Plant genome evolution in the genus Eucalyptus is driven by structural rearrangements that promote sequence divergence.

Genomes have a highly organized architecture (nonrandom organization of functional and nonfunctional genetic elements within chromosomes) that is essential for many biological functions, particularly gene expression and reproduction. Despite the need to conserve genome architecture, a high level of structural variation has been observed within species. As species separate and diverge, genome architecture also diverges, becoming increasingly poorly conserved as divergence time increases. However, within plant genomes, the processes of genome architecture divergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically diverse, wild and naturally evolving Eucalyptus species, covering 1-50 million years of diverging genome evolution to measure genome architectural conservation and describe architectural divergence. The investigation of these genomes revealed that following lineage divergence, genome architecture is highly fragmented by rearrangements. As genomes continue to diverge, the accumulation of mutations and the subsequent divergence beyond recognition of rearrangements become the primary driver of genome divergence. The loss of syntenic regions also contribute to genome divergence but at a slower pace than that of rearrangements. We hypothesize that duplications and translocations are potentially the greatest contributors to Eucalyptus genome divergence.

Genomic determinants, architecture, and constraints in drought-related traits in Corymbia calophylla.

BACKGROUND: Drought adaptation is critical to many tree species persisting under climate change, however our knowledge of the genetic basis for trees to adapt to drought is limited. This knowledge gap impedes our fundamental understanding of drought response and application to forest production and conservation. To improve our understanding of the genomic determinants, architecture, and trait constraints, we assembled a reference genome and detected ~ 6.5 M variants in 432 phenotyped individuals for the foundational tree Corymbia calophylla. RESULTS: We found 273 genomic variants determining traits with moderate heritability (h2SNP = 0.26-0.64). Significant variants were predominantly in gene regulatory elements distributed among several haplotype blocks across all chromosomes. Furthermore, traits were constrained by frequent epistatic and pleiotropic interactions. CONCLUSIONS: Our results on the genetic basis for drought traits in Corymbia calophylla have several implications for the ability to adapt to climate change: (1) drought related traits are controlled by complex genomic architectures with large haplotypes, epistatic, and pleiotropic interactions; (2) the most significant variants determining drought related traits occurred in regulatory regions; and (3) models incorporating epistatic interactions increase trait predictions. Our findings indicate that despite moderate heritability drought traits are likely constrained by complex genomic architecture potentially limiting trees response to climate change.

Stabilisation of soil organic matter with rock dust partially counteracted by plants.

Soil application of Ca- and Mg-rich silicates can capture and store atmospheric carbon dioxide as inorganic carbon but could also have the potential to stabilise soil organic matter (SOM). Synergies between these two processes have not been investigated. Here, we apply finely ground silicate rock mining residues (basalt and granite blend) to a loamy sand in a pot trial at a rate of 4% (equivalent to 50 t ha-1 ) and investigate the effects of a wheat plant and two watering regimes on soil carbon sequestration over the course of 6 months. Rock dust addition increased soil pH, electric conductivity, inorganic carbon content and soil-exchangeable Ca and Mg contents, as expected for weathering. However, it decreased exchangeable levels of micronutrients Mn and Zn, likely related to the elevated soil pH. Importantly, it increased mineral-associated organic matter by 22% due to the supply of secondary minerals and associated sites for SOM sorption. Additionally, in the nonplanted treatments, rock supply of Ca and Mg increased soil microaggregation that subsequently stabilised labile particulate organic matter as organic matter occluded in aggregates by 46%. Plants, however, reduced soil-exchangeable Mg and Ca contents and hence counteracted the silicate rock effect on microaggregates and carbon within. We suggest this cation loss might be attributed to plant exudates released to solubilise micronutrients and hence neutralise plant deficiencies. The effect of enhanced silicate rock weathering on SOM stabilisation could substantially boost its carbon sequestration potential.

Interspecies genome divergence is predominantly due to frequent small scale rearrangements in Eucalyptus.

Synteny, the ordering of sequences within homologous chromosomes, must be maintained within the genomes of sexually reproducing species for the sharing of alleles and production of viable, reproducing offspring. However, when the genomes of closely related species are compared, a loss of synteny is often observed. Unequal homologous recombination is the primary mechanism behind synteny loss, occurring more often in transposon rich regions, and resulting in the formation of chromosomal rearrangements. To examine patterns of synteny among three closely related, interbreeding, and wild Eucalyptus species, we assembled their genomes using long-read DNA sequencing and de novo assembly. We identify syntenic and rearranged regions between these genomes and estimate that ~48% of our genomes remain syntenic while ~36% is rearranged. We observed that rearrangements highly fragment microsynteny. Our results suggest that synteny between these species is primarily lost through small-scale rearrangements, not through sequence loss, gain, or sequence divergence. Further examination of identified rearrangements suggests that rearrangements may be altering the phenotypes of Eucalyptus species. Our study also underscores that the use of single reference genomes in genomic variation studies could lead to reference bias, especially given the scale at which we show potentially adaptive loci have highly diverged, deleted, duplicated and/or rearranged. This study provides an unbiased framework to look at potential speciation and adaptive loci among a rapidly radiating foundation species of woodland trees that are free from selective breeding seen in most crop species.

GWAS on multiple traits identifies mitochondrial ACONITASE3 as important for acclimation to submergence stress.

Flooding causes severe crop losses in many parts of the world. Genetic variation in flooding tolerance exists in many species; however, there are few examples for the identification of tolerance genes and their underlying function. We conducted a genome-wide association study (GWAS) in 387 Arabidopsis (Arabidopsis thaliana) accessions. Plants were subjected to prolonged submergence followed by desubmergence, and seven traits (score, water content, Fv/Fm, and concentrations of nitrate, chlorophyll, protein, and starch) were quantified to characterize their acclimation responses. These traits showed substantial variation across the range of accessions. A total of 35 highly significant single-nucleotide polymorphisms (SNPs) were identified across the 20 GWA datasets, pointing to 22 candidate genes, with functions in TCA cycle, DNA modification, and cell division. Detailed functional characterization of one candidate gene, ACONITASE3 (ACO3), was performed. Chromatin immunoprecipitation followed by sequencing showed that a single nucleotide polymorphism in the ACO3 promoter co-located with the binding site of the master regulator of retrograde signaling ANAC017, while subcellular localization of an ACO3-YFP fusion protein confirmed a mitochondrial localization during submergence. Analysis of mutant and overexpression lines determined changes in trait parameters that correlated with altered submergence tolerance and were consistent with the GWAS results. Subsequent RNA-seq experiments suggested that impairing ACO3 function increases the sensitivity to submergence by altering ethylene signaling, whereas ACO3 overexpression leads to tolerance by metabolic priming. These results indicate that ACO3 impacts submergence tolerance through integration of carbon and nitrogen metabolism via the mitochondrial TCA cycle and impacts stress signaling during acclimation to stress.

Genomic consequences of artificial selection during early domestication of a wood fibre crop.

From its origins in Australia, Eucalyptus grandis has spread to every continent, except Antarctica, as a wood crop. It has been cultivated and bred for over 100 yr in places such as South Africa. Unlike most annual crops and fruit trees, domestication of E. grandis is still in its infancy, representing a unique opportunity to interrogate the genomic consequences of artificial selection early in the domestication process. To determine how a century of artificial selection has changed the genome of E. grandis, we generated single nucleotide polymorphism genotypes for 1080 individuals from three advanced South African breeding programmes using the EUChip60K chip, and investigated population structure and genome-wide differentiation patterns relative to wild progenitors. Breeding and wild populations appeared genetically distinct. We found genomic evidence of evolutionary processes known to have occurred in other plant domesticates, including interspecific introgression and intraspecific infusion from wild material. Furthermore, we found genomic regions with increased linkage disequilibrium and genetic differentiation, putatively representing early soft sweeps of selection. This is, to our knowledge, the first study of genomic signatures of domestication in a timber species looking beyond the first few generations of cultivation. Our findings highlight the importance of intra- and interspecific hybridization during early domestication.

Genomic evidence of introgression and adaptation in a model subtropical tree species, Eucalyptus grandis.

The genetic consequences of adaptation to changing environments can be deciphered using population genomics, which may help predict species' responses to global climate change. Towards this, we used genome-wide SNP marker analysis to determine population structure and patterns of genetic differentiation in terms of neutral and adaptive genetic variation in the natural range of Eucalyptus grandis, a widely cultivated subtropical and temperate species, serving as genomic reference for the genus. We analysed introgression patterns at subchromosomal resolution using a modified ancestry mapping approach and identified provenances with extensive interspecific introgression in response to increased aridity. Furthermore, we describe potentially adaptive genetic variation as explained by environment-associated SNP markers, which also led to the discovery of what is likely a large structural variant. Finally, we show that genes linked to these markers are enriched for biotic and abiotic stress responses.

Spatial, climate and ploidy factors drive genomic diversity and resilience in the widespread grass Themeda triandra.

Global climate change poses a significant threat to natural communities around the world, with many plant species showing signs of climate stress. Grassland ecosystems are not an exception, with climate change compounding contemporary pressures such as habitat loss and fragmentation. In this study, we assess the climate resilience of Themeda triandra, a foundational species and the most widespread plant in Australia, by assessing the relative contributions of spatial, environmental and ploidy factors to contemporary genomic variation. Reduced-representation genome sequencing on 472 samples from 52 locations was used to test how the distribution of genomic variation, including ploidy polymorphism, supports adaptation to hotter and drier climates. We explicitly quantified isolation by distance (IBD) and isolation by environment (IBE) and predicted genomic vulnerability of populations to future climates based on expected deviation from current genomic composition. We found that a majority (54%) of genomic variation could be attributed to IBD, while an additional 22% (27% when including ploidy information) could be explained by two temperature and two precipitation climate variables demonstrating IBE. Ploidy polymorphisms were common within populations (31/52 populations), indicating that ploidy mixing is characteristic of T. triandra populations. Genomic vulnerabilities were found to be heterogeneously distributed throughout the landscape, and our analysis suggested that ploidy polymorphism, along with other factors linked to polyploidy, reduced vulnerability to future climates by 60% (0.25-0.10). Our data suggests that polyploidy may facilitate adaptation to hotter climates and highlight the importance of incorporating ploidy in adaptive management strategies to promote the resilience of this and other foundation species.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.

HOME: a histogram based machine learning approach for effective identification of differentially methylated regions.

BACKGROUND: The development of whole genome bisulfite sequencing has made it possible to identify methylation differences at single base resolution throughout an entire genome. However, a persistent challenge in DNA methylome analysis is the accurate identification of differentially methylated regions (DMRs) between samples. Sensitive and specific identification of DMRs among different conditions requires accurate and efficient algorithms, and while various tools have been developed to tackle this problem, they frequently suffer from inaccurate DMR boundary identification and high false positive rate. RESULTS: We present a novel Histogram Of MEthylation (HOME) based method that takes into account the inherent difference in the distribution of methylation levels between DMRs and non-DMRs to discriminate between the two using a Support Vector Machine. We show that generated features used by HOME are dataset-independent such that a classifier trained on, for example, a mouse methylome training set of regions of differentially accessible chromatin, can be applied to any other organism's dataset and identify accurate DMRs. We demonstrate that DMRs identified by HOME exhibit higher association with biologically relevant genes, processes, and regulatory events compared to the existing methods. Moreover, HOME provides additional functionalities lacking in most of the current DMR finders such as DMR identification in non-CG context and time series analysis. HOME is freely available at https://github.com/ListerLab/HOME . CONCLUSION: HOME produces more accurate DMRs than the current state-of-the-art methods on both simulated and biological datasets. The broad applicability of HOME to identify accurate DMRs in genomic data from any organism will have a significant impact upon expanding our knowledge of how DNA methylation dynamics affect cell development and differentiation.

DNA methylation profiles of diverse Brachypodium distachyon align with underlying genetic diversity.

DNA methylation, a common modification of genomic DNA, is known to influence the expression of transposable elements as well as some genes. Although commonly viewed as an epigenetic mark, evidence has shown that underlying genetic variation, such as transposable element polymorphisms, often associate with differential DNA methylation states. To investigate the role of DNA methylation variation, transposable element polymorphism, and genomic diversity, whole-genome bisulfite sequencing was performed on genetically diverse lines of the model cereal Brachypodium distachyon Although DNA methylation profiles are broadly similar, thousands of differentially methylated regions are observed between lines. An analysis of novel transposable element indel variation highlighted hundreds of new polymorphisms not seen in the reference sequence. DNA methylation and transposable element variation is correlated with the genome-wide amount of genetic variation present between samples. However, there was minimal evidence that novel transposon insertions or deletions are associated with nearby differential methylation. This study highlights unique relationships between genetic variation and DNA methylation variation within Brachypodium and provides a valuable map of DNA methylation across diverse resequenced accessions of this model cereal species.

Axe: rapid, competitive sequence read demultiplexing using a trie.

Summary: We describe a rapid algorithm for demultiplexing DNA sequence reads with in-read indices. Axe selects the optimal index present in a sequence read, even in the presence of sequencing errors. The algorithm is able to handle combinatorial indexing, indices of differing length and several mismatches per index sequence. Availability and implementation: Axe is implemented in C, and is used as a command-line program on Unix-like systems. Axe is available online at https://github.com/kdmurray91/axe, and is available in Debian/Ubuntu distributions of GNU/Linux as the package axe-demultiplexer. Supplementary information: Supplementary data are available at Bioinformatics online.

Landscape drivers of genomic diversity and divergence in woodland Eucalyptus.

Spatial genetic patterns are influenced by numerous factors, and they can vary even among coexisting, closely related species due to differences in dispersal and selection. Eucalyptus (L'Héritier 1789; the "eucalypts") are foundation tree species that provide essential habitat and modulate ecosystem services throughout Australia. Here we present a study of landscape genomic variation in two woodland eucalypt species, using whole-genome sequencing of 388 individuals of Eucalyptus albens and Eucalyptus sideroxylon. We found exceptionally high genetic diversity (π ≈ 0.05) and low genome-wide, interspecific differentiation (FST  = 0.15) and intraspecific differentiation between localities (FST  ≈ 0.01-0.02). We found no support for strong, discrete population structure, but found substantial support for isolation by geographic distance (IBD) in both species. Using generalized dissimilarity modelling, we identified additional isolation by environment (IBE). Eucalyptus albens showed moderate IBD, and environmental variables have a small but significant amount of additional predictive power (i.e. IBE). Eucalyptus sideroxylon showed much stronger IBD and moderate IBE. These results highlight the vast adaptive potential of these species and set the stage for testing evolutionary hypotheses of interspecific adaptive differentiation across environments.

Adaptation genomics: The angel is in the details.

This article comments on: Genome-wide signatures of flowering adaptation to climate temperature: Regional analyses in a highly diverse native range of Arabidopsis thaliana.

kWIP: The k-mer weighted inner product, a de novo estimator of genetic similarity.

Modern genomics techniques generate overwhelming quantities of data. Extracting population genetic variation demands computationally efficient methods to determine genetic relatedness between individuals (or "samples") in an unbiased manner, preferably de novo. Rapid estimation of genetic relatedness directly from sequencing data has the potential to overcome reference genome bias, and to verify that individuals belong to the correct genetic lineage before conclusions are drawn using mislabelled, or misidentified samples. We present the k-mer Weighted Inner Product (kWIP), an assembly-, and alignment-free estimator of genetic similarity. kWIP combines a probabilistic data structure with a novel metric, the weighted inner product (WIP), to efficiently calculate pairwise similarity between sequencing runs from their k-mer counts. It produces a distance matrix, which can then be further analysed and visualised. Our method does not require prior knowledge of the underlying genomes and applications include establishing sample identity and detecting mix-up, non-obvious genomic variation, and population structure. We show that kWIP can reconstruct the true relatedness between samples from simulated populations. By re-analysing several published datasets we show that our results are consistent with marker-based analyses. kWIP is written in C++, licensed under the GNU GPL, and is available from https://github.com/kdmurray91/kwip.

Development of the first consensus genetic map of intermediate wheatgrass (Thinopyrum intermedium) using genotyping-by-sequencing.

KEY MESSAGE: Development of the first consensus genetic map of intermediate wheatgrass gives insight into the genome and tools for molecular breeding. Intermediate wheatgrass (Thinopyrum intermedium) has been identified as a candidate for domestication and improvement as a perennial grain, forage, and biofuel crop and is actively being improved by several breeding programs. To accelerate this process using genomics-assisted breeding, efficient genotyping methods and genetic marker reference maps are needed. We present here the first consensus genetic map for intermediate wheatgrass (IWG), which confirms the species' allohexaploid nature (2n = 6x = 42) and homology to Triticeae genomes. Genotyping-by-sequencing was used to identify markers that fit expected segregation ratios and construct genetic maps for 13 heterogeneous parents of seven full-sib families. These maps were then integrated using a linear programming method to produce a consensus map with 21 linkage groups containing 10,029 markers, 3601 of which were present in at least two populations. Each of the 21 linkage groups contained between 237 and 683 markers, cumulatively covering 5061 cM (2891 cM--Kosambi) with an average distance of 0.5 cM between each pair of markers. Through mapping the sequence tags to the diploid (2n = 2x = 14) barley reference genome, we observed high colinearity and synteny between these genomes, with three homoeologous IWG chromosomes corresponding to each of the seven barley chromosomes, and mapped translocations that are known in the Triticeae. The consensus map is a valuable tool for wheat breeders to map important disease-resistance genes within intermediate wheatgrass. These genomic tools can help lead to rapid improvement of IWG and development of high-yielding cultivars of this perennial grain that would facilitate the sustainable intensification of agricultural systems.

Genomic diversity guides conservation strategies among rare terrestrial orchid species when taxonomy remains uncertain.

Background and Aims: Species are often used as the unit for conservation, but may not be suitable for species complexes where taxa are difficult to distinguish. Under such circumstances, it may be more appropriate to consider species groups or populations as evolutionarily significant units (ESUs). A population genomic approach was employed to investigate the diversity within and among closely related species to create a more robust, lineage-specific conservation strategy for a nationally endangered terrestrial orchid and its relatives from south-eastern Australia. Methods: Four putative species were sampled from a total of 16 populations in the Victorian Volcanic Plain (VVP) bioregion and one population of a sub-alpine outgroup in south-eastern Australia. Morphological measurements were taken in situ along with leaf material for genotyping by sequencing (GBS) and microsatellite analyses. Key Results: Species could not be differentiated using morphological measurements. Microsatellite and GBS markers confirmed the outgroup as distinct, but only GBS markers provided resolution of population genetic structure. The nationally endangered Diuris basaltica was indistinguishable from two related species ( D. chryseopsis and D. behrii ), while the state-protected D. gregaria showed genomic differentiation. Conclusions: Genomic diversity identified among the four Diuris species suggests that conservation of this taxonomically complex group will be best served by considering them as one ESU rather than separately aligned with species as currently recognized. This approach will maximize evolutionary potential among all species during increased isolation and environmental change. The methods used here can be applied generally to conserve evolutionary processes for groups where taxonomic uncertainty hinders the use of species as conservation units.

Isolation by distance and isolation by environment contribute to population differentiation in Protea repens (Proteaceae L.), a widespread South African species.

PREMISE OF THE STUDY: The Cape Floristic Region (CFR) of South Africa is renowned for its botanical diversity, but the evolutionary origins of this diversity remain controversial. Both neutral and adaptive processes have been implicated in driving diversification, but population-level studies of plants in the CFR are rare. Here, we investigate the limits to gene flow and potential environmental drivers of selection in Protea repens L. (Proteaceae L.), a widespread CFR species. METHODS: We sampled 19 populations across the range of P. repens and used genotyping by sequencing to identify 2066 polymorphic loci in 663 individuals. We used a Bayesian FST outlier analysis to identify single-nucleotide polymorphisms (SNPs) marking genomic regions that may be under selection; we used those SNPs to identify potential drivers of selection and excluded them from analyses of gene flow and genetic structure. RESULTS: A pattern of isolation by distance suggested limited gene flow between nearby populations. The populations of P. repens fell naturally into two or three groupings, which corresponded to an east-west split. Differences in rainfall seasonality contributed to diversification in highly divergent loci, as do barriers to gene flow that have been identified in other species. CONCLUSIONS: The strong pattern of isolation by distance is in contrast to the findings in the only other widespread species in the CFR that has been similarly studied, while the effects of rainfall seasonality are consistent with well-known patterns. Assessing the generality of these results will require investigations of other CFR species.

Population and phylogenomic decomposition via genotyping-by-sequencing in Australian Pelargonium.

Species delimitation has seen a paradigm shift as increasing accessibility of genomic-scale data enables separation of lineages with convergent morphological traits and the merging of recently diverged ecotypes that have distinguishing characteristics. We inferred the process of lineage formation among Australian species in the widespread and highly variable genus Pelargonium by combining phylogenomic and population genomic analyses along with breeding system studies and character analysis. Phylogenomic analysis and population genetic clustering supported seven of the eight currently described species but provided little evidence for differences in genetic structure within the most widely distributed group that containing P. australe. In contrast, morphometric analysis detected three deep lineages within Australian Pelargonium; with P. australe consisting of five previously unrecognized entities occupying separate geographic ranges. The genomic approach enabled elucidation of parallel evolution in some traits formerly used to delineate species, as well as identification of ecotypic morphological differentiation within recognized species. Highly variable morphology and trait convergence each contribute to the discordance between phylogenomic relationships and morphological taxonomy. Data suggest that genetic divergence among species within the Australian Pelargonium may result from allopatric speciation while morphological differentiation within and among species may be more strongly driven by environmental differences.

Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.