Current status of community resources and priorities for weed genomics research.

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Comparative genomics points to tandem duplications of SAD gene clusters as drivers of increased α-linolenic (ω-3) content in S. hispanica seeds.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Assessing the invasive potential of different source populations of ragweed (Ambrosia artemisiifolia L.) through genomically informed species distribution modelling.

The genetic composition of founding populations is likely to play a key role in determining invasion success. Individual genotypes may differ in habitat preference and environmental tolerance, so their ability to colonize novel environments can be highly variable. Despite the importance of genetic variation on invasion success, its influence on the potential distribution of invaders is rarely investigated. Here, we integrate population genomics and ecological niche models (ENMs) into a single framework to predict the distribution of globally invasive common ragweed (Ambrosia artemisiifolia) in Australia. We identified three genetic clusters for ragweed and used these to construct cluster-specific ENMs and characterize within-species niche differentiation. The potential range of ragweed in Australia depended on the genetic composition and continent of origin of the introduced population. Invaders originating from warmer, wetter climates had a broader potential distribution than those from cooler, drier ones. By quantifying this change, we identified source populations most likely to expand the ragweed distribution. As prevention remains the most effective method of invasive species management, our work provides a valuable way of ranking the threat posed by different populations to better inform management decisions.

Heterosis in crop improvement.

Heterosis, also known as hybrid vigor, is the phenomenon wherein a progeny exhibits superior traits relative to one or both parents. In terms of crop breeding, this usually refers to the yield advantage of F1 hybrids over both inbred parents. The development of high-yielding hybrid cultivars across a wider range of crops is key to meeting future food demands. However, conventional hybrid breeding strategies are proving to be exceptionally challenging to apply commercially in many self-pollinating crops, particularly wheat and barley. Currently in these crops, the relative performance advantage of hybrids over inbred line cultivars does not outweigh the cost of hybrid seed production. Here, we review the genetic basis of heterosis, discuss the challenges in hybrid breeding, and propose a strategy to recruit multiple heterosis-associated genes to develop lines with improved agronomic characteristics. This strategy leverages modern genetic engineering tools to synthesize supergenes by fusing multiple heterotic alleles across multiple heterosis-associated loci. We outline a plan to assess the feasibility of this approach to improve line performance using barley (Hordeum vulgare) as the model self-pollinating crop species, and a few heterosis-associated genes. The proposed method can be applied to all crops for which heterotic gene combinations can be identified.

Compare_Genomes: A Comparative Genomics Workflow to Streamline the Analysis of Evolutionary Divergence Across Eukaryotic Genomes.

The dawn of cost-effective genome assembly is enabling deep comparative genomics to address fundamental evolutionary questions by comparing the genomes of multiple species. However, comparative genomics analyses frequently deploy multiple, often purpose-built frameworks, limiting their transferability and replicability. Here, we present compare_genomes, a transferable and extensible comparative genomics workflow package we developed that streamlines the identification of orthologous families within and across eukaryotic genomes and tests for the presence of several mechanisms of evolution (gene family expansion or contraction and substitution rates within protein-coding sequences). The workflow is available for Linux, written as a Nextflow workflow that calls established genomics and phylogenetics tools to streamline the analysis and visualization of eukaryotic genome divergence. This workflow is freely available at https://github.com/jeffersonfparil/compare_genomes, distributed under the GNU General Public License version 3 (GPLv3). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Comparative genomics with Nextflow and Conda.

Forecasting trait responses in novel environments to aid seed provenancing under climate change.

Revegetation projects face the major challenge of sourcing optimal plant material. This is often done with limited information about plant performance and increasingly requires factoring resilience to climate change. Functional traits can be used as quantitative indices of plant performance and guide seed provenancing, but trait values expected under novel conditions are often unknown. To support climate-resilient provenancing efforts, we develop a trait prediction model that integrates the effect of genetic variation with fine-scale temperature variation. We train our model on multiple field plantings of Arabidopsis thaliana and predict two relevant fitness traits-days-to-bolting and fecundity-across the species' European range. Prediction accuracy was high for days-to-bolting and moderate for fecundity, with the majority of trait variation explained by temperature differences between plantings. Projection under future climate predicted a decline in fecundity, although this response was heterogeneous across the range. In response, we identified novel genotypes that could be introduced to genetically offset the fitness decay. Our study highlights the value of predictive models to aid seed provenancing and improve the success of revegetation projects.

Rounding up the annual ryegrass genome: High-quality reference genome of Lolium rigidum.

The genome of the major agricultural weed species, annual ryegrass (Lolium rigidum) was assembled, annotated and analysed. Annual ryegrass is a major weed in grain cropping, and has the remarkable capacity to evolve resistance to herbicides with various modes of action. The chromosome-level assembly was achieved using short- and long-read sequencing in combination with Hi-C mapping. The assembly size is 2.44 Gb with N50 = 361.79 Mb across 1,764 scaffolds where the seven longest sequences correspond to the seven chromosomes. Genome completeness assessed through BUSCO returned a 99.8% score for complete (unique and duplicated) and fragmented genes using the Viridiplantae set. We found evidence for the expansion of herbicide resistance-related gene families including detoxification genes. The reference genome of L. rigidum is a critical asset for leveraging genetic information for the management of this highly problematic weed species.

Pollen competition in hybridizing Cakile species: How does a latecomer win the race?

PREMISE: Hybridization between cross-compatible species depends on the extent of competition between alternative mates. Even if stigmatic compatibility allows for hybridization, hybridization requires the heterospecific pollen to be competitive. Here, we determined whether conspecific pollen has an advantage in the race to fertilize ovules and the potential handicap to be overcome by heterospecific pollen in invasive Cakile species. METHODS: We used fluorescence microscopy to measure pollen tube growth after conspecific and heterospecific hand-pollination treatments. We then determined siring success in the progeny relative to the timing of heterospecific pollen arrival on the stigma using CAPS markers. RESULTS: In the absence of pollen competition, pollination time and pollen recipient species had a significant effect on the ratio of pollen tube growth. In long-styled C. maritima (outcrosser), pollen tubes grew similarly in both directions. In short-styled C. edentula (selfer), conspecific and heterospecific pollen tubes grew differently. Cakile edentula pollen produced more pollen tubes, revealing the potential for a mating asymmetry whereby C. edentula pollen had an advantage relative to C. maritima. In the presence of pollen competition, siring success was equivalent when pollen deposition was synchronous. However, a moderate 1-h advantage in the timing of conspecific pollination resulted in almost complete assortative mating, while an equivalent delay in conspecific pollination resulted in substantial hybrid formation. CONCLUSIONS: Hybridization can aid the establishment of invasive species through the transfer of adaptive alleles from cross-compatible species, but also lead to extinction through demographic or genetic swamping. Time of pollen arrival on the stigma substantially affected hybridization rate, pointing to the importance of pollination timing in driving introgression and genetic swamping.

Phytochemical and Safety Evaluations of Finger Lime, Mountain Pepper, and Tamarind in Zebrafish Embryos.

Plants play a pivotal role in drug discovery, constituting 50% of modern pharmacopeia. Many human diseases, including age-related degenerative diseases, converge onto common cellular oxidative stress pathways. This provides an opportunity to develop broad treatments to treat a wide range of diseases in the ageing population. Here, we characterize and assess the toxicological effects of finger lime (Citrus australasica), mountain pepper (Tasmannia lanceolata), and small-leaved tamarind (Diploglottis australis) extracts. The characterization demonstrates that these Australian native plants have antioxidant potential and, importantly, they have high concentrations of distinct combinations of different antioxidant classes. Using zebrafish larvae as a high-throughput pre-clinical in vivo toxicology screening model, our experiment effectively discriminates which of these extracts (and at what exposure levels) are suitable for development towards future therapies. The LC50-96h for finger lime and tamarind were >480 mg/L, and 1.70 mg/L for mountain pepper. Critically, this work shows that adverse effects are not correlated to the properties of these antioxidants, thus highlighting the need for combining characterization and in vivo screening to identify the most promising plant extracts for further development. Thus, we present a high-throughput pre-clinical screening that robustly tests natural plant products to utilize the diversity of antioxidant compounds for drug development.

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.

Adaptive significance of flowering time variation across natural seasonal environments in Arabidopsis thaliana.

The relevance of flowering time variation and plasticity to climate adaptation requires a comprehensive empirical assessment. We investigated natural selection and the genetic architecture of flowering time in Arabidopsis through field experiments in Europe across multiple sites and seasons. We estimated selection for flowering time, plasticity and canalization. Loci associated with flowering time, plasticity and canalization by genome-wide association studies were tested for a geographic signature of climate adaptation. Selection favored early flowering and increased canalization, except at the northernmost site, but was rarely detected for plasticity. Genome-wide association studies revealed significant associations with flowering traits and supported a substantial polygenic inheritance. Alleles associated with late flowering, including functional FRIGIDA variants, were more common in regions experiencing high annual temperature variation. Flowering time plasticity to fall vs spring and summer environments was associated with GIGANTEA SUPPRESSOR 5, which promotes early flowering under decreasing day length and temperature. The finding that late flowering genotypes and alleles are associated with climate is evidence for past adaptation. Real-time phenotypic selection analysis, however, reveals pervasive contemporary selection for rapid flowering in agricultural settings across most of the species range. The response to this selection may involve genetic shifts in environmental cuing compared to the ancestral state.

Optimizing sampling design and sequencing strategy for the genomic analysis of quantitative traits in natural populations.

Mapping the genes underlying ecologically relevant traits in natural populations is fundamental to develop a molecular understanding of species adaptation. Current sequencing technologies enable the characterization of a species' genetic diversity across the landscape or even over its whole range. The relevant capture of the genetic diversity across the landscape is critical for a successful genetic mapping of traits and there are no clear guidelines on how to achieve an optimal sampling and which sequencing strategy to implement. Here we determine, through simulation, the sampling scheme that maximizes the power to map the genetic basis of a complex trait in an outbreeding species across an idealized landscape and draw genomic predictions for the trait, comparing individual and pool sequencing strategies. Our results show that quantitative trait locus detection power and prediction accuracy are higher when more populations over the landscape are sampled and this is more cost-effectively done with pool sequencing than with individual sequencing. Additionally, we recommend sampling populations from areas of high genetic diversity. As progress in sequencing enables the integration of trait-based functional ecology into landscape genomics studies, these findings will guide study designs allowing direct measures of genetic effects in natural populations across the environment.

Diverse phosphate and auxin transport loci distinguish phosphate tolerant from sensitive Arabidopsis accessions.

Phosphorus (P) is an essential element for plant growth often limiting agroecosystems. To identify genetic determinants of performance under variable phosphate (Pi) supply, we conducted genome-wide association studies on five highly predictive Pi starvation response traits in 200 Arabidopsis (Arabidopsis thaliana) accessions. Pi concentration in Pi-limited organs had the strongest, and primary root length had the weakest genetic component. Of 70 trait-associated candidate genes, 17 responded to Pi withdrawal. The PHOSPHATE TRANSPORTER1 gene cluster on chromosome 5 comprises PHT1;1, PHT1;2, and PHT1;3 with known impact on P status. A second locus featured uncharacterized endomembrane-associated auxin efflux carrier encoding PIN-LIKES7 (PILS7) which was more strongly suppressed in Pi-limited roots of Pi-starvation sensitive accessions. In the Col-0 background, Pi uptake and organ growth were impaired in both Pi-limited pht1;1 and two pils7 T-DNA insertion mutants, while Pi -limited pht1;2 had higher biomass and pht1;3 was indistinguishable from wild-type. Copy number variation at the PHT1 locus with loss of the PHT1;3 gene and smaller scale deletions in PHT1;1 and PHT1;2 predicted to alter both protein structure and function suggest diversification of PHT1 is a key driver for adaptation to P limitation. Haplogroup analysis revealed a phosphorylation site in the protein encoded by the PILS7 allele from stress-sensitive accessions as well as additional auxin-responsive elements in the promoter of the "stress tolerant" allele. The former allele's inability to complement the pils7-1 mutant in the Col-0 background implies the presence of a kinase signaling loop controlling PILS7 activity in accessions from P-rich environments, while survival in P-poor environments requires fine-tuning of stress-responsive root auxin signaling.

Sublethal larval exposure to imidacloprid impacts adult behaviour in Drosophila melanogaster.

Pesticides are now chronically found in numerous ecosystems incurring widespread toxic effects on multiple organisms. For insects, the larvae are very exposed to pesticide pollution and the acute effect of insecticides on larvae has been characterized in a range of species. However, the carry-on effects in adults of sublethal exposure occurring in larvae are not well characterized. Here, we use a collection of strains of Drosophila melanogaster differing in their larval resistance to a commonly used insecticide, imidacloprid, and we test the effect of larval exposure on behavioural traits at the adult stage. Focusing on locomotor activity and on courtship and mating behaviour, we observed a significant carry-on effect of imidacloprid exposure. The heritability of activity traits measured in flies exposed to imidacloprid was higher than measured in controls and in these, courtship traits were genetically less correlated from mating success. Altogether, we did not observe a significant effect of the larval insecticide resistance status on adult behavioural traits, suggesting that selection for resistance in larvae does not involve repeatable behavioural changes in adults. This lack of correlation between larval resistance and adult behaviour also suggests that resistance at the larval stage does not necessarily result in increased behavioural resilience at a later life stage. These findings imply that selection for resistance in larvae as well as for behavioural resilience to sublethal exposure in adult will combine and impose a greater evolutionary constraint. Our conclusions further substantiate the need to encompass multiple trait measures and life stages in toxicological assays to properly assess the environmental impact of pesticides.

Cis- and trans-acting variants contribute to survivorship in a naïve Drosophila melanogaster population exposed to ryanoid insecticides.

Insecticide resistance is a paradigm of microevolution, and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster Here we use a naïve population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome-wide association studies (GWAS) of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which we validate with linkage mapping and transgenic manipulation of gene expression. We propose that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliprole's mode of action. GWAS also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homolog of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naïve to this new insecticide chemistry and it foreshadows a selective response among natural populations to these chemicals.

What can genetic association panels tell us about evolutionary processes in insects?

If we are to fully comprehend the evolution of insect diversity at a genomic level we need to understand how natural selection can alter genetically encoded characters within populations. Genetic association panels have the potential to be standard bearers in this endeavour. They enable the mapping of phenotypes to genotypes at unprecedented resolution while simultaneously providing population genomic samples that can be interrogated for the tell-tale signs of selection. Analyses of these panels promise to elucidate the entanglement of gene ontologies, pathways, developmental processes and evolutionary constraints, and inform how these are shaped by adaptation.

Loss of Neogenin1 in human colorectal carcinoma cells causes a partial EMT and wound-healing response.

Neogenin1 (NEO1) is a receptor of the Deleted in Colorectal Carcinoma (DCC)/Frazzled/UNC-40 family, which regulates axon guidance but can also stabilize epithelial adherens junctions. NEO1 and DCC are also tumor suppressors that can inhibit metastasis by acting as dependence receptors. Given the role of NEO1 in maintaining adherens junctions we tested whether loss of NEO1 also promoted metastasis via an epithelial mesenchymal transition (EMT). Loss of NEO1 disrupted zonula adherens but tight junctions were unaffected. Neo1-depleted epithelial cells exhibited a more migratory morphology, had reduced F-actin rich stress-fibres and more basal lamellipodia. Microtubule density was decreased while microtubule outgrowth was faster. Live imaging showed that Neo1-depleted epithelial islands had increased lateral movement. Western blots and immunostaining revealed increased expression of mesenchymal markers such as Fibronectin and MMP1. Furthermore, RNA-seq analysis showed a striking decrease in expression of genes associated with oxidative phosphorylation, and increased expression of genes associated with EMT, locomotion, and wound-healing. In summary, loss of NEO1 in intestinal epithelial cells produces a partial EMT response, based on gene expression, cellular morphology and behaviour and cytoskeletal distribution. These results suggest that loss of NEO1 in carcinomas may contribute to metastasis by promoting a partial EMT and increased motility.

The spread of resistance to imidacloprid is restricted by thermotolerance in natural populations of Drosophila melanogaster.

Imidacloprid, the world's most used insecticide, has caused considerable controversy due to harmful effects on non-pest species and increasing evidence showing that insecticides have become the primary selective force in many insect species. The genetic response to insecticides is heterogeneous across populations and environments, leading to more complex patterns of genetic variation than previously thought. This motivated the investigation of imidacloprid resistance at different temperatures in natural populations of Drosophila melanogaster originating from four climate extremes replicated across two continents. Population and quantitative genomic analysis, supported by functional tests, have revealed a mixed genetic architecture to resistance involving major genes (Paramyosin and Nicotinic-Acetylcholine Receptor Alpha 3) and polygenes with a major trade-off with thermotolerance. Reduced genetic differentiation at resistance-associated loci indicated enhanced gene flow at these loci. Resistance alleles showed stronger evidence of positive selection in temperate populations compared to tropical populations in which chromosomal inversions In(2 L)t, In(3 R)Mo and In(3 R)Payne harbour susceptibility alleles. Polygenic architecture and ecological factors should be considered when developing sustainable management strategies for both pest and beneficial insects.

instaGraminoid, a Novel Colorimetric Method to Assess Herbicide Resistance, Identifies Patterns of Cross-Resistance in Annual Ryegrass.

Herbicide resistance in agricultural weeds is a global problem with an increasing understanding that it is caused by multiple genes leading to quantitative resistance. These quantitative patterns of resistance are not easy to decipher with mortality assays alone, and there is a need for straightforward and unbiased protocols to accurately assess quantitative herbicide resistance. instaGraminoid-a computer vision and statistical analysis package-was developed as an automated and scalable method for quantifying herbicide resistance. The package was tested in rigid ryegrass (Lolium rigidum), the most noxious and highly resistant weed in Australia and the Mediterranean region. This method provides quantitative measures of the degree of chlorosis and necrosis of individual plants which was shown to accurately reflect herbicide resistance. We were able to reliably characterise resistance to four herbicides with different sites of action (glyphosate, sulfometuron, terbuthylazine, and trifluralin) in two L. rigidum populations from Southeast Australia. Cross-validation of the method across populations and herbicide treatments showed high repeatability and transferability. Significant positive correlations in resistance of individual plants were observed across herbicides, which suggest either the accumulation of herbicide-specific resistance alleles in single genotypes (multiple stacked resistance) or the presence of general broad-effects resistance alleles (cross-resistance). We used these quantitative estimates of cross-resistance to simulate how resistance development under an herbicide rotation strategy is likely to be higher than expected.

Structural Variants and Selective Sweep Foci Contribute to Insecticide Resistance in the Drosophila Genetic Reference Panel.

Patterns of nucleotide polymorphism within populations of Drosophila melanogaster suggest that insecticides have been the selective agents driving the strongest recent bouts of positive selection. However, there is a need to explicitly link selective sweeps to the particular insecticide phenotypes that could plausibly account for the drastic selective responses that are observed in these non-target insects. Here, we screen the Drosophila Genetic Reference Panel with two common insecticides; malathion (an organophosphate) and permethrin (a pyrethroid). Genome-wide association studies map survival on malathion to two of the largest sweeps in the D. melanogaster genome; Ace and Cyp6g1 Malathion survivorship also correlates with lines which have high levels of Cyp12d1, Jheh1 and Jheh2 transcript abundance. Permethrin phenotypes map to the largest cluster of P450 genes in the Drosophila genome, however in contrast to a selective sweep driven by insecticide use, the derived allele seems to be associated with susceptibility. These results underscore previous findings that highlight the importance of structural variation to insecticide phenotypes: Cyp6g1 exhibits copy number variation and transposable element insertions, Cyp12d1 is tandemly duplicated, the Jheh loci are associated with a Bari1 transposable element insertion, and a Cyp6a17 deletion is associated with susceptibility.