Variation and plasticity in life-history traits and fitness of wild Arabidopsis thaliana populations are not related to their genotypic and ecological diversity.

BACKGROUND: Despite its implications for population dynamics and evolution, the relationship between genetic and phenotypic variation in wild populations remains unclear. Here, we estimated variation and plasticity in life-history traits and fitness of the annual plant Arabidopsis thaliana in two common garden experiments that differed in environmental conditions. We used up to 306 maternal inbred lines from six Iberian populations characterized by low and high genotypic (based on whole-genome sequences) and ecological (vegetation type) diversity. RESULTS: Low and high genotypic and ecological diversity was found in edge and core Iberian environments, respectively. Given that selection is expected to be stronger in edge environments and that ecological diversity may enhance both phenotypic variation and plasticity, we expected genotypic diversity to be positively associated with phenotypic variation and plasticity. However, maternal lines, irrespective of the genotypic and ecological diversity of their population of origin, exhibited a substantial amount of phenotypic variation and plasticity for all traits. Furthermore, all populations harbored maternal lines with canalization (robustness) or sensitivity in response to harsher environmental conditions in one of the two experiments. CONCLUSIONS: Overall, we conclude that the environmental attributes of each population probably determine their genotypic diversity, but all populations maintain substantial phenotypic variation and plasticity for all traits, which represents an asset to endure in changing environments.

Genetic basis of Arabidopsis thaliana responses to infection by naïve and adapted isolates of turnip mosaic virus.

Plant viruses account for enormous agricultural losses worldwide, and the most effective way to combat them is to identify genetic material conferring plant resistance to these pathogens. Aiming to identify genetic associations with responses to infection, we screened a large panel of Arabidopsis thaliana natural inbred lines for four disease-related traits caused by infection by A. thaliana-naïve and -adapted isolates of the natural pathogen turnip mosaic virus (TuMV). We detected a strong, replicable association in a 1.5 Mb region on chromosome 2 with a 10-fold increase in relative risk of systemic necrosis. The region contains several plausible causal genes as well as abundant structural variation, including an insertion of a Copia transposon into a Toll/interleukin receptor (TIR-NBS-LRR) coding for a gene involved in defense, that could be either a driver or a consequence of the disease-resistance locus. When inoculated with TuMV, loss-of-function mutant plants of this gene exhibited different symptoms than wild-type plants. The direction and severity of symptom differences depended on the adaptation history of the virus. This increase in symptom severity was specific for infections with the adapted isolate. Necrosis-associated alleles are found worldwide, and their distribution is consistent with a trade-off between resistance during viral outbreaks and a cost of resistance otherwise, leading to negative frequency-dependent selection.

Population-level annotation of lncRNAs in Arabidopsis reveals extensive expression variation associated with transposable element-like silencing.

Long noncoding RNAs (lncRNAs) are understudied and underannotated in plants. In mammals, lncRNA loci are nearly as ubiquitous as protein-coding genes, and their expression is highly variable between individuals of the same species. Using Arabidopsis thaliana as a model, we aimed to elucidate the true scope of lncRNA transcription across plants from different regions and study its natural variation. We used transcriptome deep sequencing data sets spanning hundreds of natural accessions and several developmental stages to create a population-wide annotation of lncRNAs, revealing thousands of previously unannotated lncRNA loci. While lncRNA transcription is ubiquitous in the genome, most loci appear to be actively silenced and their expression is extremely variable between natural accessions. This high expression variability is largely caused by the high variability of repressive chromatin levels at lncRNA loci. High variability was particularly common for intergenic lncRNAs (lincRNAs), where pieces of transposable elements (TEs) present in 50% of these lincRNA loci are associated with increased silencing and variation, and such lncRNAs tend to be targeted by the TE silencing machinery. We created a population-wide lncRNA annotation in Arabidopsis and improve our understanding of plant lncRNA genome biology, raising fundamental questions about what causes transcription and silencing across the genome.

Cell cycle status of male and female gametes during Arabidopsis reproduction.

Fertilization in Arabidopsis (Arabidopsis thaliana) is a highly coordinated process that begins with a pollen tube delivering the 2 sperm cells into the embryo sac. Each sperm cell can then fertilize either the egg or the central cell to initiate embryo or endosperm development, respectively. The success of this double fertilization process requires a tight cell cycle synchrony between the male and female gametes to allow karyogamy (nuclei fusion). However, the cell cycle status of the male and female gametes during fertilization remains elusive as DNA quantification and DNA replication assays have given conflicting results. Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all phases of the cell cycle. We show that male and female Arabidopsis gametes are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.

Long noncoding RNAs contribute to DNA damage resistance in Arabidopsis thaliana.

Efficient repair of DNA lesions is essential for the faithful transmission of genetic information between somatic cells and for genome integrity across generations. Plants have multiple, partially redundant, and overlapping DNA repair pathways, probably due to the less constricted germline and the inevitable exposure to light including higher energy wavelengths. Many proteins involved in DNA repair and their mode of actions are well described. In contrast, a role for DNA damage-associated RNA components, evident from many other organisms, is less well understood. Here, we have challenged young Arabidopsis thaliana plants with two different types of genotoxic stress and performed de novo assembly and transcriptome analysis. We identified three long noncoding RNAs (lncRNAs) that are lowly or not expressed under regular conditions but up-regulated or induced by DNA damage. We generated CRISPR/Cas deletion mutants and found that the absence of the lncRNAs impairs the recovery capacity of the plants from genotoxic stress. The genetic loci are highly conserved among world-wide distributed Arabidopsis accessions and within related species in the Brassicaceae group. Together, these results suggest that the lncRNAs have a conserved function in connection with DNA damage and provide a basis for mechanistic analysis of their role.

Metabolome plasticity in 241 Arabidopsis thaliana accessions reveals evolutionary cold adaptation processes.

Acclimation and adaptation of metabolism to a changing environment are key processes for plant survival and reproductive success. In the present study, 241 natural accessions of Arabidopsis (Arabidopsis thaliana) were grown under two different temperature regimes, 16 °C and 6 °C, and growth parameters were recorded, together with metabolite profiles, to investigate the natural genome × environment effects on metabolome variation. The plasticity of metabolism, which was captured by metabolic distance measures, varied considerably between accessions. Both relative growth rates and metabolic distances were predictable by the underlying natural genetic variation of accessions. Applying machine learning methods, climatic variables of the original growth habitats were tested for their predictive power of natural metabolic variation among accessions. We found specifically habitat temperature during the first quarter of the year to be the best predictor of the plasticity of primary metabolism, indicating habitat temperature as the causal driver of evolutionary cold adaptation processes. Analyses of epigenome- and genome-wide associations revealed accession-specific differential DNA-methylation levels as potentially linked to the metabolome and identified FUMARASE2 as strongly associated with cold adaptation in Arabidopsis accessions. These findings were supported by calculations of the biochemical Jacobian matrix based on variance and covariance of metabolomics data, which revealed that growth under low temperatures most substantially affects the accession-specific plasticity of fumarate and sugar metabolism. Our findings indicate that the plasticity of metabolic regulation is predictable from the genome and epigenome and driven evolutionarily by Arabidopsis growth habitats.

On the causes of gene-body methylation variation in Arabidopsis thaliana.

Gene-body methylation (gbM) refers to sparse CG methylation of coding regions, which is especially prominent in evolutionarily conserved house-keeping genes. It is found in both plants and animals, but is directly and stably (epigenetically) inherited over multiple generations in the former. Studies in Arabidopsis thaliana have demonstrated that plants originating from different parts of the world exhibit genome-wide differences in gbM, which could reflect direct selection on gbM, but which could also reflect an epigenetic memory of ancestral genetic and/or environmental factors. Here we look for evidence of such factors in F2 plants resulting from a cross between a southern Swedish line with low gbM and a northern Swedish line with high gbM, grown at two different temperatures. Using bisulfite-sequencing data with nucleotide-level resolution on hundreds of individuals, we confirm that CG sites are either methylated (nearly 100% methylation across sampled cells) or unmethylated (approximately 0% methylation across sampled cells), and show that the higher level of gbM in the northern line is due to more sites being methylated. Furthermore, methylation variants almost always show Mendelian segregation, consistent with their being directly and stably inherited through meiosis. To explore how the differences between the parental lines could have arisen, we focused on somatic deviations from the inherited state, distinguishing between gains (relative to the inherited 0% methylation) and losses (relative to the inherited 100% methylation) at each site in the F2 generation. We demonstrate that deviations predominantly affect sites that differ between the parental lines, consistent with these sites being more mutable. Gains and losses behave very differently in terms of the genomic distribution, and are influenced by the local chromatin state. We find clear evidence for different trans-acting genetic polymorphism affecting gains and losses, with those affecting gains showing strong environmental interactions (G×E). Direct effects of the environment were minimal. In conclusion, we show that genetic and environmental factors can change gbM at a cellular level, and hypothesize that these factors can also lead to transgenerational differences between individuals via the inclusion of such changes in the zygote. If true, this could explain genographic pattern of gbM with selection, and would cast doubt on estimates of epimutation rates from inbred lines in constant environments.

Cycles of satellite and transposon evolution in Arabidopsis centromeres.

Centromeres are critical for cell division, loading CENH3 or CENPA histone variant nucleosomes, directing kinetochore formation and allowing chromosome segregation1,2. Despite their conserved function, centromere size and structure are diverse across species. To understand this centromere paradox3,4, it is necessary to know how centromeric diversity is generated and whether it reflects ancient trans-species variation or, instead, rapid post-speciation divergence. To address these questions, we assembled 346 centromeres from 66 Arabidopsis thaliana and 2 Arabidopsis lyrata accessions, which exhibited a remarkable degree of intra- and inter-species diversity. A. thaliana centromere repeat arrays are embedded in linkage blocks, despite ongoing internal satellite turnover, consistent with roles for unidirectional gene conversion or unequal crossover between sister chromatids in sequence diversification. Additionally, centrophilic ATHILA transposons have recently invaded the satellite arrays. To counter ATHILA invasion, chromosome-specific bursts of satellite homogenization generate higher-order repeats and purge transposons, in line with cycles of repeat evolution. Centromeric sequence changes are even more extreme in comparison between A. thaliana and A. lyrata. Together, our findings identify rapid cycles of transposon invasion and purging through satellite homogenization, which drive centromere evolution and ultimately contribute to speciation.

Extensive sequence duplication in Arabidopsis revealed by pseudo-heterozygosity.

BACKGROUND: It is apparent that genomes harbor much structural variation that is largely undetected for technical reasons. Such variation can cause artifacts when short-read sequencing data are mapped to a reference genome. Spurious SNPs may result from mapping of reads to unrecognized duplicated regions. Calling SNP using the raw reads of the 1001 Arabidopsis Genomes Project we identified 3.3 million (44%) heterozygous SNPs. Given that Arabidopsis thaliana (A. thaliana) is highly selfing, and that extensively heterozygous individuals have been removed, we hypothesize that these SNPs reflected cryptic copy number variation. RESULTS: The heterozygosity we observe consists of particular SNPs being heterozygous across individuals in a manner that strongly suggests it reflects shared segregating duplications rather than random tracts of residual heterozygosity due to occasional outcrossing. Focusing on such pseudo-heterozygosity in annotated genes, we use genome-wide association to map the position of the duplicates. We identify 2500 putatively duplicated genes and validate them using de novo genome assemblies from six lines. Specific examples included an annotated gene and nearby transposon that transpose together. We also demonstrate that cryptic structural variation produces highly inaccurate estimates of DNA methylation polymorphism. CONCLUSIONS: Our study confirms that most heterozygous SNP calls in A. thaliana are artifacts and suggest that great caution is needed when analyzing SNP data from short-read sequencing. The finding that 10% of annotated genes exhibit copy-number variation, and the realization that neither gene- nor transposon-annotation necessarily tells us what is actually mobile in the genome suggests that future analyses based on independently assembled genomes will be very informative.

Conditional GWAS of non-CG transposon methylation in Arabidopsis thaliana reveals major polymorphisms in five genes.

Genome-wide association studies (GWAS) have revealed that the striking natural variation for DNA CHH-methylation (mCHH; H is A, T, or C) of transposons has oligogenic architecture involving major alleles at a handful of known methylation regulators. Here we use a conditional GWAS approach to show that CHG-methylation (mCHG) has a similar genetic architecture-once mCHH is statistically controlled for. We identify five key trans-regulators that appear to modulate mCHG levels, and show that they interact with a previously identified modifier of mCHH in regulating natural transposon mobilization.

Plant genetic effects on microbial hubs impact host fitness in repeated field trials.

Although complex interactions between hosts and microbial associates are increasingly well documented, we still know little about how and why hosts shape microbial communities in nature. In addition, host genetic effects on microbial communities vary widely depending on the environment, obscuring conclusions about which microbes are impacted and which plant functions are important. We characterized the leaf microbiota of 200 Arabidopsis thaliana genotypes in eight field experiments and detected consistent host effects on specific, broadly distributed microbial species (operational taxonomic unit [OTUs]). Host genetic effects disproportionately influenced central ecological hubs, with heritability of particular OTUs declining with their distance from the nearest hub within the microbial network. These host effects could reflect either OTUs preferentially associating with specific genotypes or differential microbial success within them. Host genetics associated with microbial hubs explained over 10% of the variation in lifetime seed production among host genotypes across sites and years. We successfully cultured one of these microbial hubs and demonstrated its growth-promoting effects on plants in sterile conditions. Finally, genome-wide association mapping identified many putatively causal genes with small effects on the relative abundance of microbial hubs across sites and years, and these genes were enriched for those involved in the synthesis of specialized metabolites, auxins, and the immune system. Using untargeted metabolomics, we corroborate the consistent association between variation in specialized metabolites and microbial hubs across field sites. Together, our results reveal that host genetic variation impacts the microbial communities in consistent ways across environments and that these effects contribute to fitness variation among host genotypes.

Locally adaptive temperature response of vegetative growth in Arabidopsis thaliana.

We investigated early vegetative growth of natural Arabidopsis thaliana accessions in cold, nonfreezing temperatures, similar to temperatures these plants naturally encounter in fall at northern latitudes. We found that accessions from northern latitudes produced larger seedlings than accessions from southern latitudes, partly as a result of larger seed size. However, their subsequent vegetative growth when exposed to colder temperatures was slower. The difference was too large to be explained by random population differentiation, and is thus suggestive of local adaptation, a notion that is further supported by substantial transcriptome and metabolome changes in northern accessions. We hypothesize that the reduced growth of northern accessions is an adaptive response and a consequence of reallocating resources toward cold acclimation and winter survival.

Gradual evolution of allopolyploidy in Arabidopsis suecica.

Most diploid organisms have polyploid ancestors. The evolutionary process of polyploidization is poorly understood but has frequently been conjectured to involve some form of 'genome shock', such as genome reorganization and subgenome expression dominance. Here we study polyploidization in Arabidopsis suecica, a post-glacial allopolyploid species formed via hybridization of Arabidopsis thaliana and Arabidopsis arenosa. We generated a chromosome-level genome assembly of A. suecica and complemented it with polymorphism and transcriptome data from all species. Despite a divergence around 6 million years ago (Ma) between the ancestral species and differences in their genome composition, we see no evidence of a genome shock: the A. suecica genome is colinear with the ancestral genomes; there is no subgenome dominance in expression; and transposon dynamics appear stable. However, we find changes suggesting gradual adaptation to polyploidy. In particular, the A. thaliana subgenome shows upregulation of meiosis-related genes, possibly to prevent aneuploidy and undesirable homeologous exchanges that are observed in synthetic A. suecica, and the A. arenosa subgenome shows upregulation of cyto-nuclear processes, possibly in response to the new cytoplasmic environment of A. suecica, with plastids maternally inherited from A. thaliana. These changes are not seen in synthetic hybrids, and thus are likely to represent subsequent evolution.

Revisiting a GWAS peak in Arabidopsis thaliana reveals possible confounding by genetic heterogeneity.

Genome-wide association studies (GWAS) have become a standard approach for exploring the genetic basis of phenotypic variation. However, correlation is not causation, and only a tiny fraction of all associations have been experimentally confirmed. One practical problem is that a peak of association does not always pinpoint a causal gene, but may instead be tagging multiple causal variants. In this study, we reanalyze a previously reported peak associated with flowering time traits in Swedish Arabidopsis thaliana population. The peak appeared to pinpoint the AOP2/AOP3 cluster of glucosinolate biosynthesis genes, which is known to be responsible for natural variation in herbivore resistance. Here we propose an alternative hypothesis, by demonstrating that the AOP2/AOP3 flowering association can be wholly accounted for by allelic variation in two flanking genes with clear roles in regulating flowering: NDX1, a regulator of the main flowering time controller FLC, and GA1, which plays a central role in gibberellin synthesis and is required for flowering under some conditions. In other words, we propose that the AOP2/AOP3 flowering-time association may be yet another example of a spurious, "synthetic" association, arising from trying to fit a single-locus model in the presence of two statistically associated causative loci. We conclude that caution is needed when using GWAS for fine-mapping.

Global Genetic Heterogeneity in Adaptive Traits.

Understanding the genetic architecture of complex traits is a major objective in biology. The standard approach for doing so is genome-wide association studies (GWAS), which aim to identify genetic polymorphisms responsible for variation in traits of interest. In human genetics, consistency across studies is commonly used as an indicator of reliability. However, if traits are involved in adaptation to the local environment, we do not necessarily expect reproducibility. On the contrary, results may depend on where you sample, and sampling across a wide range of environments may decrease the power of GWAS because of increased genetic heterogeneity. In this study, we examine how sampling affects GWAS in the model plant species Arabidopsis thaliana. We show that traits like flowering time are indeed influenced by distinct genetic effects in local populations. Furthermore, using gene expression as a molecular phenotype, we show that some genes are globally affected by shared variants, whereas others are affected by variants specific to subpopulations. Remarkably, the former are essentially all cis-regulated, whereas the latter are predominately affected by trans-acting variants. Our result illustrate that conclusions about genetic architecture can be extremely sensitive to sampling and population structure.

Arabidopsis shoot stem cells display dynamic transcription and DNA methylation patterns.

In plants, aerial organs originate continuously from stem cells in the center of the shoot apical meristem. Descendants of stem cells in the subepidermal layer are progenitors of germ cells, giving rise to male and female gametes. In these cells, mutations, including insertions of transposable elements or viruses, must be avoided to preserve genome integrity across generations. To investigate the molecular characteristics of stem cells in Arabidopsis, we isolated their nuclei and analyzed stage-specific gene expression and DNA methylation in plants of different ages. Stem cell expression signatures are largely defined by developmental stage but include a core set of stem cell-specific genes, among which are genes implicated in epigenetic silencing. Transiently increased expression of transposable elements in meristems prior to flower induction correlates with increasing CHG methylation during development and decreased CHH methylation, before stem cells enter the reproductive lineage. These results suggest that epigenetic reprogramming may occur at an early stage in this lineage and could contribute to genome protection in stem cells during germline development.

Adaptive reduction of male gamete number in the selfing plant Arabidopsis thaliana.

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.

AraPheno and the AraGWAS Catalog 2020: a major database update including RNA-Seq and knockout mutation data for Arabidopsis thaliana.

Genome-wide association studies (GWAS) are integral for studying genotype-phenotype relationships and gaining a deeper understanding of the genetic architecture underlying trait variation. A plethora of genetic associations between distinct loci and various traits have been successfully discovered and published for the model plant Arabidopsis thaliana. This success and the free availability of full genomes and phenotypic data for more than 1,000 different natural inbred lines led to the development of several data repositories. AraPheno (https://arapheno.1001genomes.org) serves as a central repository of population-scale phenotypes in A. thaliana, while the AraGWAS Catalog (https://aragwas.1001genomes.org) provides a publicly available, manually curated and standardized collection of marker-trait associations for all available phenotypes from AraPheno. In this major update, we introduce the next generation of both platforms, including new data, features and tools. We included novel results on associations between knockout-mutations and all AraPheno traits. Furthermore, AraPheno has been extended to display RNA-Seq data for hundreds of accessions, providing expression information for over 28 000 genes for these accessions. All data, including the imputed genotype matrix used for GWAS, are easily downloadable via the respective databases.

Common alleles of CMT2 and NRPE1 are major determinants of CHH methylation variation in Arabidopsis thaliana.

DNA cytosine methylation is an epigenetic mark associated with silencing of transposable elements (TEs) and heterochromatin formation. In plants, it occurs in three sequence contexts: CG, CHG, and CHH (where H is A, T, or C). The latter does not allow direct inheritance of methylation during DNA replication due to lack of symmetry, and methylation must therefore be re-established every cell generation. Genome-wide association studies (GWAS) have previously shown that CMT2 and NRPE1 are major determinants of genome-wide patterns of TE CHH methylation. Here we instead focus on CHH methylation of individual TEs and TE-families, allowing us to identify the pathways involved in CHH methylation simply from natural variation and confirm the associations by comparing them with mutant phenotypes. Methylation at TEs targeted by the RNA-directed DNA methylation (RdDM) pathway is unaffected by CMT2 variation, but is strongly affected by variation at NRPE1, which is largely responsible for the longitudinal cline in this phenotype. In contrast, CMT2-targeted TEs are affected by both loci, which jointly explain 7.3% of the phenotypic variation (13.2% of total genetic effects). There is no longitudinal pattern for this phenotype, however, because the geographic patterns appear to compensate for each other in a pattern suggestive of stabilizing selection.