Soil variation among natural habitats alters glucosinolate content in a wild perennial mustard.

Baseline levels of glucosinolates-important defensive phytochemicals in brassicaceous plants-are determined by both genotype and environment. However, the ecological causes of glucosinolate plasticity are not well characterized. Fertilization is known to alter glucosinolate content of Brassica crops, but the effect of naturally occurring soil variation on glucosinolate content of wild plants is unknown. Here, we conducted greenhouse experiments using Boechera stricta to ask (i) whether soil variation among natural habitats shapes leaf and root glucosinolate profiles; (ii) whether such changes are caused by abiotic soil properties, soil microbes, or both; and (iii) whether soil-induced glucosinolate plasticity is genetically variable. Total glucosinolate quantity differed up to 2-fold between soils from different natural habitats, while the relative amounts of different compounds were less responsive. This effect was due to physico-chemical soil properties rather than microbial communities. We detected modest genetic variation for glucosinolate plasticity in response to soil. In addition, glucosinolate composition, but not quantity, of field-grown plants could be accurately predicted from measurements from greenhouse-grown plants. In summary, soil alone is sufficient to cause plasticity of baseline glucosinolate levels in natural plant populations, which may have implications for the evolution of this important trait across complex landscapes.

An epigenetic pathway in rice connects genetic variation to anaerobic germination and seedling establishment.

Rice production is shifting from transplanting seedlings to direct sowing of seeds. Following heavy rains, directly sown seeds may need to germinate under anaerobic environments, but most rice (Oryza sativa) genotypes cannot survive these conditions. To identify the genetic architecture of complex traits, we quantified percentage anaerobic germination (AG) in 2,700 (wet-season) and 1,500 (dry-season) sequenced rice genotypes and performed genome-wide association studies (GWAS) using 693,502 single nucleotide polymorphisms. This was followed by post-GWAS analysis with a generalized SNP-to-gene set analysis, meta-analysis, and network analysis. We determined that percentage AG is intermediate-to-high among indica subpopulations, and AG is a polygenic trait associated with transcription factors linked to ethylene responses or genes involved in metabolic processes that are known to be associated with AG. Our post-GWAS analysis identified several genes involved in a wide variety of metabolic processes. We subsequently performed functional analysis focused on the small RNA and methylation pathways. We selected CLASSY 1 (CLSY1), a gene involved in the RNA-directed DNA methylation (RdDm) pathway, for further analyses under AG and found several lines of evidence that CLSY1 influences AG. We propose that the RdDm pathway plays a role in rice responses to water status during germination and seedling establishment developmental stages.

The Boechera model system for evolutionary ecology.

Model systems in biology expand the research capacity of individuals and the community. Closely related to Arabidopsis, the genus Boechera has emerged as an important ecological model owing to the ability to integrate across molecular, functional, and eco-evolutionary approaches. Boechera species are broadly distributed in relatively undisturbed habitats predominantly in western North America and provide one of the few experimental systems for identification of ecologically important genes through genome-wide association studies and investigations of selection with plants in their native habitats. The ecologically, evolutionarily, and agriculturally important trait of apomixis (asexual reproduction via seeds) is common in the genus, and field experiments suggest that abiotic and biotic environments shape the evolution of sex. To date, population genetic studies have focused on the widespread species B. stricta, detailing population divergence and demographic history. Molecular and ecological studies show that balancing selection maintains genetic variation in ~10% of the genome, and ecological trade-offs contribute to complex trait variation for herbivore resistance, flowering phenology, and drought tolerance. Microbiome analyses have shown that host genotypes influence leaf and root microbiome composition, and the soil microbiome influences flowering phenology and natural selection. Furthermore, Boechera offers numerous opportunities for investigating biological responses to global change. In B. stricta, climate change has induced a shift of >2 weeks in the timing of first flowering since the 1970s, altered patterns of natural selection, generated maladaptation in previously locally-adapted populations, and disrupted life history trade-offs. Here we review resources and results for this eco-evolutionary model system and discuss future research directions.

The ecological, genetic and genomic architecture of local adaptation and population differentiation in Boechera stricta.

Differential local adaptation restricts gene flow between populations inhabiting distinct environments, resulting in isolation by adaptation. In addition to the statistical inferences of genotype-environment associations, an integrative approach is needed to investigate the effect of local adaptation on population divergence at the ecological, genetic and genomic scale. Here, we combine reciprocal transplant, genome-environment association and QTL mapping to investigate local adaptation in Boechera stricta (Drummond's rockcress). With reciprocal transplant experiment, we found local genetic groups exhibit phenotypic characteristics corresponding to the distinct selection forces from different water availability. At the genetic level, the local allele of a major fitness QTL confers higher and sturdier flowering stalks, maximizing the fecundity fitness component under sufficient water supply, and its genetic variation is associated with precipitation across the landscape. At the genomewide scale, we further showed that multiple loci associated with precipitation are highly differentiated between genetic groups, suggesting that local adaptation has a widespread effect on reducing gene flow. This study provides one of the few comprehensive examples demonstrating how local adaptation facilitates population divergence at the trait, gene and genome level.

Genetic architecture and adaptation of flowering time among environments.

The genetic basis of flowering time changes across environments, and pleiotropy may limit adaptive evolution of populations in response to local conditions. However, little information is known about how genetic architecture changes among environments. We used genome-wide association studies (GWAS) in Boechera stricta (Graham) Al-Shehbaz, a relative of Arabidopsis, to examine flowering variation among environments and associations with climate conditions in home environments. Also, we used molecular population genetics to search for evidence of historical natural selection. GWAS found 47 significant quantitative trait loci (QTLs) that influence flowering time in one or more environments, control plastic changes in phenology between experiments, or show associations with climate in sites of origin. Genetic architecture of flowering varied substantially among environments. We found that some pairs of QTLs showed similar patterns of pleiotropy across environments. A large-effect QTL showed molecular signatures of adaptive evolution and is associated with climate in home environments. The derived allele at this locus causes later flowering and predominates in sites with greater water availability. This work shows that GWAS of climate associations and ecologically important traits across diverse environments can be combined with molecular signatures of natural selection to elucidate ecological genetics of adaptive evolution.

DIACYLGLYCEROL ACYLTRANSFERASE1 Contributes to Freezing Tolerance.

Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Adaptive evolution: evaluating empirical support for theoretical predictions.

Adaptive evolution is shaped by the interaction of population genetics, natural selection and underlying network and biochemical constraints. Variation created by mutation, the raw material for evolutionary change, is translated into phenotypes by flux through metabolic pathways and by the topography and dynamics of molecular networks. Finally, the retention of genetic variation and the efficacy of selection depend on population genetics and demographic history. Emergent high-throughput experimental methods and sequencing technologies allow us to gather more evidence and to move beyond the theory in different systems and populations. Here we review the extent to which recent evidence supports long-established theoretical principles of adaptation.

Unifying genetic canalization, genetic constraint, and genotype-by-environment interaction: QTL by genomic background by environment interaction of flowering time in Boechera stricta.

Natural populations exhibit substantial variation in quantitative traits. A quantitative trait is typically defined by its mean and variance, and to date most genetic mapping studies focus on loci altering trait means but not (co)variances. For single traits, the control of trait variance across genetic backgrounds is referred to as genetic canalization. With multiple traits, the genetic covariance among different traits in the same environment indicates the magnitude of potential genetic constraint, while genotype-by-environment interaction (GxE) concerns the same trait across different environments. While some have suggested that these three attributes of quantitative traits are different views of similar concepts, it is not yet clear, however, whether they have the same underlying genetic mechanism. Here, we detect quantitative trait loci (QTL) influencing the (co)variance of phenological traits in six distinct environments in Boechera stricta, a close relative of Arabidopsis. We identified nFT as the QTL altering the magnitude of phenological trait canalization, genetic constraint, and GxE. Both the magnitude and direction of nFT's canalization effects depend on the environment, and to our knowledge, this reversibility of canalization across environments has not been reported previously. nFT's effects on trait covariance structure (genetic constraint and GxE) likely result from the variable and reversible canalization effects across different traits and environments, which can be explained by the interaction among nFT, genomic backgrounds, and environmental stimuli. This view is supported by experiments demonstrating significant nFT by genomic background epistatic interactions affecting phenological traits and expression of the candidate gene, FT. In contrast to the well-known canalization gene Hsp90, the case of nFT may exemplify an alternative mechanism: Our results suggest that (at least in traits with major signal integrators such as flowering time) genetic canalization, genetic constraint, and GxE may have related genetic mechanisms resulting from interactions among major QTL, genomic backgrounds, and environments.

3D phenotyping and quantitative trait locus mapping identify core regions of the rice genome controlling root architecture.

Identification of genes that control root system architecture in crop plants requires innovations that enable high-throughput and accurate measurements of root system architecture through time. We demonstrate the ability of a semiautomated 3D in vivo imaging and digital phenotyping pipeline to interrogate the quantitative genetic basis of root system growth in a rice biparental mapping population, Bala × Azucena. We phenotyped >1,400 3D root models and >57,000 2D images for a suite of 25 traits that quantified the distribution, shape, extent of exploration, and the intrinsic size of root networks at days 12, 14, and 16 of growth in a gellan gum medium. From these data we identified 89 quantitative trait loci, some of which correspond to those found previously in soil-grown plants, and provide evidence for genetic tradeoffs in root growth allocations, such as between the extent and thoroughness of exploration. We also developed a multivariate method for generating and mapping central root architecture phenotypes and used it to identify five major quantitative trait loci (r(2) = 24-37%), two of which were not identified by our univariate analysis. Our imaging and analytical platform provides a means to identify genes with high potential for improving root traits and agronomic qualities of crops.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

Microgeographic Patterns of Genetic Divergence and Adaptation across Environmental Gradients in Boechera stricta (Brassicaceae).

Abiotic and biotic conditions often vary continuously across the landscape, imposing divergent selection on local populations. We used a provenance trial approach to examine microgeographic variation in local adaptation in Boechera stricta (Brassicaceae), a perennial forb native to the Rocky Mountains. In montane ecosystems, environmental conditions change considerably over short spatial scales, such that neighboring populations can be subject to different selective pressures. Using accessions from southern (Colorado) and northern (Idaho) populations, we characterized spatial variation in genetic similarity via microsatellite markers. We then transplanted genotypes from multiple local populations into common gardens in both regions. Continuous variation in local adaptation emerged for several components of fitness. In Idaho, genotypes from warmer environments (low-elevation or south-facing sites) were poorly adapted to the north-facing garden. In high- and low-elevation Colorado gardens, susceptibility to insect herbivory increased with source elevation. In the high-elevation Colorado garden, germination success peaked for genotypes that evolved at elevations similar to that of the garden and decreased for genotypes from higher and lower elevations. We also found evidence for local maladaptation in survival and fecundity components of fitness in the low-elevation Colorado garden. This approach is a first step in predicting how global change could affect evolutionary dynamics.

Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative.

Plant phenology is known to depend on many different environmental variables, but soil microbial communities have rarely been acknowledged as possible drivers of flowering time. Here, we tested separately the effects of four naturally occurring soil microbiomes and their constituent soil chemistries on flowering phenology and reproductive fitness of Boechera stricta, a wild relative of Arabidopsis. Flowering time was sensitive to both microbes and the abiotic properties of different soils; varying soil microbiota also altered patterns of selection on flowering time. Thus, soil microbes potentially contribute to phenotypic plasticity of flowering time and to differential selection observed between habitats. We also describe a method to dissect the microbiome into single axes of variation that can help identify candidate organisms whose abundance in soil correlates with flowering time. This approach is broadly applicable to search for microbial community members that alter biological characteristics of interest.

Evolution of flux control in the glucosinolate pathway in Arabidopsis thaliana.

Network characteristics of biochemical pathways are believed to influence the rate of evolutionary change in constituent enzymes. One characteristic that may affect rate heterogeneity is control of the amount of product produced by a biochemical pathway or flux control. In particular, theoretical analyses suggest that adaptive substitutions should be concentrated in the enzyme(s) that exert the greatest control over flux. Although a handful of studies have found a correlation between position in a pathway and evolutionary rate, these investigations have not examined the relationship between evolutionary rate and flux control. Given that genes with greater control will experience stronger selection and that the probability of fixation is proportional to the selective advantage, we ask the following: 1) do upstream enzymes have majority flux control, 2) do enzymes with majority flux control accumulate adaptive substitutions, and 3) are upstream enzymes under higher selective constraint? First, by perturbing the enzymes in the aliphatic glucosinolate pathway in Arabidopsis thaliana with gene insertion lines, we show that flux control is focused in the first enzyme in the pathway. Next, by analyzing several sequence signatures of selection, we also show that this enzyme is the only one in the pathway that shows convincing evidence of selection. Our results support the hypothesis that natural selection preferentially acts on enzymes with high flux control.

Evolutionary genetics of plant adaptation.

Plants provide unique opportunities to study the mechanistic basis and evolutionary processes of adaptation to diverse environmental conditions. Complementary laboratory and field experiments are important for testing hypotheses reflecting long-term ecological and evolutionary history. For example, these approaches can infer whether local adaptation results from genetic tradeoffs (antagonistic pleiotropy), where native alleles are best adapted to local conditions, or if local adaptation is caused by conditional neutrality at many loci, where alleles show fitness differences in one environment, but not in a contrasting environment. Ecological genetics in natural populations of perennial or outcrossing plants can also differ substantially from model systems. In this review of the evolutionary genetics of plant adaptation, we emphasize the importance of field studies for understanding the evolutionary dynamics of model and nonmodel systems, highlight a key life history trait (flowering time) and discuss emerging conservation issues.

Identification of quantitative trait loci and a candidate locus for freezing tolerance in controlled and outdoor environments in the overwintering crucifer Boechera stricta.

Development of chilling and freezing tolerance is complex and can be affected by photoperiod, temperature and photosynthetic performance; however, there has been limited research on the interaction of these three factors. We evaluated 108 recombinant inbred lines of Boechera stricta, derived from a cross between lines originating from Montana and Colorado, under controlled long day (LD), short-day (SD) and in an outdoor environment (OE). We measured maximum quantum yield of photosystem II, lethal temperature for 50% survival and electrolyte leakage of leaves. Our results revealed significant variation for chilling and freezing tolerance and photosynthetic performance in different environments. Using both single- and multi-trait analyses, three main-effect quantitative trait loci (QTL) were identified. QTL on linkage group (LG)3 were SD specific, whereas QTL on LG4 were found under both LD and SD. Under all conditions, QTL on LG7 were identified, but were particularly predictive for the outdoor experiment. The co-localization of photosynthetic performance and freezing tolerance effects supports these traits being co-regulated. Finally, the major QTL on LG7 is syntenic to the Arabidopsis C-repeat binding factor locus, known regulators of chilling and freezing responses in Arabidopsis thaliana and other species.

Environmental adaptation contributes to gene polymorphism across the Arabidopsis thaliana genome.

The level of within-species polymorphism differs greatly among genes in a genome. Many genomic studies have investigated the relationship between gene polymorphism and factors such as recombination rate or expression pattern. However, the polymorphism of a gene is affected not only by its physical properties or functional constraints but also by natural selection on organisms in their environments. Specifically, if functionally divergent alleles enable adaptation to different environments, locus-specific polymorphism may be maintained by spatially heterogeneous natural selection. To test this hypothesis and estimate the extent to which environmental selection shapes the pattern of genome-wide polymorphism, we define the "environmental relevance" of a gene as the proportion of genetic variation explained by environmental factors, after controlling for population structure. We found substantial effects of environmental relevance on patterns of polymorphism among genes. In addition, the correlation between environmental relevance and gene polymorphism is positive, consistent with the expectation that balancing selection among heterogeneous environments maintains genetic variation at ecologically important genes. Comparison of the gene ontology annotations shows that genes with high environmental relevance are enriched in unknown function categories. These results suggest an important role for environmental factors in shaping genome-wide patterns of polymorphism and indicate another direction of genomic study.

Selection on QTL and complex traits in complex environments.

Understanding genetic variation for complex traits in heterogeneous environments is a fundamental problem in biology. In this issue of Molecular Ecology, Fournier-Level et al. (2013) analyse quantitative trait loci (QTL)influencing ecologically important phenotypes in mapping populations of Arabidopsis thaliana grown in four habitats across its native European range. They used causal modelling to quantify the selective consequences of life history and morphological traits and QTL on components of fitness. They found phenology QTL colocalizing with known flowering time genes as well as novel loci. Most QTL influenced fitness via life history and size traits, rather than QTL having direct effects on fitness.Comparison of phenotypes among environments found no evidence for genetic trade-offs for phenology or growth traits, but genetic trade-offs for fitness resulted because flowering time had opposite fitness effects in different environments. These changes in QTL effects and selective consequences may maintain genetic variation among populations.

Complex trait divergence contributes to environmental niche differentiation in ecological speciation of Boechera stricta.

Ecological factors may contribute to reproductive isolation if differential local adaptation causes immigrant or hybrid fitness reduction. Because local adaptation results from the interaction between natural selection and adaptive traits, it is crucial to investigate both to understand ecological speciation. Previously, we used niche modelling to identify local water availability as an environmental correlate of incipient ecological speciation between two subspecies in Boechera stricta, a close relative of Arabidopsis. Here, we performed several large-scale glasshouse experiments to investigate the divergence of various physiological, phenological and morphological traits. Although we found no significant difference in physiological traits, the Western subspecies has significantly faster growth rate, larger leaf area, less succulent leaves, delayed reproductive time and longer flowering duration. These trait differences are concordant with previous results that habitats of the Western genotypes have more consistent water availability, while Eastern genotypes inhabit locations with more ephemeral water supplies. In addition, by comparing univariate and multivariate divergence of complex traits (Q(ST)) to the genomewide distribution of SNP FST , we conclude that the aspects of phenology and morphology (but not physiology) are under divergent selection. In addition, we also identified several highly diverged traits without obvious water-related functions.

Genetic trade-offs and conditional neutrality contribute to local adaptation.

Divergent natural selection promotes local adaptation and can lead to reproductive isolation of populations in contrasting environments; however, the genetic basis of local adaptation remains largely unresolved in natural populations. Local adaptation might result from antagonistic pleiotropy, where alternate alleles are favoured in distinct habitats, and polymorphism is maintained by selection. Alternatively, under conditional neutrality some alleles may be favoured in one environment but neutral at other locations. Antagonistic pleiotropy maintains genetic variation across the landscape; however, there is a systematic bias against discovery of antagonistic pleiotropy because the fitness benefits of local alleles need to be significant in at least two environments. Here, we develop a generally applicable method to investigate polygenic local adaptation and identify loci that are the targets of selection. This approach evaluates allele frequency changes after selection at loci across the genome to distinguish antagonistic pleiotropy from conditional neutrality and deleterious variation. We investigate local adaptation at the qualitative trait loci (QTL) level in field experiments, in which we expose 177 F(6) recombinant inbred lines and parental lines of Boechera stricta (Brassicaceae) to their parental environments over two seasons. We demonstrate polygenic selection for native alleles in both environments, with 2.8% of the genome exhibiting antagonistic pleiotropy and 8% displaying conditional neutrality. Our study strongly supports antagonistic pleiotropy at one large-effect flowering phenology QTL (nFT): native homozygotes had significantly greater probabilities of flowering than foreign homozygotes in both parental environments. Such large-scale field studies are essential to elucidate the genetic basis of adaptation in natural populations.

Which evolutionary processes influence natural genetic variation for phenotypic traits?

Although many studies provide examples of evolutionary processes such as adaptive evolution, balancing selection, deleterious variation and genetic drift, the relative importance of these selective and stochastic processes for phenotypic variation within and among populations is unclear. Theoretical and empirical studies from humans as well as natural animal and plant populations have made progress in examining the role of these evolutionary forces within species. Tentative generalizations about evolutionary processes across species are beginning to emerge, as well as contrasting patterns that characterize different groups of organisms. Furthermore, recent technical advances now allow the combination of ecological measurements of selection in natural environments with population genetic analysis of cloned QTLs, promising advances in identifying the evolutionary processes that influence natural genetic variation.