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The mechanisms underlying trait conservation over long evolutionary time scales are poorly known. These mechanisms fall into the two broad and nonmutually exclusive categories of constraint and selection. A variety of factors have been hypothesized to constrain trait evolution. Alternatively, selection can maintain similar trait values across many species if the causes of selection are also relatively conserved, while many sources of constraint may be overcome over longer periods of evolutionary divergence. An example of deep trait conservation is tetradynamy in the large family Brassicaceae, where the four medial stamens are longer than the two lateral stamens. Previous work has found selection to maintain this difference in lengths, which we call anther separation, in wild radish,Raphanus raphanistrum.

Here, we test the constraint hypothesis using five generations of artificial selection to reduce anther separation in wild radish.

We found a rapid linear response to this selection, with no evidence for depletion of genetic variation and correlated responses to this selection in only four of 15 other traits, suggesting a lack of strong constraint.

Taken together, available evidence suggests that tetradynamy is likely to be conserved due to selection, but the function of this trait remains unclear.

 

The study of adaptation helps explain biodiversity and predict future evolution. Yet the process of adaptation can be difficult to observe due to limited phenotypic variation in contemporary populations. Furthermore, the scarcity of male fitness estimates has made it difficult to both understand adaptation and evaluate sexual conflict hypotheses. We addressed both issues in our study of two anther position traits in wild radish (Raphanus raphanistrum): anther exsertion (long filament − corolla tube lengths) and anther separation (long − short filament lengths). These traits affect pollination efficiency and are particularly interesting due to the unusually high correlations among their component traits. We measured selection through male and female fitness on wild radish plants from populations artificially selected to recreate ancestral variation in each anther trait. We found little evidence for conflicts between male and female function. We found strong evidence for stabilizing selection on anther exsertion and disruptive selection on anther separation, indicating positive and negative correlational selection on the component traits. Intermediate levels of exsertion are likely an adaptation to best contact small bees. The function of anther separation is less clear, but future studies might investigate pollen placement on pollinators and compare species possessing multiple stamen types.

 

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

 

Revealing the contributions of genes to plant phenotype is frequently challenging because loss‐of‐function effects may be subtle or masked by varying degrees of genetic redundancy. Such effects can potentially be detected by measuring plant fitness, which reflects the cumulative effects of genetic changes over the lifetime of a plant. However, fitness is challenging to measure accurately, particularly in species with high fecundity and relatively small propagule sizes such asArabidopsis thaliana.

An image segmentation‐based method using the software ImageJ and an object detection‐based method using the Faster Region‐based Convolutional Neural Network (R‐CNN) algorithm were used for measuring two Arabidopsis fitness traits: seed and fruit counts.

The segmentation‐based method was error‐prone (correlation between true and predicted seed counts,r² = 0.849) because seeds touching each other were undercounted. By contrast, the object detection‐based algorithm yielded near perfect seed counts (r² = 0.9996) and highly accurate fruit counts (r² = 0.980). Comparing seed counts for wild‐type and 12 mutant lines revealed fitness effects for three genes; fruit counts revealed the same effects for two genes.

Our study provides analysis pipelines and models to facilitate the investigation of Arabidopsis fitness traits and demonstrates the importance of examining fitness traits when studying gene functions.

 

Plant metabolites from diverse pathways are important for plant survival, human nutrition and medicine. The pathway memberships of most plant enzyme genes are unknown. While co‐expression is useful for assigning genes to pathways, expression correlation may exist only under specific spatiotemporal and conditional contexts.

Utilising > 600 tomato (Solanum lycopersicum) expression data combinations, three strategies for predicting memberships in 85 pathways were explored.

Optimal predictions for different pathways require distinct data combinations indicative of pathway functions. Naive prediction (i.e. identifying pathways with the most similarly expressed genes) is error prone. In 52 pathways, unsupervised learning performed better than supervised approaches, possibly due to limited training data availability. Using gene‐to‐pathway expression similarities led to prediction models that outperformed those based simply on expression levels. Using 36 experimental validated genes, the pathway‐best model prediction accuracy is 58.3%, significantly better compared with that for predicting annotated genes without experimental evidence (37.0%) or random guess (1.2%), demonstrating the importance of data quality.

Our study highlights the need to extensively explore expression‐based features and prediction strategies to maximise the accuracy of metabolic pathway membership assignment. The prediction framework outlined here can be applied to other species and serves as a baseline model for future comparisons.

 

Phenotypic plasticity is the primary mechanism of organismal resilience to abiotic and biotic stress, and genetic differentiation in plasticity can evolve if stresses differ among populations. Inducible defence is a common form of adaptive phenotypic plasticity, and long‐standing theory predicts that its evolution is shaped by costs of the defensive traits, costs of plasticity and a trade‐off in allocation to constitutive versus induced traits. We used a common garden to study the evolution of defence in two native populations of wild arugulaEruca sativa(Brassicaceae) from contrasting desert and Mediterranean habitats that differ in attack by caterpillars and aphids. We report genetic differentiation and additive genetic variance for phenology, growth and three defensive traits (toxic glucosinolates, anti‐nutritive protease inhibitors and physical trichome barriers) as well their inducibility in response to the plant hormone jasmonic acid. The two populations were strongly differentiated for plasticity in nearly all traits. There was little evidence for costs of defence or plasticity, but constitutive and induced traits showed a consistent additive genetic trade‐off within each population for the three defensive traits. We conclude that these populations have evolutionarily diverged in inducible defence and retain ample potential for the future evolution of phenotypic plasticity in defence.

 

Extensive transcriptional activity occurring in intergenic regions of genomes has raised the question whether intergenic transcription represents the activity of novel genes or noisy expression. To address this, we evaluated cross-species and post-duplication sequence and expression conservation of intergenic transcribed regions (ITRs) in four Poaceae species. Among 43,301 ITRs across the four species, 34,460 (80%) are species-specific. ITRs found across species tend to be more divergent in expression and have more recent duplicates compared to annotated genes. To assess if ITRs are functional (under selection), machine learning models were established inOryza sativa(rice) that could accurately distinguish between phenotype genes and pseudogenes (area under curve-receiver operating characteristic = 0.94). Based on the models, 584 (8%) and 4391 (61%) rice ITRs are classified as likely functional and nonfunctional with high confidence, respectively. ITRs with conserved expression and ancient retained duplicates, features that were not part of the model, are frequently classified as likely-functional, suggesting these characteristics could serve as pragmatic rules of thumb for identifying candidate sequences likely to be under selection. This study also provides a framework to identify novel genes using comparative transcriptomic data to improve genome annotation that is fundamental for connecting genotype to phenotype in crop and model systems.

 

Abstract Background Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively. Results To understand what the causes may be for such uneven coverage, we first established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have higher simple sequence repeat and tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available tomato long-read based assembly and found that 27.8% (1.41 Mb) of high coverage regions were potentially misassembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a predictive model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements. Conclusions Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to plant genome misassembly when using short reads and the generality of these causes and factors should be tested further in other species.