Cécile Godé

The Genetic Basis of Zinc Tolerance in the Metallophyte Arabidopsis halleri ssp. halleri (Brassicaceae): An Analysis of Quantitative Trait Loci

The species Arabidopsis halleri, an emerging model for the study of heavy metal tolerance and accumulation in plants, has evolved a high level of constitutive zinc tolerance. Mapping of quantitative trait loci (QTL) was used to investigate the genetic architecture of zinc tolerance in this species. A first-generation backcross progeny of A. halleri ssp. halleri from a highly contaminated industrial site and its nontolerant relative A. lyrata ssp. petraea was produced and used for QTL mapping of zinc tolerance. A genetic map covering most of the A. halleri genome was constructed using 85 markers. Among these markers, 65 were anchored in A. thaliana and revealed high synteny with other Arabidopsis genomes. Three QTL of comparable magnitude on three different linkage groups were identified. At all QTL positions zinc tolerance was enhanced by A. halleri alleles, indicating directional selection for higher zinc tolerance in this species. The two-LOD support intervals associated with these QTL cover 24, 4, and 13 cM. The importance of each of these three regions is emphasized by their colocalization with HMA4, MTP1-A, and MTP1-B, respectively, three genes well known to be involved in metal homeostasis and tolerance in plants.

Genetic architecture of zinc hyperaccumulation in Arabidopsis halleri: the essential role of QTL × environment interactions.

SUMMARY This study sought to determine the main genomic regions that control zinc (Zn) hyperaccumulation in Arabidopsis halleri and to examine genotype x environment effects on phenotypic variance. To do so, quantitative trait loci (QTLs) were mapped using an interspecific A. halleri x Arabidopsis lyrata petraea F(2) population. *The F(2) progeny as well as representatives of the parental populations were cultivated on soils at two different Zn concentrations. A linkage map was constructed using 70 markers. *In both low and high pollution treatments, zinc hyperaccumulation showed high broad-sense heritability (81.9 and 74.7%, respectively). Five significant QTLs were detected: two QTLs specific to the low pollution treatment (chromosomes 1 and 4), and three QTLs identified at both treatments (chromosomes 3, 6 and 7). These QTLs explained 50.1 and 36.5% of the phenotypic variance in low and high pollution treatments, respectively. Two QTLs identified at both treatments (chromosomes 3 and 6) showed significant QTL x environment interactions. *The QTL on chromosome 3 largely colocalized with a major QTL previously identified for Zn and cadmium (Cd) tolerance. This suggests that Zn tolerance and hyperaccumulation share, at least partially, a common genetic basis and may have simultaneously evolved on heavy metal-contaminated soils.

Nuclear and chloroplast DNA phylogeography reveals vicariance among European populations of the model species for the study of metal tolerance, Arabidopsis halleri (Brassicaceae)

Arabidopsis halleri is a pseudometallophyte involved in numerous molecular studies of the adaptation to anthropogenic metal stress. In order to test the representativeness of genetic accessions commonly used in these studies, we investigated the A. halleri population genetic structure in Europe. Microsatellite and nucleotide polymorphisms from the nuclear and chloroplast genomes, respectively, were used to genotype 65 populations scattered over Europe. The large-scale population structure was characterized by a significant phylogeographic signal between two major genetic units. The localization of the phylogeographic break was assumed to result from vicariance between large populations isolated in southern and central Europe, on either side of ice sheets covering the Alps during the Quaternary ice ages. Genetic isolation was shown to be maintained in western Europe by the high summits of the Alps, whereas admixture was detected in the Carpathians. Considering the phylogeographic literature, our results suggest a distinct phylogeographic pattern for European species occurring in both mountain and lowland habitats. Considering the evolution of metal adaptation in A. halleri, it appears that recent adaptations to anthropogenic metal stress that have occurred within either phylogeographic unit should be regarded as independent events that potentially have involved the evolution of a variety of genetic mechanisms.

Polymerase chain reaction–single strand conformation polymorphism analyses of nuclear and chloroplast DNA provide evidence for recombination, multiple introductions and nascent speciation in the Caulerpa taxifolia complex

Independent lines of evidence support an Australian origin for the Mediterranean populations of the tropical alga Caulerpa taxifolia. To complement previous biogeographical studies based on nuclear rDNA internal transcribed spacer (ITS), a new chloroplast marker was developed — the cp 16S rDNA intron‐2. Sequence variability for both nuclear and chloroplast markers were assessed in 110 individuals using single strand conformation polymorphism. Comparison of intrapopulation genetic diversity between invasive Mediterranean and ‘native’ Australian populations revealed the occurrence of two divergent and widespread clades. The first clade grouped nontropical invasive populations with inshore‐mainland populations from Australia, while the second clustered all offshore‐island populations studied so far. Despite our finding of nine distinct nuclear and five distinct chloroplast profiles, a single nucleocytoplasmic combination was characteristic of the invasive populations and sexual reproduction was found to be very rare. C. taxifolia is clearly a complex of genetically and ecologically differentiated sibling species or subspecies.

Fine‐scale geographical structure of genetic diversity in inland wild beet populations

Introgression arising from crop‐to‐wild gene flow provides novel sources of genetic variation in plant species complexes. Hybridization within the Beta vulgaris species complex is of immediate concern; crop lineages (B. vulgaris ssp. vulgaris) hybridize easily with their wild relatives (B. vulgaris ssp. maritima) thereby threatening wild beet gene diversity with genetic swamping. Hybridization ‘hotspots’ occur in European seed production areas because inland ruderal wild beets occur and reproduce in sympatry with cultivated beets. We studied gene flow occurring between seed‐producing cultivars and ruderal wild B. vulgaris in southwestern France to determine whether feral beets, arising from unharvested cultivated seed, represent an opportunity for crop‐to‐wild gene flow. We surveyed 42 inland ruderal beet populations located near seed production fields for nucleo‐cytoplasmic variation and used a cytoplasmic marker diagnostic of cultivated lines. Occurrence of cultivated‐type cytoplasm within ruderal populations clearly reflected events of crop seed escape. However, we found no genetic signatures of nuclear cultivated gene introgression, which suggests past introgression of cultivated cytoplasm into a wild nuclear background through seed escape rather than recent direct pollen flow. Overall, patterns of genetic structure suggested that inland ruderal wild beet populations act as a metapopulation, with founding events involving a few sib groups, followed by low rates of seed or pollen gene flow after populations are established. Altogether, our results indicate that a long‐lived seed bank plays a key role in maintaining cultivated‐type cytoplasm in the wild and highlight the need for careful management of seed production areas where wild and cultivated relatives co‐occur.

The use of comparative genome analysis and syntenic relationships allows extrapolating the position of Zn tolerance QTL regions from Arabidopsis halleri into Arabidopsis thaliana

Arabidopsis halleri is a species that has undergone natural selection for zinc (Zn) tolerance. Isolation of the quantitative trait loci (QTL) associated with this trait holds great promise for the identification of the main genes responsible for this adaptation. Using a segregating progeny produced by an interspecific cross, we previously constructed a genetic linkage map of A. halleri × A. lyrata petraea and mapped the three main QTL that confer Zn tolerance in A. halleri (Willems et al.). The goal of the present study is to compare the genetic linkage map of A. halleri × A. l. petraea to the annotated A. thaliana genome sequence to generate a tool for A. halleri genomic approaches. To achieve this aim, we constructed a genetic linkage map with 81 markers anchored on A. thaliana, including 23 genes known to be involved in metal homeostasis. First, this provided an extensive overview of the chromosomal rearrangements that have occurred since the divergence between A. thaliana and its closest relative A. halleri. Second, on the basis of the syntenic relationships assessed experimentally through this work, we transferred the QTL confidence intervals for Zn tolerance to the A. thaliana physical map, allowing access to all the genes localized in the corresponding regions. Third, we validated from the 23 genes involved in metal homeostasis the three ones localized in the QTL regions that can be considered the best candidates for conferring Zn tolerance.

Selfish male‐determining element favors the transition from hermaphroditism to androdioecy

According to the current, widely accepted paradigm, the evolutionary transition from hermaphroditism toward separate sexes occurs in two successive steps: an initial, intermediate step in which unisexual individuals, male or female, sterility mutants coexist with hermaphrodites and a final step that definitively establishes dioecy. Two nonexclusive processes can drive this transition: inbreeding avoidance and reallocation of resources from one sexual function to the other. Here, we report results of controlled crosses between males and hermaphrodites in Phillyrea angustifolia, an androdioecious species with two mutually intercompatible, but intraincompatible groups of hermaphrodites. We observed different segregation patterns that can be explained by: (1) epistatic interactions between two unlinked diallelic loci, determining sex and mating compatibility, and (2) a mutation with pleiotropic effects: female sterility, full compatibility of males with both hermaphrodite incompatibility groups, and complete male‐biased sex‐ratio distortion in one of the two groups. Modeling shows that these mechanisms can explain the high frequency of males in populations of P. angustifolia and can promote the maintenance of androdioecy without requiring inbreeding depression or resource reallocation. We thus argue that segregation distortion establishes the right conditions for the evolution of cryptic dioecy and potentially initiates the evolution toward separate sexes.

Patterns of genetic divergence among populations of the pseudometallophyte Biscutella laevigata from southern Poland

Background and aimsPseudometallophytes are model organisms for adaptation and population differentiation because they persist in contrasting edaphic conditions of metalliferous and non-metalliferous habitats. We examine patterns of genetic divergence and local adaptation of Biscutella laevigata to assess historical and evolutionary processes shaping its genetic structure.MethodsWe sampled all known populations of B. laevigata in Poland and analyzed respective soil metal concentrations. For genotyping we used nine nuclear microsatellite loci. Population genetic pools were identified (Bayesian clustering) and we estimated genetic parameters and demographic divergence between metallicolous and non-metallicolous populations (ABC-approach).ResultsPopulations clustered into two groups which corresponded to their edaphic origin and diverged 1,200 generations ago. We detected a significant decrease in genetic diversity and evidence for a recent bottleneck in metallicolous populations. Genetic structure was unrelated to site distribution but is rather influenced by environmental conditions (i.e. soil metal concentration).ConclusionsThe intriguing disjunctive distribution of B. laevigata in Poland results from a fragmentation of the species range during the Holocene, rather than recent long-distance-dispersal events. The genetic structure of populations, however, continues to be modified by microevolutionary processes at anthropogenic sites. These clear divergence patterns promote B. laevigata as a model species for plant adaptation to polluted environments.

Nuclear microsatellite loci for Arabidopsis halleri (Brassicaceae), a model species to study plant adaptation to heavy metals

PREMISE OF THE STUDY Arabidopsis halleri is a model species to study the adaptation of plants to soils contaminated by zinc, cadmium, and lead. To provide a neutral genetic background with which adaptive genetic markers could be compared, we developed highly polymorphic neutral microsatellite markers. METHODS AND RESULTS Using a microsatellite-enriched library method, we identified 120 microsatellite loci for quantitative trait locus (QTL) mapping analysis, of which eight primer pairs were developed in a single multiplex for population genetic studies. Analyses were performed on 508 individuals from 26 populations. All loci were polymorphic with six to 23 alleles per locus. Genetic diversity varied between 0.56 and 0.76. CONCLUSIONS Our results demonstrated the value of these eight microsatellite markers to investigate neutral population genetic structure in A. halleri. To increase the resolution of population genetic analyses, we suggest adding them to the 11 markers previously developed independently.

Lineages of Silene nutans developed rapid, strong, asymmetric postzygotic reproductive isolation in allopatry

Reproductive isolation can rise either as a consequence of genomic divergence in allopatry or as a byproduct of divergent selection in parapatry. To determine whether reproductive isolation in gynodioecious Silene nutans results from allopatric divergence or from ecological adaptation following secondary contact, we investigated the pattern of postzygotic reproductive isolation and hybridization in natural populations using two phylogeographic lineages, western (W1) and eastern (E1). Experimental crosses between the lineages identified strong, asymmetric postzygotic isolation between the W1 and the E1 lineages, independent of geographic overlap. The proportion of ovules fertilized, seeds aborted, and seeds germinated revealed relatively little effect on the fitness of hybrids. In contrast, hybrid mortality was high and asymmetric: while half of the hybrid seedlings with western lineage mothers died, nearly all hybrid seedlings with E1 mothers died. This asymmetric mortality mirrored the proportion of chlorotic seedlings, and is congruent with cytonuclear incompatibility. We found no evidence of hybridization between the lineages in regions of co‐occurrence using nuclear and plastid markers. Together, our results are consistent with the hypothesis that strong postzygotic reproductive isolation involving cytonuclear incompatibilities arose in allopatry. We argue that the dynamics of cytonuclear gynodioecy could facilitate the evolution of reproductive isolation.