Predicting the unpredictable: the regulatory nature and promiscuity of herbicide cross resistance.

The emergence of herbicide-resistant weeds is a significant threat to modern agriculture. Cross resistance, a phenomenon where resistance to one herbicide confers resistance to another, is a particular concern owing to its unpredictability. Nontarget-site (NTS) cross resistance is especially challenging to predict, as it arises from genes that encode enzymes that do not directly involve the herbicide target site and can affect multiple herbicides. Recent advancements in genomic and structural biology techniques could provide new venues for predicting NTS resistance in weed species. In this review, we present an overview of the latest approaches that could be used. We discuss the use of genomic and epigenomics techniques such as ATAC-seq and DAP-seq to identify transcription factors and cis-regulatory elements associated with resistance traits. Enzyme/protein structure prediction and docking analysis are discussed as an initial step for predicting herbicide binding affinities with key enzymes to identify candidates for subsequent in vitro validation. We also provide example analyses that can be deployed toward elucidating cross resistance and its regulatory patterns. Ultimately, our review provides important insights into the latest scientific advancements and potential directions for predicting and managing herbicide cross resistance in weeds. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Repeated evolution of herbicide resistance in Lolium multiflorum revealed by haplotype-resolved analysis of acetyl-CoA carboxylase.

Herbicide resistance in weeds is one of the greatest challenges in modern food production. The grass species Lolium multiflorum is an excellent model species to investigate evolution under similar selection pressure because populations have repeatedly evolved resistance to many herbicides, utilizing a multitude of mechanisms to neutralize herbicide damage. In this work, we investigated the gene that encodes acetyl-CoA carboxylase (ACCase), the target site of the most successful herbicide group available for grass weed control. We sampled L. multiflorum populations from agricultural fields with history of intense herbicide use, and studied their response to three ACCase-inhibiting herbicides. To elucidate the mechanisms of herbicide resistance and the genetic relationship among populations, we resolved the haplotypes of 97 resistant and susceptible individuals by sequencing ACCase amplicons using long-read DNA sequencing technologies. Our dose-response data indicated the existence of many, often unpredictable, resistance patterns to ACCase-inhibiting herbicides, where populations exhibited as much as 37-fold reduction in herbicide response. The majority of the populations exhibited resistance to all three herbicides studied. Phylogenetic and molecular genetic analyses revealed multiple evolutionary origins of resistance-endowing ACCase haplotypes, as well as widespread admixture in the region regardless of cropping system. The amplicons generated were diverse, with haplotypes exhibiting 26-110 polymorphisms. Polymorphisms included insertions and deletions 1-31 bp in length, none of which were associated with the resistance phenotype based on an association analysis. We also found evidence that some populations have multiple mechanisms of resistance. Our results highlight the astounding genetic diversity in L. multiflorum populations, and the potential for repeated evolution of herbicide resistance across the landscape that challenges weed management approaches and jeopardizes sustainable weed control practices. We provide an in-depth discussion of the evolutionary and practical implications of our results.

Comparative transcriptomic analysis of male and females in the dioecious weeds Amaranthus palmeri and Amaranthus tuberculatus.

BACKGROUND: Waterhemp (Amaranthus tuberculatus (Moq.) Sauer) and Palmer amaranth (Amaranthus palmeri S. Wats.) are two dioecious and important weed species in the world that can rapidly evolve herbicide-resistance traits. Understanding these two species' dioecious and sex-determination mechanisms could open opportunities for new tools to control them. This study aims to identify the differential expression patterns between males and females in A. tuberculatus and A. palmeri. Multiple analyses, including differential expression, co-expression, and promoter analyses, used RNA-seq data from multiple tissue types to identify putative essential genes for sex determination in both dioecious species. RESULTS: Genes were identified as potential key players for sex determination in A. palmeri. Genes PPR247, WEX, and ACD6 were differentially expressed between the sexes and located at scaffold 20 within or near the male-specific Y (MSY) region. Multiple genes involved with flower development were co-expressed with these three genes. For A. tuberculatus, no differentially expressed gene was identified within the MSY region; however, multiple autosomal class B and C genes were identified as differentially expressed and possible candidate genes. CONCLUSIONS: This is the first study comparing the global expression profile between males and females in dioecious weedy Amaranthus species. Results narrow down putative essential genes for sex-determination in A. palmeri and A. tuberculatus and also strengthen the hypothesis of two different evolutionary events for dioecy within the genus.

Comparative analysis of dioecious Amaranthus plastomes and phylogenomic implications within Amaranthaceae s.s.

BACKGROUND: The genus Amaranthus L. consists of 70-80 species distributed across temperate and tropical regions of the world. Nine species are dioecious and native to North America; two of which are agronomically important weeds of row crops. The genus has been described as taxonomically challenging and relationships among species including the dioecious ones are poorly understood. In this study, we investigated the phylogenetic relationships among the dioecious amaranths and sought to gain insights into plastid tree incongruence. A total of 19 Amaranthus species' complete plastomes were analyzed. Among these, seven dioecious Amaranthus plastomes were newly sequenced and assembled, an additional two were assembled from previously published short reads sequences and 10 other plastomes were obtained from a public repository (GenBank). RESULTS: Comparative analysis of the dioecious Amaranthus species' plastomes revealed sizes ranged from 150,011 to 150,735 bp and consisted of 112 unique genes (78 protein-coding genes, 30 transfer RNAs and 4 ribosomal RNAs). Maximum likelihood trees, Bayesian inference trees and splits graphs support the monophyly of subgenera Acnida (7 dioecious species) and Amaranthus; however, the relationship of A. australis and A. cannabinus to the other dioecious species in Acnida could not be established, as it appears a chloroplast capture occurred from the lineage leading to the Acnida + Amaranthus clades. Our results also revealed intraplastome conflict at some tree branches that were in some cases alleviated with the use of whole chloroplast genome alignment, indicating non-coding regions contribute valuable phylogenetic signals toward shallow relationship resolution. Furthermore, we report a very low evolutionary distance between A. palmeri and A. watsonii, indicating that these two species are more genetically related than previously reported. CONCLUSIONS: Our study provides valuable plastome resources as well as a framework for further evolutionary analyses of the entire Amaranthus genus as more species are sequenced.

Genomic profiling of dioecious Amaranthus species provides novel insights into species relatedness and sex genes.

BACKGROUND: Amaranthus L. is a diverse genus consisting of domesticated, weedy, and non-invasive species distributed around the world. Nine species are dioecious, of which Amaranthus palmeri S. Watson and Amaranthus tuberculatus (Moq.) J.D. Sauer are troublesome weeds of agronomic crops in the USA and elsewhere. Shallow relationships among the dioecious Amaranthus species and the conservation of candidate genes within previously identified A. palmeri and A. tuberculatus male-specific regions of the Y (MSYs) in other dioecious species are poorly understood. In this study, seven genomes of dioecious amaranths were obtained by paired-end short-read sequencing and combined with short reads of seventeen species in the family Amaranthaceae from NCBI database. The species were phylogenomically analyzed to understand their relatedness. Genome characteristics for the dioecious species were evaluated and coverage analysis was used to investigate the conservation of sequences within the MSY regions. RESULTS: We provide genome size, heterozygosity, and ploidy level inference for seven newly sequenced dioecious Amaranthus species and two additional dioecious species from the NCBI database. We report a pattern of transposable element proliferation in the species, in which seven species had more Ty3 elements than copia elements while A. palmeri and A. watsonii had more copia elements than Ty3 elements, similar to the TE pattern in some monoecious amaranths. Using a Mash-based phylogenomic analysis, we accurately recovered taxonomic relationships among the dioecious Amaranthus species that were previously identified based on comparative morphology. Coverage analysis revealed eleven candidate gene models within the A. palmeri MSY region with male-enriched coverages, as well as regions on scaffold 19 with female-enriched coverage, based on A. watsonii read alignments. A previously reported FLOWERING LOCUS T (FT) within A. tuberculatus MSY contig was also found to exhibit male-enriched coverages for three species closely related to A. tuberculatus but not for A. watsonii reads. Additional characterization of the A. palmeri MSY region revealed that 78% of the region is made of repetitive elements, typical of a sex determination region with reduced recombination. CONCLUSIONS: The results of this study further increase our understanding of the relationships among the dioecious species of the Amaranthus genus as well as revealed genes with potential roles in sex function in the species.

Inhibition profile of trifludimoxazin towards PPO2 target site mutations.

BACKGROUND: Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides. RESULTS: Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations. CONCLUSION: Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Repeated origins, widespread gene flow, and allelic interactions of target-site herbicide resistance mutations.

Causal mutations and their frequency in agricultural fields are well-characterized for herbicide resistance. However, we still lack understanding of their evolutionary history: the extent of parallelism in the origins of target-site resistance (TSR), how long these mutations persist, how quickly they spread, and allelic interactions that mediate their selective advantage. We addressed these questions with genomic data from 19 agricultural populations of common waterhemp (Amaranthus tuberculatus), which we show to have undergone a massive expansion over the past century, with a contemporary effective population size estimate of 8 x 107. We found variation at seven characterized TSR loci, two of which had multiple amino acid substitutions, and three of which were common. These three common resistance variants show extreme parallelism in their mutational origins, with gene flow having shaped their distribution across the landscape. Allele age estimates supported a strong role of adaptation from de novo mutations, with a median age of 30 suggesting that most resistance alleles arose soon after the onset of herbicide use. However, resistant lineages varied in both their age and evidence for selection over two different timescales, implying considerable heterogeneity in the forces that govern their persistence. Two such forces are intra- and inter-locus allelic interactions; we report a signal of extended haplotype competition between two common TSR alleles, and extreme linkage with genome-wide alleles with known functions in resistance adaptation. Together, this work reveals a remarkable example of spatial parallel evolution in a metapopulation, with important implications for the management of herbicide resistance.

Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

Genomic-based epidemiology reveals independent origins and gene flow of glyphosate resistance in Bassia scoparia populations across North America.

Genomic-based epidemiology can provide insight into the origins and spread of herbicide resistance mechanisms in weeds. We used kochia (Bassia scoparia) populations resistant to the herbicide glyphosate from across western North America to test the alternative hypotheses that (i) a single EPSPS gene duplication event occurred initially in the Central Great Plains and then subsequently spread to all other geographical areas now exhibiting glyphosate-resistant kochia populations or that (ii) gene duplication occurred multiple times in independent events in a case of parallel evolution. We used qPCR markers previously developed for measuring the structure of the EPSPS tandem duplication to investigate whether all glyphosate-resistant individuals had the same EPSPS repeat structure. We also investigated population structure using simple sequence repeat markers to determine the relatedness of kochia populations from across the Central Great Plains, Northern Plains and the Pacific Northwest. We found that the original EPSPS duplication genotype was predominant in the Central Great Plains where glyphosate resistance was first reported. We identified two additional EPSPS duplication genotypes, one having geographical associations with the Northern Plains and the other with the Pacific Northwest. The EPSPS duplication genotype from the Pacific Northwest seems likely to represent a second, independent evolutionary origin of a resistance allele. We found evidence of gene flow across populations and a general lack of population structure. The results support at least two independent evolutionary origins of glyphosate resistance in kochia, followed by substantial and mostly geographically localized gene flow to spread the resistance alleles into diverse genetic backgrounds.

Genetic architecture underlying HPPD-inhibitor resistance in a Nebraska Amaranthus tuberculatus population.

BACKGROUND: Amaranthus tuberculatus is a problematic weed species in Midwest USA agricultural systems. Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) are an important chemistry for weed management in numerous cropping systems. Here, we characterize the genetic architecture underlying the HPPD-inhibitor resistance trait in an A. tuberculatus population (NEB). RESULTS: Dose-response studies of an F1 generation identified HPPD-inhibitor resistance as a dominant trait with a resistance factor of 15.0-21.1 based on dose required for 50% growth reduction. Segregation analysis in a pseudo-F2 generation determined the trait is moderately heritable (H2  = 0.556) and complex. Bulk segregant analysis and validation with molecular markers identified two quantitative trait loci (QTL), one on each of Scaffold 4 and 12. CONCLUSIONS: Resistance to HPPD inhibitors is a complex, largely dominant trait within the NEB population. Two large-effect QTL were identified controlling HPPD-inhibitor resistance in A. tuberculatus. This is the first QTL mapping study to characterize herbicide resistance in a weedy species.

Evolution of Glyphosate-Resistant Weeds.

Widespread adoption of glyphosate-resistant crops and concomitant reliance on glyphosate for weed control set an unprecedented stage for the evolution of herbicide-resistant weeds. There are now 48 weed species that have evolved glyphosate resistance. Diverse glyphosate-resistance mechanisms have evolved, including single, double, and triple amino acid substitutions in the target-site gene, duplication of the gene encoding the target site, and others that are rare or nonexistent for evolved resistance to other herbicides. This review summarizes these resistance mechanisms, discusses what is known about their evolution, and concludes with some of the impacts glyphosate-resistant weeds have had on weed management.

The genetic architecture and population genomic signatures of glyphosate resistance in Amaranthus tuberculatus.

Much of what we know about the genetic basis of herbicide resistance has come from detailed investigations of monogenic adaptation at known target-sites, despite the increasingly recognized importance of polygenic resistance. Little work has been done to characterize the broader genomic basis of herbicide resistance, including the number and distribution of genes involved, their effect sizes, allele frequencies and signatures of selection. In this work, we implemented genome-wide association (GWA) and population genomic approaches to examine the genetic architecture of glyphosate (Round-up) resistance in the problematic agricultural weed Amaranthus tuberculatus. A GWA was able to correctly identify the known target-gene but statistically controlling for two causal target-site mechanisms revealed an additional 250 genes across all 16 chromosomes associated with non-target-site resistance (NTSR). The encoded proteins had functions that have been linked to NTSR, the most significant of which is response to chemicals, but also showed pleiotropic roles in reproduction and growth. Compared to an empirical null that accounts for complex population structure, the architecture of NTSR was enriched for large effect sizes and low allele frequencies, suggesting the role of pleiotropic constraints on its evolution. The enrichment of rare alleles also suggested that the genetic architecture of NTSR may be population-specific and heterogeneous across the range. Despite their rarity, we found signals of recent positive selection on NTSR-alleles by both window- and haplotype-based statistics, and an enrichment of amino acid changing variants. In our samples, genome-wide single nucleotide polymorphisms explain a comparable amount of the total variation in glyphosate resistance to monogenic mechanisms, even in a collection of individuals where 80% of resistant individuals have large-effect TSR mutations, indicating an underappreciated polygenic contribution to the evolution of herbicide resistance in weed populations.

Future efficacy of pre-emergence herbicides in corn (Zea mays) is threatened by more variable weather.

BACKGROUND: By 2050, weather is expected to become more variable with a shift towards higher temperatures and more erratic rainfall throughout the U.S. Corn Belt. The effects of this predicted weather change on pre-emergence (PRE) herbicide efficacy have been inadequately explored. Using an extensive database, spanning 252 unique weather environments, the efficacy of atrazine, acetochlor, S-metolachlor, and mesotrione, applied PRE alone and in combinations, was modeled on common weed species in corn (Zea mays L.). RESULTS: Adequate rainfall to dissolve the herbicide into soil water solution so that it could be absorbed by developing weed seedlings within the first 15 days after PRE application was essential for effective weed control. Across three annual weed species, the probability of effective control increased as rainfall increased and was maximized when rainfall was 10 cm or more. When rainfall was less than 10 cm, increasing soil temperatures had either a positive or negative effect on the probability of effective control, depending on the herbicide(s) and weed species. Herbicide combinations required less rainfall to maximize the probability of effective control and had higher odds of successfully controlling weeds compared with the herbicides applied individually. CONCLUSIONS: Results of this study highlight the importance of rainfall following PRE herbicide application. As rainfall becomes more variable in future, the efficacy of common PRE herbicides will likely decline. However, utilizing combinations of PRE herbicides along with additional cultural, mechanical, biological, and chemical weed control methods will create a more sustainable integrated weed management system and help U.S. corn production adapt to more extreme weather. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Herbicide resistance in Amaranthus tuberculatus†.

Amaranthus tuberculatus is the major weed species in many midwestern US row-crop production fields, and it is among the most problematic weeds in the world in terms of its ability to evolve herbicide resistance. It has now evolved resistance to herbicides spanning seven unique sites of action, with populations and even individual plants often possessing resistance to several herbicides/herbicide groups. Historically, herbicide target-site changes accounted for most of the known resistance mechanisms in this weed; however, over the last few years, non-target-site mechanisms, particularly enhanced herbicide detoxification, have become extremely common in A. tuberculatus. Unravelling the genetics and molecular details of non-target-site resistance mechanisms, understanding the extent to which they confer cross resistance to other herbicides, and understanding how they evolve remain as critical research endeavors. Transcriptomic and genomics approaches are already facilitating such studies, the results of which hopefully will inform better resistance-mitigation strategies. The largely unprecedented level of herbicide resistance in A. tuberculatus is not only a fascinating example of evolution in action, but it is a serious and growing threat to the sustainability of midwestern US cropping systems. © 2020 Society of Chemical Industry.

Male-specific Y-chromosomal regions in waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri).

Amaranthus tuberculatus and Amaranthus palmeri are agronomically important weed species, both with stable dioecious reproductive systems. An understanding of the genetic basis of sex determination may lead to new methods of managing these troublesome weeds. Previous research identified genomic sequences associated with maleness in each species. Male-specific sequences were used to identify genomic regions in both species that are believed to contain sex-determining genes, i.e. the male-specific Y (MSY) region. These regions were compared to understand if sex determination is controlled via the same physiological pathway and if dioecy evolved independently. A contiguously assembled candidate MSY region identified in Amaranthus palmeri is approximately 1.3 Mb with 121 predicted gene models. In Amaranthus tuberculatus, several contigs, with combined length of 4.6 Mb and with 147 gene models, were identified as belonging to the MSY region. Synteny was not detected between the two species' candidate MSY regions but they shared two predicted genes. With lists of candidate genes for sex determination containing fewer than 200 in each species, future research can address whether sex determination is controlled via similar physiological pathways and whether dioecy has indeed evolved independently in these species.

Genetic variation associated with PPO-inhibiting herbicide tolerance in sorghum.

Herbicide application is crucial for weed management in most crop production systems, but for sorghum herbicide options are limited. Sorghum is sensitive to residual protoporphyrinogen oxidase (PPO)-inhibiting herbicides, such as fomesafen, and a long re-entry period is required before sorghum can be planted after its application. Improving sorghum for tolerance to such residual herbicides would allow for increased sorghum production and the expansion of herbicide options for growers. In this study, we observed sorghum tolerance to residual fomesafen. To investigate the underlying tolerance mechanism a genome-wide association mapping study was conducted using field-collected sorghum biomass panel (SBP) data, and a greenhouse assay was developed to confirm the field phenotypes. A total of 26 significant SNPs (FDR<0.05), spanning a 215.3 kb region on chromosome 3, were detected. The ten most significant SNPs included two in genic regions (Sobic.003G136800, and Sobic.003G136900) and eight SNPs in the intergenic region encompassing the genes Sobic.003G136700, Sobic.003G136800, Sobic.003G137000, Sobic.003G136900, and Sobic.003G137100. The gene Sobic.003G137100 (PPXI), which encodes the PPO1 enzyme, one of the targets of PPO-inhibiting herbicides, was located 12kb downstream of the significant SNP S03_13152838. We found that PPXI is highly conserved in sorghum and expression does not significantly differ between tolerant and sensitive sorghum lines. Our results suggest that PPXI most likely does not underlie the observed herbicide tolerance. Instead, the mechanism underlying herbicide tolerance in the SBP is likely metabolism-based resistance, possibly regulated by the action of multiple genes. Further research is necessary to confirm candidate genes and their functions.

Coexpression Clusters and Allele-Specific Expression in Metabolism-Based Herbicide Resistance.

In the last decade, Amaranthus tuberculatus has evolved resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-hydroxyphenylpyruvate dioxygenase inhibitors in multiple states across the midwestern United States. Two populations resistant to both mode-of-action groups, one from Nebraska (NEB) and one from Illinois (CHR), were studied using an RNA-seq approach on F2 mapping populations to identify the genes responsible for resistance. Using both an A. tuberculatus transcriptome assembly and a high-quality grain amaranth (A. hypochondriacus) genome as references, differential transcript and gene expression analyses were conducted to identify genes that were significantly over- or underexpressed in resistant plants. When these differentially expressed genes (DEGs) were mapped on the A. hypochondriacus genome, physical clustering of the DEGs was apparent along several of the 16 A. hypochondriacus scaffolds. Furthermore, single-nucleotide polymorphism calling to look for resistant-specific (R) variants, and subsequent mapping of these variants, also found similar patterns of clustering. Specifically, regions biased toward R alleles overlapped with the DEG clusters. Within one of these clusters, allele-specific expression of cytochrome  P450  81E8 was observed for 2,4-D resistance in both the CHR and NEB populations, and phylogenetic analysis indicated a common evolutionary origin of this R allele in the two populations.

Draft Genomes of Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri.

Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri are agronomically important weed species. Here, we present the most contiguous draft assemblies of these three species to date. We utilized a combination of Pacific Biosciences long-read sequencing and chromatin contact mapping information to assemble and order sequences of A. palmeri to near-chromosome-level resolution, with scaffold N50 of 20.1 Mb. To resolve the issues of heterozygosity and coassembly of alleles in diploid species, we adapted the trio binning approach to produce haplotype assemblies of A. tuberculatus and A. hybridus. This approach resulted in an improved assembly of A. tuberculatus, and the first genome assembly for A. hybridus, with contig N50s of 2.58 and 2.26 Mb, respectively. Species-specific transcriptomes and information from related species were used to predict transcripts within each assembly. Syntenic comparisons of these species and Amaranthus hypochondriacus identified sites of genomic rearrangement, including duplication and translocation, whereas genetic map construction within A. tuberculatus highlighted the need for further ordering of the A. hybridus and A. tuberculatus contigs. These multiple reference genomes will accelerate genomic studies in these species to further our understanding of weedy evolution within Amaranthus.

Mechanisms of evolved herbicide resistance.

The widely successful use of synthetic herbicides over the past 70 years has imposed strong and widespread selection pressure, leading to the evolution of herbicide resistance in hundreds of weed species. Both target-site resistance (TSR) and nontarget-site resistance (NTSR) mechanisms have evolved to most herbicide classes. TSR often involves mutations in genes encoding the protein targets of herbicides, affecting the binding of the herbicide either at or near catalytic domains or in regions affecting access to them. Most of these mutations are nonsynonymous SNPs, but polymorphisms in more than one codon or entire codon deletions have also evolved. Some herbicides bind multiple proteins, making the evolution of TSR mechanisms more difficult. Increased amounts of protein target, by increased gene expression or by gene duplication, are an important, albeit less common, TSR mechanism. NTSR mechanisms include reduced absorption or translocation and increased sequestration or metabolic degradation. The mechanisms that can contribute to NTSR are complex and often involve genes that are members of large gene families. For example, enzymes involved in herbicide metabolism-based resistances include cytochromes P450, GSH S-transferases, glucosyl and other transferases, aryl acylamidase, and others. Both TSR and NTSR mechanisms can combine at the individual level to produce higher resistance levels. The vast array of herbicide-resistance mechanisms for generalist (NTSR) and specialist (TSR and some NTSR) adaptations that have evolved over a few decades illustrate the evolutionary resilience of weed populations to extreme selection pressures. These evolutionary processes drive herbicide and herbicide-resistant crop development and resistance management strategies.

Multiple modes of convergent adaptation in the spread of glyphosate-resistant Amaranthus tuberculatus.

The selection pressure exerted by herbicides has led to the repeated evolution of herbicide resistance in weeds. The evolution of herbicide resistance on contemporary timescales in turn provides an outstanding opportunity to investigate key questions about the genetics of adaptation, in particular the relative importance of adaptation from new mutations, standing genetic variation, or geographic spread of adaptive alleles through gene flow. Glyphosate-resistant Amaranthus tuberculatus poses one of the most significant threats to crop yields in the Midwestern United States, with both agricultural populations and herbicide resistance only recently emerging in Canada. To understand the evolutionary mechanisms driving the spread of resistance, we sequenced and assembled the A. tuberculatus genome and investigated the origins and population genomics of 163 resequenced glyphosate-resistant and susceptible individuals from Canada and the United States. In Canada, we discovered multiple modes of convergent evolution: in one locality, resistance appears to have evolved through introductions of preadapted US genotypes, while in another, there is evidence for the independent evolution of resistance on genomic backgrounds that are historically nonagricultural. Moreover, resistance on these local, nonagricultural backgrounds appears to have occurred predominantly through the partial sweep of a single haplotype. In contrast, resistant haplotypes arising from the Midwestern United States show multiple amplification haplotypes segregating both between and within populations. Therefore, while the remarkable species-wide diversity of A. tuberculatus has facilitated geographic parallel adaptation of glyphosate resistance, more recently established agricultural populations are limited to adaptation in a more mutation-limited framework.