Joëlle Ronfort

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.

Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a breeding program, using SSR markers.

Alfalfa (Medicago sativa) is an autotetraploid, allogamous and heterozygous species whose cultivars are synthetic populations. The breeders apply selection pressure for some agronomic traits within a breeding pool to increase the frequency of favorable individuals. The objective of this study was to investigate the differentiation level among seven cultivars originating from one breeding program, and between these cultivars and the breeding pool, with eight SSR markers. These highly polymorphic and codominant markers, together with recent population genetic statistics extended to autotetraploids, offer tools to analyse genetic diversity in alfalfa. The number of alleles per locus varied between 3 and 24. All loci were at a panmictic equilibrium in the cultivars, except one, probably because of null alleles. With seven SSR loci, each cultivar was at panmictic equilibrium. The mean gene diversity was high, ranging from 0.665 to 0.717 in the cultivars. The parameter FST indicated a low but significant diversity among cultivars. Among 21 pairs of cultivars, 15 were significantly different. The breeding pool also had a high diversity, and was significantly different from each cultivar except the most recent one. Considering the characteristics of the breeding program and the mode of cultivar elaboration, we found that they were unable to generate a large variety differentiation. Estimation of population genetics parameters at SSR loci can be applied for assessing the differences between cultivars or populations, either for variety distinction or the management of genetic resources.

Spatial effects and rare outcrossing events in Medicago truncatula (Fabaceae)

In order to elucidate the mechanisms underlying the large amount of RAPD polymorphism found in 1990 in a population of the selfing annual Medicago truncatula GAERTN. (Fabaceae), we have analysed most of the individuals (n = 363) from the same population 6 years later using microsatellite loci. We confirm the result of the earlier study, namely that this population is very polymorphic and highly subdivided, with approximately 37% of the variance distributed among subpopulations, only 50 m apart one from another. We use standard F‐statistics analyses, linkage disequilibria, minimum spanning network, multilocus assignment tests and spatial autocorrelation analyses to test the hypotheses that spatial structure and outcrossing events are involved in maintaining the large amount of genetic diversity at the level of each subpopulation. Interestingly, fine‐scale spatial structure could be observed in only one subpopulation suggesting that other mechanisms are acting elsewhere. To the best of our knowledge, this is the first study of fine spatial genetic structure in a predominantly selfing species.

Differentiation between natural and cultivated populations of Medicago sativa (Leguminosae) from Spain: analysis with random amplified polymorphic DNA (RAPD) markers and comparison to allozymes

The conservation of a crop’s wild relatives as genetic resources requires an understanding of the way genetic diversity is maintained in their populations, notably the effect of crop‐to‐wild gene flow. In this study, the amount of differentiation between natural and cultivated populations of Medicago sativa was analysed using random amplified polymorphic DNA (RAPD) markers and an extension of the amova procedure adapted to autotetraploid organisms. Simulations of structured populations were performed to test whether amova provides estimates of population structure in autotetraploids that can be directly compared to those obtained for allozyme data. Simulations showed that φ‐statistics allow a good estimation of population differentiation when unbiased allelic frequencies are used to correct the conditional expectations of squared genetic distances. But such unbiased estimates can not be practically guaranteed, and population structure is notably overestimated when some populations are fixed for the presence of amplified fragments. However, removing fixed loci from the data set improves the statistical power of the test for population structure. The genetic variation of 15 natural and six cultivated populations of M. sativa was analysed at 25 RAPD loci and compared to estimates computed with allozymes on the same set of populations. Although RAPD markers revealed less within‐population genetic diversity than allozymes, the quantitative and qualitative patterns of population structure were in full agreement with allozymes. This confirmed the conclusions drawn from the allozymic survey: crop‐to‐wild gene flow occurred in many locations, but some other mechanisms opposed cultivated traits to be maintained into natural 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.

Domestication history in the Medicago sativa species complex: inferences from nuclear sequence polymorphism.

DNA sequence polymorphism carries genealogical information and allows for testing hypotheses on selection and population history, especially through coalescent‐based analysis. Understanding the evolutionary forces at work in plant domestication and subsequent selection is of critical importance for the management of genetic resources. In this study, we surveyed DNA sequence diversity at two assumed neutral nuclear loci in the wild–domesticated species complex of alfalfa (Medicago sativa L.). A high level of polymorphism was detected. The domesticated pool contains on average 31% less diversity than the wild pool, but with a high heterogeneity among loci. Coalescent simulations of the domestication process showed that this result cannot be explained by assuming a constant population size but is rather consistent with a demographic bottleneck during domestication. A very low level of divergence was detected between the wild and the domesticated forms as well as between the related subspecies of the M. sativa species complex. However, the originality of the Spanish wild populations, already observed based on mitochondrial DNA polymorphism, was confirmed. These results, together with patterns of intrapopulation polymorphism, suggest that nuclear sequence polymorphism could be a promising tool, complementary to mitochondrial DNA and phenotypic evaluations, to investigate historical demographic and evolutionary processes.

Investigation of the demographic and selective forces shaping the nucleotide diversity of genes involved in nod factor signaling in Medicago truncatula.

Symbiotic nitrogen-fixing rhizobia are able to trigger root deformation in their Fabaceae host plants, allowing their intracellular accommodation. They do so by delivering molecules called Nod factors. We analyzed the patterns of nucleotide polymorphism of five genes controlling early Nod factor perception and signaling in the Fabaceae Medicago truncatula to understand the selective forces shaping the evolution of these genes. We used 30 M. truncatula genotypes sampled in a genetically homogeneous region of the species distribution range. We first sequenced 24 independent loci and detected a genomewide departure from the hypothesis of neutrality and demographic equilibrium that suggests a population expansion. These data were used to estimate parameters of a simple demographic model incorporating population expansion. The selective neutrality of genes controlling Nod factor perception was then examined using a combination of two complementary neutrality tests, Tajimas D and Fay and Wus standardized H. The joint distribution of D and H expected under neutrality was obtained under the fitted population expansion model. Only the gene DMI1, which is expected to regulate the downstream signal, shows a pattern consistent with a putative selective event. In contrast, the receptor-encoding genes NFP and NORK show no significant signatures of selection. Among the genes that we analyzed, only DMI1 should be viewed as a candidate for adaptation in the recent history of M. truncatula.

How multilocus genotypic pattern helps to understand the history of selfing populations: a case study in Medicago truncatula.

The occurrence of populations exhibiting high genetic diversity in predominantly selfing species remains a puzzling question, since under regular selfing genetic diversity is expected to be depleted at a faster rate than under outcrossing. Fine-scale population genetics approaches may help to answer this question. Here we study a natural population of the legume Medicago truncatula in which both the fine-scale spatial structure and the selfing rate are characterized using three different methods. Selfing rate estimates were very high (∼99%) irrespective of the method used. A clear pattern of isolation by distance reflecting small seed dispersal distances was detected. Combining genotypic data over loci, we could define 34 multilocus genotypes. Among those, six highly inbred genotypes (lines) represented more than 75% of the individuals studied and harboured all the allelic variation present in the population. We also detected a large set of multilocus genotypes resembling recombinant inbred lines between the most frequent lines occurring in the population. This finding illustrates the importance of rare recombination in redistributing available allelic diversity into new genotypic combinations. This study shows how multilocus and fine-scale spatial analyses may help to understand the population history of self-fertilizing species, especially to make inferences about the relative role of foundation/migration and recombination events in such populations.

Morphologic and Agronomic Diversity of Wild Genetic Resources of Medicago sativa L. Collected in Spain

One hundred and three natural populations of Medicago L. were collected in Spain, mainly from roadsides, non-irrigated or grazed lands. This germplasm was evaluated at Montpellier (France) with control cultivars and Spanish landraces under completely random block design, replicated four times, and observed for 64 qualitative or quantitative characters. Differences between natural populations and cultivated controls are highly significant, but gene flow occurs between wild and cultivated compartment and hybrid populations were identified. On the basis of multivariable analysis, the accessions were grouped into four clusters depending on their proximity with the cultivated pool to facilitate their management and ex situ conservation. The relationship between environment of the site of collection and phenotypic characteristics of the natural populations was also discussed. Different policies of conservation of these genetic resources are suggested to avoid their disappearance even when they disappeared from the other regions of the western Mediterranean. Spanish wild pool of alfalfa, also called ‘Mielga’, appears of great interest for the breeding of alfalfa because it contains a large diversity of characteristics (prostrate habit, rhizomes) linked to tolerance to grazing or drought environment. With the need for more sustainable systems in agriculture, the erosion of natural habitats and the necessity for rehabilitation of degraded ecosystems, the importance of this wild pool is really inestimable.

Spatial structure of genetic variation and primary succession in the pioneer tree species Antirhea borbonica on La Réunion.

In habitats where colonization and extinction are recurrent, the distribution of gene frequencies among patches of suitable habitat may reflect the age structure of different populations. In this study, we quantify population genetic structure for a pioneer tree species, Antirhea borbonica, in a chrono‐sequence of primary succession on the lava flows of the Piton de La Fournaise volcano (La Réunion). Using microsatellite loci and amplified fragment length polymorphism (AFLP) markers, we quantified genetic variation within and among populations for early‐ and late‐succession populations in a landscape where extinction and recolonization are recurrent (the ‘Grand Brûlé’) and for late‐succession populations in a more stable landscape. This study produced three main results. First, we detected no evidence that founder events increase genetic differentiation among colonizing populations; FST values among early‐ and among late‐succession populations were similar. Second, we found no evidence for isolation by distance; genetic distance was not correlated with spatial distance within and among populations. Third, FIS values are consistently high in all populations, despite the fact that A. borbonica populations are functionally close to dioecy and thus expected to have an outcrossing mating system. Multiple colonization events from different sources may limit differentiation among young populations and spatial isolation may enhance differentiation among late‐succession populations. Ecological processes acting during colonization may create the conditions for spatial aggregation within pioneer populations, and thus contribute to the high FIS values.