A role for an endosperm-localized subtilase in the control of seed size in legumes.

Here, we report a subtilase gene (SBT1.1) specifically expressed in the endosperm of Medicago truncatula and Pisum sativum seeds during development, which is located at a chromosomal position coinciding with a seed weight quantitative trait locus (QTL). Association studies between SBT1.1 polymorphisms and seed weights in ecotype collections provided further evidence for linkage disequilibrium between the SBT1.1 locus and a seed weight locus. To investigate the possible contribution of SBT1.1 to the control of seed weight, a search for TILLING (Targeting Induced Local Lesions in Genomes) mutants was performed. An inspection of seed phenotype revealed a decreased weight and area of the sbt1.1 mutant seeds, thus inferring a role of SBT1.1 in the control of seed size in the forage and grain legume species. Microscopic analyses of the embryo, representing the major part of the seed, revealed a reduced number of cells in the MtP330S mutant, but no significant variation in cell size. SBT1.1 is therefore most likely to be involved in the control of cotyledon cell number, rather than cell expansion, during seed development. This raises the hypothesis of a role of SBT1.1 in the regulation of seed size by providing molecules that can act as signals to control cell division within the embryo.

Using a model-based framework for analysing genetic diversity during germination and heterotrophic growth of Medicago truncatula.

BACKGROUND AND AIMS: The framework provided by an emergence model was used: (1) for phenotyping germination and heterotrophic growth of Medicago truncatula in relation to two major environmental factors, temperature and water potential; and (2) to evaluate the extent of genetic differences in emergence-model parameters. METHODS: Eight cultivars and natural accessions of M. trunculata were studied. Germination was recorded from 5 to 30 degrees C and from 0 to -0.75 MPa, and seedling growth from 10 to 20 degrees C. KEY RESULTS: Thermal time to reach 50 % germination was very short (15 degrees Cd) and almost stable between genotypes, while base temperature (2-3 degrees C) and base water potential for germination (-0.7 to -1.3 MPa) varied between genotypes. Only 35 degrees Cd after germination were required to reach 30 mm hypocotyl length with significant differences among genotypes. Base temperature for elongation varied from 5.5 to 7.5 degrees C. Low temperatures induced a general shortening of the seedling, with some genotypes more responsive than others. No relationship with initial seed mass or seed reserve distribution was observed, which might have explained differences between genotypes and the effects of low temperatures. CONCLUSIONS: The study provides a set of reference values for M. trunculata users. The use of the ecophysiological model allows comparison of these values between such non-crop species and other crops. It has enabled phenotypic variability in response to environmental conditions related to the emergence process to be identified. The model will allow simulation of emergence differences between genotypes in a range of environments using these parameter values. Genomic tools available for the model species M. trunculata will make it possible to analyse the genetic and molecular determinants of these differences.

AER1, a major gene conferring resistance to Aphanomyces euteiches in Medicago truncatula.

Aphanomyces euteiches is a major soilborne oomycete pathogen that infects various legume species, including pea and alfalfa. The model legume Medicago truncatula has recently emerged as a valuable genetic system for understanding the genetic basis of resistance to A. euteiches in leguminous crops. The objective of this study was to identify genetic determinants of resistance to a broad host-range pea-infecting strain of A. euteiches in M. truncatula. Two M. truncatula segregating populations of 178 F(5) recombinant inbred lines and 200 F(3) families from the cross F83005.5 (susceptible) x DZA045.5 (resistant) were screened for resistance to A. euteiches. Phenotypic distributions observed suggested a dominant monogenic control of resistance. A major locus associated with resistance to A. euteiches, namely AER1, was mapped by bulk segregant analysis to a terminal end of chromosome 3 in M. truncatula and explained 88% of the phenotypic variation. AER1 was identified in a resistance-gene-rich region, where resistance gene analogs and genes associated with disease resistance phenotypes have been identified. Discovery of AER1 opens up new prospects for improving resistance to A. euteiches in cultivated legumes using a comparative genomics approach.

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 ( approximately 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.

Genetic markers and quantitative genetic variation in Medicago truncatula (Leguminosae): a comparative analysis of population structure.

Two populations of the selfing annual Medicago truncatula Gaertn. (Leguminoseae), each subdivided into three subpopulations, were studied for both metric traits (quantitative characters) and genetic markers (random amplified polymorphic DNA and one morphological, single-locus marker). Hierarchical analyses of variance components show that (1) populations are more differentiated for quantitative characters than for marker loci, (2) the contribution of both within and among subpopulations components of variance to overall genetic variance of these characters is reduced as compared to markers, and (3) at the population level, within population structure is slightly but not significantly larger for markers than for quantitative traits. Under the hypothesis that most markers are neutral, such comparisons may be used to make hypotheses about the strength and heterogeneity of natural selection in the face of genetic drift and gene flow. We thus suggest that in these populations, quantitative characters are under strong divergent selection among populations, and that gene flow is restricted among populations and subpopulations.

Molecular phylogenetic approach for studying life-history evolution: the ambiguous example of the genus Medicago L.

We present a molecular phylogeny including most species of the genus Medicago L. (Fabaceae). Based on the consensus of the 48 most parsimonious trees, life-history and mating-system characters are mapped, and a putative history of the genus is suggested. The most parsimonious reconstruction suggests an ancestral annual and selfing state, and recurrent evolution towards perenniality and outcrossing. Based on theoretical predictions and classical hypotheses of the history of the genus, different assumptions about the ancestral state and different weighting schemes of evolution between the character states are made. Assuming an outcrossing, perennial ancestral state (partly supported by morphological features) does not fundamentally change the reconstruction. To meet theoretical expectations, various weighting schemes favouring evolution towards annuality and selfing are applied. Influence and validity of such weighting schemes are discussed with regard to other studies.

Effective population size associated with self-fertilization: lessons from temporal changes in allele frequencies in the selfing annual Medicago truncatula.

Despite its significance in evolutionary and conservation biology, few estimates of effective population size (N(e)) are available in plant species. Self-fertilization is expected to affect N(e), through both its effect on homozygosity and population dynamics. Here, we estimated N(e) using temporal variation in allele frequencies for two contrasted populations of the selfing annual Medicago truncatula: a large and continuous population and a subdivided population. Estimated N(e) values were around 5-10% of the population census size suggesting that other factors than selfing must contribute to variation in allele frequencies. Further comparisons between monolocus allelic variation and changes in the multilocus genotypic composition of the populations show that the local dynamics of inbred lines can play an important role in the fluctuations of allele frequencies. Finally, comparing N(e) estimates and levels of genetic variation suggest that H(e) is a poor estimator of the contemporaneous variance effective population size.

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.

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 straight phi-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.

Evolution of annual species of the genus Medicago: a molecular phylogenetic approach.

We performed a molecular phylogenetic study based on the nuclear ribosomal internal and external transcribed spacer (ITS and ETS). Thirty-one annual Medicago species were included in the study, representing more than half of the genus and 85% of the annuals of the genus. Major incongruences were found between phylogenetic relationships and morphological classification of the genus. Morphological and cytological traits were mapped onto the phylogeny. The most parsimonious reconstruction suggested an ancestral spiny state and a recurrent transition from spiny to spineless state. From the ancestral state of 2n=16, three loss events of chromosomes must have occurred leading to the same specific number of 14 chromosomes whereas species having 30 chromosomes form a monophyletic clade.

Evidence for gene flow between wild and cultivated Medicago sativa (Leguminosae)based on allozyme markers andquantitative traits.

Genetic differentiation between co-occurring crops and their wild relatives will be greatly modified by crop-to-weed gene flow and variation between human and natural selective pressures. The maintenance of original morphological features in most natural populations of Medicago sativa in Spain questions the relative extent of these antagonistic forces. In this paper, we measured and compared the pattern of population differentiation within and among the wild and cultivated gene pool with respect to both allozymes and quantitative traits. Patterns of diversity defined three kinds of natural populations. First, some populations were intermediate with respect to both allozymes and quantitative traits. This suggests that crop-to-weed gene flow may have created hybrid populations in some locations. Second, some populations were different from all the cultivated landraces with respect to both allozymes and quantitative traits. This probably results from variable gene flow in space and in time, due to demographic stochasticity in either natural or cultivated populations. Third, differentiation from cultivated landraces was only achieved for the quantitative traits but not for allozymes in two populations. This suggests that natural selection in some locations may oppose gene flow to establish cultivated traits into the natural introgressed populations.

Inferences from mitochondrial DNA patterns on the domestication history of alfalfa (Medicago sativa).

The evolutionary history of a wild-cultivated complex is the outcome of different factors, among which are the domestication of the cultivated form, its geographical and demographic expansion, as well as gene flow between natural and cultivated populations. To clarify this history for alfalfa (Medicago sativa), we adopted a phylogeographical approach based on a sample of natural and cultivated populations of the M. sativa species complex. This sample was characterized for mtDNA variation through restriction fragment length polymorphisms. Twenty-two mitotypes were identified in the whole data set (155 individuals). In the wild pool from the presumed area of origin (Near East to Central Asia), the diversity was high but the absence of geographical differentiation hinders a more precise location of the centre of domestication. Within the cultivated alfalfa, the geographical structure suggests strongly the existence of at least two independent routes of dissemination of alfalfa from its centre of origin. In particular, original mitotypes detected in some regions suggest that wild populations not sampled in this study, and located outside the presumed area of domestication, may have contributed to the diversity of the cultivated pool. Finally, analysis of the mtDNA variation shows that natural populations collected in Spain constitute an endemic wild pool now introgressed partially by cultivated alfalfa.

How mitochondrial DNA diversity can help to understand the dynamics of wild-cultivated complexes. The case of Medicago sativa in Spain.

In order to clarify the relationships (genetic exchange and shared ancestry) between natural and cultivated populations of alfalfa (Medicago sativa L.) in Spain, we investigated the patterns of mitochondrial DNA variation (characterized through restriction fragment length polymorphism) for 248 individuals in seven natural and six cultivated populations of this species. Mitochondrial variation was evidenced in both natural and cultivated populations of M. sativa. Among the seven mitotypes identified in the species, two were specific of the natural populations, a result attesting the fact that the Spanish wild form of M. sativa is an original genetic pool compared to the cultivated one. Other mitotypes were observed in both natural and cultivated populations, suggesting the occurrence of gene flow through seeds from cultivated towards natural populations. Comparisons with previously gathered nuclear and phenotypic data give insights into the different evolutionary forces acting on the different kinds of Spanish natural populations examined so far.