Deciphering the genetics of flowering time by an association study on candidate genes in bread wheat (Triticum aestivum L.).

Earliness is very important for the adaptation of wheat to environmental conditions and the achievement of high grain yield. A detailed knowledge of key genetic components of the life cycle would enable an easier control by the breeders. The objective of the study was to investigate the effect of candidate genes on flowering time. Using a collection of hexaploid wheat composed of 235 lines from diverse geographical origins, we conducted an association study for six candidate genes for flowering time and its components (vernalization sensitivity and earliness per se). The effect on the variation of earliness components of polymorphisms within the copies of each gene was tested in ANOVA models accounting for the underlying genetic structure. The collection was structured in five groups that minimized the residual covariance. Vernalization requirement and lateness tend to increase according to the mean latitude of each group. Heading date for an autumnal sowing was mainly determined by the earliness per se. Except for the Constans (CO) gene orthologous of the barley HvCO3, all gene polymorphisms had a significant impact on earliness components. The three traits used to quantify vernalization requirement were primarily associated with polymorphisms at Vrn-1 and then at Vrn-3 and Luminidependens (LD) genes. We found a good correspondence between spring/winter types and genotypes at the three homeologous copies of Vrn-1. Earliness per se was mainly explained by polymorphisms at Vrn-3 and to a lesser extent at Vrn-1, Hd-1 and Gigantea (GI) genes. Vernalization requirement and earliness as a function of geographical origin, as well as the possible role of the breeding practices in the geographical distribution of the alleles and the hypothetical adaptive value of the candidate genes, are discussed.

High-throughput single nucleotide polymorphism genotyping in wheat (Triticum spp.).

Over the past few years, considerable progress has been made in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, largely through the investment of the human genetics community. These technologies are well adapted to diploid species. For plant breeding purposes, it is important to determine whether these genotyping methods are adapted to polyploidy, as most major crops are former or recent polyploids. To address this problem, we tested the capacity of the multiplex technology SNPlex with a set of 47 wheat SNPs to genotype DNAs of 1314 lines that were organized in four 384-well plates. These lines represented different taxa of tetra- and hexaploid Triticum species and their wild diploid relatives. We observed 40 markers which gave less than 20% missing data. Different methods, based on either Sanger sequencing or the MassARRAY genotyping technology, were then used to validate the genotypes obtained by SNPlex for 11 markers. The concordance of the genotypes obtained by SNPlex with the results obtained by the different validation methods was 96%, except for one discarded marker. Furthermore, a mapping study on six markers showed the expected genetic positions previously described. To conclude, this study showed that high-throughput genotyping technologies developed for diploid species can be used successfully in polyploids, although there is a need for manual reading. For the first time in wheat species, a core of 39 SNPs is available that can serve as the basis for the development of a complete SNPlex set of 48 markers.

Insight into the genetic bases of climatic adaptation in experimentally evolving wheat populations.

Experimental populations evolving under natural selection represent an interesting tool to study genetic bases of adaptation. Evolution of genes possibly involved in adaptive response can be followed together with the corresponding phenotypic traits. Using experimental populations of hexaploid wheat, we studied the evolution of flowering time, a major adaptive trait that synchronizes the initiation of reproduction and the occurrence of favourable environmental conditions. During 12 generations, three populations were grown in contrasted environments (Vervins North France, Le Moulon near Paris, Toulouse South France) under the influence of natural selection, drift, mutation and recombination. Evolution of diversity at the major gene VRN-1 involved in wheat vernalization response has been analysed jointly with earliness estimated in controlled conditions. Whatever the population, rapid phenotypic changes as well as parallel genotypic variations were observed in the first seven generations, probably as the result of selection acting on this major gene which explains 80% of the trait variation overall. Different allelic combinations at physically unlinked copies of VRN-1 located on distinct genomes (A, B and D) were selected between populations. As theoretically expected, due to population differentiation, a high level of genetic diversity was maintained overall in generation 12. Surprisingly, in two populations out of three, the emergence of new alleles by mutation or migration, coupled with temporal variable selection or frequency-dependent selection, allowed to maintain within-population diversity despite local genetic drift and natural selection. This result may plead for an evolutionary approach of wheat genetic resource conservation.

Do Arabidopsis halleri from nonmetallicolous populations accumulate zinc and cadmium more effectively than those from metallicolous populations?

• The ability of metallicolous and nonmetallicolous populations of Arabidopsis halleri to accumulate zinc (Zn), cadmium (Cd) and lead (Pb) is compared here in order to explore the extent and variability of this trait in wild A. halleri plants. • Aerial plant parts and the soil around the harvested plants were collected and analysed for metal concentrations or total and extractable metal concentrations, respectively, for 20 metallicolous and 13 nonmetallicolous populations. • Results show that metallicolous and nonmetallicolous populations have the same ability to accumulate Zn and Cd but that neither population type is able to accumulate Pb. Between populations within type, an homogenous accumulating response is observed for Zn, whereas the ability to accumulate Cd is variable. • Zn and Cd accumulation to very high concentrations is a constitutive property of the species. The Zn and Cd hyperaccumulator trait of A. halleri from contaminated sites was confirmed. Interestingly, nonmetallicolous plants are Zn and Cd hyperaccumulators. The possibility of using A. halleri in phytoremediation is discussed.

Evolution of flowering time in experimental wheat populations: a comprehensive approach to detect genetic signatures of natural selection.

In annual plant species, flowering time is a major adaptive trait that synchronizes the initiation of reproduction with favorable environmental conditions. Here, we aimed at studying the evolution of flowering time in three experimental populations of bread wheat, grown in contrasting environments (Northern to Southern France) for 12 generations. By comparing the distribution of phenotypic and presumably neutral variation, we first showed that flowering time responded to selection during the 12 generations of the experiment. To get insight into the genetic architecture of that trait, we then tested whether the distribution of genetic polymorphisms at six candidate genes, presumably involved in the trait expression, departed from neutral expectation. To that end, we focused on the temporal variation during the course of the experiment, and on the spatial differentiation at the end of the experiment, using previously published methods adapted to our experimental design. Only those genes that were strongly associated with flowering time variation were detected as responding to selection. For genes that had low-to-moderate phenotypic effects, or when there was interaction across different genes, we did not find evidence of selection using methods based on the distribution of temporal or spatial variation. In such cases, it might be more informative to consider multilocus and multiallelic combinations across genes, which could be the targets of selection.

FT genome A and D polymorphisms are associated with the variation of earliness components in hexaploid wheat.

The transition from vegetative to floral meristems in higher plants is determined by the coincidence of internal and environmental signals. Contrary to the photoperiod pathway, convergent evolution of the cold-dependent pathway has implicated different genes between dicots and monocots. Whereas no association between natural variation in vernalization requirement and Flowering time locus T (FT) gene polymorphism has been described in Arabidopsis, recent studies in Triticeae suggest implication of orthologous copies of FT in the cold response. In our study, we show that nucleotide polymorphisms on A and D copies of the wheat FT gene were associated with variations for heading date in a collection of 239 lines representing diverse geographical origins and status (landraces, old or recent cultivars). Interestingly, polymorphisms in the non-coding intronic region were strongly associated to flowering variation observed on plants grown without vernalization. But differently from VRN1, no epistatic interaction between FT homeologous copies was revealed. In agreement with the results of association study, the A and D copies of FT were mapped in regions including major QTLs for earliness traits in hexaploid wheat. This work, by identifying additional homeoalleles involved in wheat vernalization pathway, will contribute to a better understanding of the control of flowering, hence providing tools for the breeding of varieties with enhanced adaptation to changing environments.

Rapid differentiation of experimental populations of wheat for heading time in response to local climatic conditions.

BACKGROUND AND AIMS: Dynamic management (DM) of genetic resources aims at maintaining genetic variability between different populations evolving under natural selection in contrasting environments. In 1984, this strategy was applied in a pilot experiment on wheat (Triticum aestivum). Spatio-temporal evolution of earliness and its components (partial vernalization sensitivity, daylength sensitivity and earliness per se that determines flowering time independently of environmental stimuli) was investigated in this multisite and long-term experiment. METHODS: Heading time of six populations from the tenth generation was evaluated under different vernalization and photoperiodic conditions. KEY RESULTS: Although temporal evolution during ten generations was not significant, populations of generation 10 were genetically differentiated according to a north-south latitudinal trend for two components out of three: partial vernalization sensitivity and narrow-sense earliness. CONCLUSIONS: It is concluded that local climatic conditions greatly influenced the evolution of population earliness, thus being a major factor of differentiation in the DM system. Accordingly, a substantial proportion (approximately 25 %) of genetic variance was distributed among populations, suggesting that diversity was on average conserved during evolution but was differently distributed by natural selection (and possibly drift). Earliness is a complex trait and each genetic factor is controlled by multiple homeoalleles; the next step will be to look for spatial divergence in allele frequencies.

A broad-scale analysis of population differentiation for Zn tolerance in an emerging model species for tolerance study: Arabidopsis halleri (Brassicaceae).

Although current knowledge about the overall distribution of zinc (Zn) tolerance in Arabidopsis halleri populations is scarce, the species is an emerging model for the study of heavy metal tolerance in plants. We attempted to improve this knowledge by testing the Zn tolerance of scattered European metallicolous (M) and nonmetallicolous (NM) populations of A. h. subsp. halleri and A. h. subsp. ovirensis in hydroponic culture. The occurrence of constitutive tolerance was unconditionally established in A. h. halleri and tolerance was extended to the subspecies ovirensis. M populations were the most tolerant but there was a continuous range of variation in tolerance from NM to M populations. Finally, relatively high levels of tolerance were detected in some NM populations, suggesting that enhanced tolerance could be present at high frequency in populations that have not experienced metal exposure. We used our results to argue the evolutionary dynamics and origin of Zn tolerance in A. halleri.

Multiple origin of metallicolous populations of the pseudometallophyte Arabidopsis halleri (Brassicaceae) in central Europe: the cpDNA testimony.

The population structure of the pseudo-metallophyte herb, Arabidopsis halleri, was studied using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) on chloroplast DNA (cpDNA). The history of metallicolous (M) populations showing increased zinc tolerance was investigated. Eight primer-enzyme combinations out of 72 tested were applied to a total of 625 individuals from 28 widespread populations, 14 of them being M. Eleven distinct chlorotypes were found: five were common to nonmetallicolous (NM) and M populations, whereas six were only observed in one edaphic type (five in NM and one in M). No difference in chlorotype diversity between edaphic types was detected. Computed on the basis of chlorotype frequencies, the level of population differentiation was high but remained the same when taking into account levels of molecular divergence between chlorotypes. Isolation by distance was largely responsible for population differentiation. Geographically isolated groups of M populations were more genetically related to their closest NM populations than to each other. Our results suggest that M populations have been founded separately from distinct NM populations without suffering founding events and that the evolution towards increased tolerance observed in the distinct M population groups occurred independently.

Spatial genetic structure within a metallicolous population of Arabidopsis halleri, a clonal, self-incompatible and heavy-metal-tolerant species.

Arabidopsis halleri, a close wild relative of A. thaliana, is a clonal, insect-pollinated herb tolerant to heavy metals (Zn, Pd, Cd) and a hyperaccumulator of Zn and Cd. It is of particular interest in the study of evolutionary processes and phytoremediation. However, little is known about its population gene flow patterns and the structure of its genetic diversity. We used five microsatellite loci to investigate the genetic structure at a fine spatial scale (10 cm to 500 m) in a metallicolous population of A. halleri. We also studied the contributions made by clonal propagation and sexual reproduction (seed and pollen dispersal) to the genetic patterns. Clonal diversity was high (D(G) > 0.9). Clonal spread occurs only at short distances (< 1 m). Both clonal spread and limited dispersal, associated with sexual reproduction, contribute to the significant spatial genetic structure revealed by spatial autocorrelation analysis. The shape of the autocorrelogram suggests that seed dispersal is restricted and pollen flow extensive, which may be related to intense activity by insect pollinators. Clonal spread was more extensive in the lowly polluted zone than in the highly polluted zone. This cannot be interpreted as a strategy for promoting the propagation of adapted genotypes under the harshest ecological constraints (highest heavy metal concentrations). The higher fine-scale spatial genetic structure found in the lowly polluted zone can be ascribed to plant densities that were lower than in the highly polluted zone. No evidence of genetic divergence due to spatial heavy metal heterogeneity was found between lowly and highly polluted zones.

Population structure of an endangered species living in contrasted habitats: Parnassia palustris (Saxifragaceae).

In endangered species, it is critical to analyse the level at which populations interact (i.e. dispersal) as well as the levels of inbreeding and local adaptation to set up conservation policies. These parameters were investigated in the endangered species Parnassia palustris living in contrasted habitats. We analysed population structure in 14 populations of northern France for isozymes, cpDNA markers and phenotypic traits related to fitness. Within population genetic diversity and inbreeding coefficients were not correlated to population size. Populations seem not to have undergone severe recent bottleneck. Conversely to pollen migration, seed migration seems limited at a regional scale, which could prevent colonization of new sites even if suitable habitats appear. Finally, the habitat type affects neither within-population genetic diversity nor genetic and phenotypic differentiation among populations. Thus, even if unnoticed local adaptation to habitats exists, it does not influence gene flow between populations.