with empirical data on populations of Medicago truncatula confirms the pertinence of our simulation framework.

How to optimize the precision of allele and haplotype frequency estimates using pooled-sequencing data.

Sequencing pools of individuals rather than individuals separately reduces the costs of estimating allele frequencies at many loci in many populations. Theoretical and empirical studies show that sequencing pools comprising a limited number of individuals (typically fewer than 50) provides reliable allele frequency estimates, provided that the DNA pooling and DNA sequencing steps are carefully controlled. Unequal contributions of different individuals to the DNA pool and the mean and variance in sequencing depth both can affect the standard error of allele frequency estimates. To our knowledge, no study separately investigated the effect of these two factors on allele frequency estimates; so that there is currently no method to a priori estimate the relative importance of unequal individual DNA contributions independently of sequencing depth. We develop a new analytical model for allele frequency estimation that explicitly distinguishes these two effects. Our model shows that the DNA pooling variance in a pooled sequencing experiment depends solely on two factors: the number of individuals within the pool and the coefficient of variation of individual DNA contributions to the pool. We present a new method to experimentally estimate this coefficient of variation when planning a pooled sequencing design where samples are either pooled before or after DNA extraction. Using this analytical and experimental framework, we provide guidelines to optimize the design of pooled sequencing experiments. Finally, we sequence replicated pools of inbred lines of the plant Medicago truncatula and show that the predictions from our model generally hold true when estimating the frequency of known multilocus haplotypes using pooled sequencing.

Spatial and Temporal Variation in Selection of Genes Associated with Pearl Millet Varietal Quantitative Traits In situ.

Ongoing global climate changes imply new challenges for agriculture. Whether plants and crops can adapt to such rapid changes is still a widely debated question. We previously showed adaptation in the form of earlier flowering in pearl millet at the scale of a whole country over three decades. However, this analysis did not deal with variability of year to year selection. To understand and possibly manage plant and crop adaptation, we need more knowledge of how selection acts in situ. Is selection gradual, abrupt, and does it vary in space and over time? In the present study, we tracked the evolution of allele frequency in two genes associated with pearl millet phenotypic variation in situ. We sampled 17 populations of cultivated pearl millet over a period of 2 years. We tracked changes in allele frequencies in these populations by genotyping more than seven thousand individuals. We demonstrate that several allele frequencies changes are compatible with selection, by correcting allele frequency changes associated with genetic drift. We found marked variation in allele frequencies from year to year, suggesting a variable selection effect in space and over time. We estimated the strength of selection associated with variations in allele frequency. Our results suggest that the polymorphism maintained at the genes we studied is partially explained by the spatial and temporal variability of selection. In response to environmental changes, traditional pearl millet varieties could rapidly adapt thanks to this available functional variability.

Adaptation to climate through flowering phenology: a case study in Medicago truncatula.

Local climatic conditions likely constitute an important selective pressure on genes underlying important fitness-related traits such as flowering time, and in many species, flowering phenology and climatic gradients strongly covary. To test whether climate shapes the genetic variation on flowering time genes and to identify candidate flowering genes involved in the adaptation to environmental heterogeneity, we used a large Medicago truncatula core collection to examine the association between nucleotide polymorphisms at 224 candidate genes and both climate variables and flowering phenotypes. Unlike genome-wide studies, candidate gene approaches are expected to enrich for the number of meaningful trait associations because they specifically target genes that are known to affect the trait of interest. We found that flowering time mediates adaptation to climatic conditions mainly by variation at genes located upstream in the flowering pathways, close to the environmental stimuli. Variables related to the annual precipitation regime reflected selective constraints on flowering time genes better than the other variables tested (temperature, altitude, latitude or longitude). By comparing phenotype and climate associations, we identified 12 flowering genes as the most promising candidates responsible for phenological adaptation to climate. Four of these genes were located in the known flowering time QTL region on chromosome 7. However, climate and flowering associations also highlighted largely distinct gene sets, suggesting different genetic architectures for adaptation to climate and flowering onset.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. • A high-density (≈5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. • GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (σ(A)²= 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. • These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

Pedigree-free estimates of heritability in the wild: promising prospects for selfing populations.

Estimating the genetic variance available for traits informs us about a population's ability to evolve in response to novel selective challenges. In selfing species, theory predicts a loss of genetic diversity that could lead to an evolutionary dead-end, but empirical support remains scarce. Genetic variability in a trait is estimated by correlating the phenotypic resemblance with the proportion of the genome that two relatives share identical by descent ('realized relatedness'). The latter is traditionally predicted from pedigrees (Φ A : expected value) but can also be estimated using molecular markers (average number of alleles shared). Nevertheless, evolutionary biologists, unlike animal breeders, remain cautious about using marker-based relatedness coefficients to study complex phenotypic traits in populations. In this paper, we review published results comparing five different pedigree-free methods and use simulations to test individual-based models (hereafter called animal models) using marker-based relatedness coefficients, with a special focus on the influence of mating systems. Our literature review confirms that Ritland's regression method is unreliable, but suggests that animal models with marker-based estimates of relatedness and genomic selection are promising and that more testing is required. Our simulations show that using molecular markers instead of pedigrees in animal models seriously worsens the estimation of heritability in outcrossing populations, unless a very large number of loci is available. In selfing populations the results are less biased. More generally, populations with high identity disequilibrium (consanguineous or bottlenecked populations) could be propitious for using marker-based animal models, but are also more likely to deviate from the standard assumptions of quantitative genetics models (non-additive variance).

Mating system, Haldane's sieve, and the domestication process.

Mating systems are expected to have a strong influence on both the dynamic of adaptation and the genetic architecture of adaptive traits. In particular, the bias toward the fixation of dominant or partially dominant beneficial mutations predicted under outcrossing (Haldane's sieve) is expected to be reduced under self-fertilization. To test this prediction in plants, we considered domestication as an example of adaptation. We compiled data from studies reporting the degree of dominance of quantitative trait loci (QTL) involved in the domestication syndrome. We found that adaptation to cultivation mostly proceeded through the selection of recessive and partially recessive genes in predominantly selfing species whereas a much larger fraction of domestication-related QTL were dominant or partially dominant in outcrossers, as expected under Haldane's sieve. Our study also showed that levels of dominance in mixed mating crop species resemble those observed in selfers, suggesting that recessive alleles can contribute to adaptation even under moderate selfing rates. Although these results rely on a particular example of adaptation, they constitute one of the first attempts to test theoretical expectations on the level of dominance of genes involved in plant adaptation.

Detecting selection along environmental gradients: analysis of eight methods and their effectiveness for outbreeding and selfing populations.

Thanks to genome-scale diversity data, present-day studies can provide a detailed view of how natural and cultivated species adapt to their environment and particularly to environmental gradients. However, due to their sensitivity, up-to-date studies might be more sensitive to undocumented demographic effects such as the pattern of migration and the reproduction regime. In this study, we provide guidelines for the use of popular or recently developed statistical methods to detect footprints of selection. We simulated 100 populations along a selective gradient and explored different migration models, sampling schemes and rates of self-fertilization. We investigated the power and robustness of eight methods to detect loci potentially under selection: three designed to detect genotype-environment correlations and five designed to detect adaptive differentiation (based on F(ST) or similar measures). We show that genotype-environment correlation methods have substantially more power to detect selection than differentiation-based methods but that they generally suffer from high rates of false positives. This effect is exacerbated whenever allele frequencies are correlated, either between populations or within populations. Our results suggest that, when the underlying genetic structure of the data is unknown, a number of robust methods are preferable. Moreover, in the simulated scenario we used, sampling many populations led to better results than sampling many individuals per population. Finally, care should be taken when using methods to identify genotype-environment correlations without correcting for allele frequency autocorrelation because of the risk of spurious signals due to allele frequency correlations between populations.

Adaptation and maladaptation in selfing and outcrossing species: new mutations versus standing variation.

Evolution of selfing from outcrossing recurrently occurred in many lineages, especially in flowering plants. Evolution of selfing induces dramatic changes in the population genetics functioning but its consequences on the dynamics of adaptation have been overlooked. We studied a simple one-locus model of adaptation where a population experiences an environmental change at a given time. We first determined the effect of the mating system on the genetic bases and the speed of adaptation, focusing on the dominance of beneficial mutations and the respective part of standing variation and new mutations. Then, we assumed that the environmental change is associated with population decline to determine the effect of the mating system on the probability of population extinction. Extending previous results, we found that adaptation is more efficient and extinction less likely in outcrossers when beneficial mutations are dominant and codominant and when standing variation plays a significant role in adaptation. However, given adaptation does occur, it is usually more rapid in selfers than in outcrossers. Our results bear implications for the evolution of the selfing syndrome, the dynamics of the domestication process, and the dead-end hypothesis that posits that selfing lineages are doomed to extinction on the long run.

Single-nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula.

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single-nucleotide polymorphism (SNP) markers in M. truncatula through large-scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.

Contrasted patterns of selective pressure in three recent paralogous gene pairs in the Medicago genus (L.).

BACKGROUND: Gene duplications are a molecular mechanism potentially mediating generation of functional novelty. However, the probabilities of maintenance and functional divergence of duplicated genes are shaped by selective pressures acting on gene copies immediately after the duplication event. The ratio of non-synonymous to synonymous substitution rates in protein-coding sequences provides a means to investigate selective pressures based on genic sequences. Three molecular signatures can reveal early stages of functional divergence between gene copies: change in the level of purifying selection between paralogous genes, occurrence of positive selection, and transient relaxed purifying selection following gene duplication. We studied three pairs of genes that are known to be involved in an interaction with symbiotic bacteria and were recently duplicated in the history of the Medicago genus (Fabaceae). We sequenced two pairs of polygalacturonase genes (Pg11-Pg3 and Pg11a-Pg11c) and one pair of auxine transporter-like genes (Lax2-Lax4) in 17 species belonging to the Medicago genus, and sought for molecular signatures of differentiation between copies. RESULTS: Selective histories revealed by these three signatures of molecular differentiation were found to be markedly different between each pair of paralogs. We found sites under positive selection in the Pg11 paralogs while Pg3 has mainly evolved under purifying selection. The most recent paralogs examined Pg11a and Pg11c, are both undergoing positive selection and might be acquiring new functions. Lax2 and Lax4 paralogs are both under strong purifying selection, but still underwent a temporary relaxation of purifying selection immediately after duplication. CONCLUSIONS: This study illustrates the variety of selective pressures undergone by duplicated genes and the effect of age of the duplication. We found that relaxation of selective constraints immediately after duplication might promote adaptive divergence.

"Ménage à trois": the presence/absence of thyme shapes the mutualistic interaction between the host plant Medicago truncatula (Fabaceae) and its symbiotic bacterium Sinorhizobium meliloti.

The long-term maintenance of specialized mutualisms remains an evolutionary puzzle. Recent focus has been on factors governing the stability of these mutualisms, including sanctions by the host, partner choice, and coevolutionary constraint, that is, the genetic correlation (r(G)) between fitness of both partners. So far these studies have been typically carried out in a single environment. Here, we ask if the genetic correlation between fitness of the host plant Medicago truncatula (Fabaceae) and its bacterial symbiont Sinorhizobium meliloti is affected by the presence/absence of a monoterpene (carvacrol) leached into the soil by Thymus vulgaris-a common plant of the Mediterranean vegetation, often co-occuring with Medicago. We show that the presence of carvacrol in the soil dramatically affects fitness of the rhizobial partner and increases the magnitude of r(G) between plant and rhizobia fitness (r(G) = 0.02 ± 0.05 vs. r(G) = 0.57 ± 0.02). This finding emphasizes the importance of heterogeneity in the biotic environment for understanding the evolution of species interactions.

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume-rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The population-scaled recombination rate is approximately one-third of the mutation rate, consistent with expectations for a species with a high selfing rate. Linkage disequilibrium, however, is not extensive, and therefore, the low recombination rate is likely not a major constraint to adaptation. Nucleotide diversity in 100-kb windows was negatively correlated with gene density, which is expected if diversity is shaped by selection acting against slightly deleterious mutations. Among putative coding regions, members of four gene families harbor significantly higher diversity than the genome-wide average. Three of these families are involved in resistance against pathogens; one of these families, the nodule-specific, cysteine-rich gene family, is specific to the galegoid legumes and is involved in control of rhizobial differentiation. The more than 3 million SNPs that we detected, approximately one-half of which are present in more than one accession, are a valuable resource for genome-wide association mapping of genes responsible for phenotypic diversity in legumes, especially traits associated with symbiosis and nodulation.