The ancestral karyotype of the Heliantheae Alliance, herbicide resistance, and human allergens: Insights from the genomes of common and giant ragweed.

Ambrosia artemisiifolia and Ambrosia trifida (Asteraceae) are important pest species and the two greatest sources of aeroallergens globally. Here, we took advantage of a hybrid to simplify genome assembly and present chromosome-level assemblies for both species. These assemblies show high levels of completeness with Benchmarking Universal Single-Copy Ortholog (BUSCO) scores of 94.5% for A. artemisiifolia and 96.1% for A. trifida and long terminal repeat (LTR) Assembly Index values of 26.6 and 23.6, respectively. The genomes were annotated using RNA data identifying 41,642 genes in A. artemisiifolia and 50,203 in A. trifida. More than half of the genome is composed of repetitive elements, with 62% in A. artemisiifolia and 69% in A. trifida. Single copies of herbicide resistance-associated genes PPX2L, HPPD, and ALS were found, while two copies of the EPSPS gene were identified; this latter observation may reveal a possible mechanism of resistance to the herbicide glyphosate. Ten of the 12 main allergenicity genes were also localized, some forming clusters with several copies, especially in A. artemisiifolia. The evolution of genome structure has differed among these two species. The genome of A. trifida has undergone greater rearrangement, possibly the result of chromoplexy. In contrast, the genome of A. artemisiifolia retains a structure that makes the allotetraploidization of the most recent common ancestor of the Heliantheae Alliance the clearest feature of its genome. When compared to other Heliantheae Alliance species, this allowed us to reconstruct the common ancestor's karyotype-a key step for furthering of our understanding of the evolution and diversification of this economically and allergenically important group.

Global biodiversity data suggest allopolyploid plants do not occupy larger ranges or harsher conditions compared with their progenitors.

Understanding the factors determining species' geographical and environmental range is a central question in evolution and ecology, and key for developing conservation and management practices. Shortly after the discovery of polyploidy, just over 100 years ago, it was suggested that polyploids generally have greater range sizes and occur in more extreme conditions than their diploid congeners. This suggestion is now widely accepted in the literature and is attributed to polyploids having an increased capacity for genetic diversity that increases their potential for adaptation and invasiveness. However, the data supporting this idea are mixed. Here, we compare the niche of allopolyploid plants to their progenitor species to determine whether allopolyploidization is associated with increased geographic range or extreme environmental tolerance. Our analysis includes 123 allopolyploid species that exist as only one known ploidy level, with at least one known progenitor species, and at least 50 records in the Global Biodiversity Information Facility (GBIF) database. We used GBIF occurrence data and range modeling tools to quantify the geographic and environmental distribution of these allopolyploids relative to their progenitors. We find no indication that allopolyploid plants occupy more extreme conditions or larger geographic ranges than their progenitors. Data evaluated here generally indicate no significant difference in range between allopolyploids and progenitors, and where significant differences do occur, the progenitors are more likely to exist in extreme conditions. We concluded that the evidence from these data indicate allopolyploidization does not result in larger or more extreme ranges. Thus, allopolyploidization does not have a consistent effect on species distribution, and we conclude it is more likely the content of an allopolyploid's genome rather than polyploidy per se that determines the potential for invasiveness.

Insights from the genomes of 4 diploid Camelina spp.

Plant evolution has been a complex process involving hybridization and polyploidization making understanding the origin and evolution of a plant's genome challenging even once a published genome is available. The oilseed crop, Camelina sativa (Brassicaceae), has a fully sequenced allohexaploid genome with 3 unknown ancestors. To better understand which extant species best represent the ancestral genomes that contributed to C. sativa's formation, we sequenced and assembled chromosome level draft genomes for 4 diploid members of Camelina: C. neglecta C. hispida var. hispida, C. hispida var. grandiflora, and C. laxa using long and short read data scaffolded with proximity data. We then conducted phylogenetic analyses on regions of synteny and on genes described for Arabidopsis thaliana, from across each nuclear genome and the chloroplasts to examine evolutionary relationships within Camelina and Camelineae. We conclude that C. neglecta is closely related to C. sativa's sub-genome 1 and that C. hispida var. hispida and C. hispida var. grandiflora are most closely related to C. sativa's sub-genome 3. Further, the abundance and density of transposable elements, specifically Helitrons, suggest that the progenitor genome that contributed C. sativa's sub-genome 3 maybe more similar to the genome of C. hispida var. hispida than that of C. hispida var. grandiflora. These diploid genomes show few structural differences when compared to C. sativa's genome indicating little change to chromosome structure following allopolyploidization. This work also indicates that C. neglecta and C. hispida are important resources for understanding the genetics of C. sativa and potential resources for crop improvement.

Update on the Brassicaceae species checklist.

BACKGROUND: Here we present a revised species checklist for the Brassicaceae, updated from Warwick SI, Francis, A, Al-Shehbaz IA (2006), Brassicaceae: Species checklist and database on CD-ROM, Plant Systematics and Evolution 259: 249─25. This update of the checklist was initiated, based on recent taxonomic and molecular studies on the Brassicaceae that have resulted in new species names, combinations and associated synonyms. NEW INFORMATION: New data have been added indicating tribal affiliations within the family and where type specimens have been designated. In addition, information from many early publications has been checked and added to the database. The database now includes information on 14983 taxa, 4636 of which are currently accepted and divided into 340 genera and 52 tribes. A selected bibliography of recent publications on the Brassicaceae is included.

High gene flow maintains genetic diversity following selection for high EPSPS copy number in the weed kochia (Amaranthaceae).

Kochia, a major weed species, has evolved resistance to four herbicide modes of action. Herbicide resistance appears to spread quickly, which could result in diminished standing genetic variation, reducing the ability of populations to adapt further. Here we used double digest restriction enzyme associated sequencing to determine the level of gene flow among kochia populations and whether selection for glyphosate resistance reduces genetic variation. Canadian Prairie populations show little to no genetic differentiation (FST = 0.01) and no correlation between genetic and geographic distance (r2 = - 0.02 p = 0.56), indicating high gene flow and no population structure. There is some evidence that kochia populations are genetically depauperate compared to other weed species, but genetic diversity did not differ between glyphosate susceptible and resistant populations or individuals. The inbreeding coefficients suggest there are 23% fewer heterozygotes in these populations than expected with random mating, and no variation was found within the chloroplast. These results indicate that any alleles for herbicide resistance can be expected to spread quickly through kochia populations, but there is little evidence this spread will reduce the species' genetic variation or limit the species' ability to respond to further selection pressure.

A Chromosome-Scale Assembly of the Garden Orach (Atriplex hortensis L.) Genome Using Oxford Nanopore Sequencing.

Atriplex hortensis (2n = 2x = 18, 1C genome size ∼1.1 gigabases), also known as garden orach and mountain-spinach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae alliance (Chenopodiaceae sensu stricto, subfam. Chenopodioideae) that has spread in cultivation from its native primary domestication area in Eurasia to other temperate and subtropical regions worldwide. Atriplex L. is a highly complex but, as understood now, a monophyletic group of mainly halophytic and/or xerophytic plants, of which A. hortensis has been a vegetable of minor importance in some areas of Eurasia (from Central Asia to the Mediterranean) at least since antiquity. Nonetheless, it is a crop with tremendous nutritional potential due primarily to its exceptional leaf and seed protein quantities (approaching 30%) and quality (high levels of lysine). Although there is some literature describing the taxonomy and production of A. hortensis, there is a general lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic positioning, and future potential of the species. Here, we report the assembly of the first high-quality, chromosome-scale reference genome for A. hortensis cv. "Golden." Long-read data from Oxford Nanopore's MinION DNA sequencer was assembled with the program Canu and polished with Illumina short reads. Contigs were scaffolded to chromosome scale using chromatin-proximity maps (Hi-C) yielding a final assembly containing 1,325 scaffolds with a N50 of 98.9 Mb - with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-six percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy-(32%) and Copia-like (11%) long-terminal repeats. The annotation was completed using MAKER which identified 37,083 gene models and 2,555 tRNA genes. Completeness of the genome, assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) metric, identified 97.5% of the conserved orthologs as complete, with only 2.2% being duplicated, reflecting the diploid nature of A. hortensis. A resequencing panel of 21 wild, unimproved and cultivated A. hortensis accessions revealed three distinct populations with little variation within subpopulations. These resources provide vital information to better understand A. hortensis and facilitate future study.

A chromosome-scale draft sequence of the Canada fleabane genome.

BACKGROUND: Due to the accessibility of underlying technologies the 'Omics', in particular genomics, are becoming commonplace in several fields of research, including the study of agricultural pests. The weed community is starting to embrace these approaches; genome sequences have been made available in the past years, with several other sequencing projects underway, as promoted by the International Weed Genome Consortium. Chromosome-scale sequences are essential to fully exploit the power of genetics and genomics. RESULTS: We report such an assembly for Conyza canadensis, an important agricultural weed. Third-generation sequencing technology was used to create a genome assembly of 426 megabases, of which nine chromosome-scale scaffolds cover more than 98% of the entire assembled sequence. As this weed was the first to be identified with glyphosate resistance, and since we do not have a firm handle on the genetic mechanisms responsible for several herbicide resistances in the species, the genome sequence was annotated with genes known to be associated with herbicide resistance. A high number of ABC-type transporters, cytochrome P450 and glycosyltransferases (159, 352 and 181, respectively) were identified among the list of ab initio predicted genes. CONCLUSION: As C. canadensis has a small genome that is syntenic with other Asteraceaes, has a short life cycle and is relatively easy to cross, it has the potential to become a model weed species and, with the chromosome-scale genome sequence, contribute to a paradigm shift in the way non-target site resistance is studied. © 2020 Her Majesty the Queen in Right of CanadaPest Management Science © 2020 Society of Chemical Industry.

Population Genomic Approaches for Weed Science.

Genomic approaches are opening avenues for understanding all aspects of biological life, especially as they begin to be applied to multiple individuals and populations. However, these approaches typically depend on the availability of a sequenced genome for the species of interest. While the number of genomes being sequenced is exploding, one group that has lagged behind are weeds. Although the power of genomic approaches for weed science has been recognized, what is needed to implement these approaches is unfamiliar to many weed scientists. In this review we attempt to address this problem by providing a primer on genome sequencing and provide examples of how genomics can help answer key questions in weed science such as: (1) Where do agricultural weeds come from; (2) what genes underlie herbicide resistance; and, more speculatively, (3) can we alter weed populations to make them easier to control? This review is intended as an introduction to orient weed scientists who are thinking about initiating genome sequencing projects to better understand weed populations, to highlight recent publications that illustrate the potential for these methods, and to provide direction to key tools and literature that will facilitate the development and execution of weed genomic projects.

Polyploidization for the Genetic Improvement of Cannabis sativa.

Cannabis sativa L. is a diploid species, cultivated throughout the ages as a source of fiber, food, and secondary metabolites with therapeutic and recreational properties. Polyploidization is considered as a valuable tool in the genetic improvement of crop plants. Although this method has been used in hemp-type Cannabis, it has never been applied to drug-type strains. Here, we describe the development of tetraploid drug-type Cannabis lines and test whether this transformation alters yield or the profile of important secondary metabolites: Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), or terpenes. The mitotic spindle inhibitor oryzalin was used to induce polyploids in a THC/CBD balanced drug-type strain of Cannabis sativa. Cultured axillary bud explants were exposed to a range of oryzalin concentrations for 24 h. Flow cytometry was used to assess the ploidy of regenerated shoots. Treatment with 20-40 µM oryzalin produced the highest number of tetraploids. Tetraploid clones were assessed for changes in morphology and chemical profile compared to diploid control plants. Tetraploid fan leaves were larger, with stomata about 30% larger and about half as dense compared to diploids. Trichome density was increased by about 40% on tetraploid sugar leaves, coupled with significant changes in the terpene profile and a 9% increase in CBD that was significant in buds. No significant increase in yield of dried bud or THC content was observed. This research lays important groundwork for the breeding and development of new Cannabis strains with diverse chemical profiles, of benefit to medical and recreational users.

Hybridization rate and hybrid fitness for Camelina microcarpa Andrz. ex DC (♀) and Camelina sativa (L.) Crantz(Brassicaceae) (♂).

Hybridization between crops and their wild relatives has the potential to introduce novel variation into wild populations. Camelina (Camelina sativa) is a promising oilseed and cultivars with modified seed characteristics and herbicide resistance are in development, prompting a need to evaluate the potential for novel trait introgression into weedy relatives. Little-podded false flax (littlepod; Camelina microcarpa) is a naturalized weed in Canada and the USA. Here we evaluated the hybridization rate between the three cytotypes of littlepod (♀) and camelina (♂), assessed characteristics of hybrids, and evaluated the fitness of hexaploid littlepod and camelina hybrids in the glasshouse and field. In total we conducted, 1,005 manual crosses with diploid littlepod, 1, 172 crosses with tetraploid littlepod, and 896 crosses with hexaploid littlepod. Hybrids were not produced by the diploids, but were produced by the tetraploids and hexaploids at rates of one hybrid for 2,000 ovules pollinated and 24 hybrids for 25 ovules pollinated, respectively. Hybrids between tetraploid littlepod and camelina showed low pollen fertility and produced a small number of seeds. In the glasshouse, hybrids between hexaploid littlepod and camelina also showed significantly lower pollen fertility and seed production than parental lines, but their seeds showed high viability. A similar pattern was observed in field trials, with hybrids showing earlier flowering, reduced biomass, seed production and seed weight. However, seed produced by the hybrids showed greater viability than that produced by hexaploid littlepod and is potentially the result of a shortened lifecycle. The introgression of lifecycle traits into littlepod populations may facilitate range expansion and contribute to crop gene persistence. Consequently, future work should evaluate the hybridization rate in the field, the fitness of advanced generation backcrosses, and the role of time to maturity in limiting hexaploid littlepod's distribution.

Assessment of Antinutritional Compounds and Chemotaxonomic Relationships between Camelina sativa and Its Wild Relatives.

We compared the secondary metabolite composition in seeds of Camelina sativa and its wild relatives to identify potential germplasm with reduced levels of antinutritional compounds. Twenty Camelina accessions, from five different species, were analyzed by liquid chromatography mass spectrometry and subjected to principal component analysis, which revealed that Camelina spp. separated into distinct chemotaxonomic groups. Three major glucosinolates (GSs) were identified in our study, namely, 9-methylsulfinylnonyl GS (GS9), 10-methylsulfinyldecyl GS (GS10), and 11-methylsulfinylundecyl GS (GS11). While there were differences in total GS levels, species-specific patterns for GS9 and GS11 were noted. Sinapine content ranged between 1.4 and 5.6 mg/g FW, with the lowest levels observed in  C. laxa and C. sativa. Lignin levels were also lowest in C. sativa, with most accessions containing less than 6 mg/g FW. Our results show that wild Camelina spp. have distinct metabolomes, and based on their levels of major antinutritionals, some could be incorporated into breeding programs with C. sativa.

flowPloidy: An R package for genome size and ploidy assessment of flow cytometry data.

PREMISE OF THE STUDY: Despite advantages in terms of reproducibility, histogram analysis based on nonlinear regression is rarely used in genome size assessments in plant biology. This is due in part to the lack of a freely available program to implement the procedure. We have developed such a program, the R package flowPloidy. METHODS AND RESULTS: flowPloidy builds on the existing statistical tools provided with the R environment. This base provides tools for importing flow cytometry data, fitting nonlinear regressions, and interactively visualizing data. flowPloidy adds tools for building flow cytometry models, fitting the models to histogram data, and producing visual and tabular summaries of the results. CONCLUSIONS: flowPloidy fills an important gap in the study of plant genome size. This package will enable plant scientists to apply a more powerful statistical technique for assessing genome size. flowPloidy improves on existing software options by providing a no-cost workflow streamlined for genome size and ploidy determination.

Glyphosate resistance reduces kochia fitness: Comparison of segregating resistant and susceptible F2 populations.

Glyphosate is considered the world's most important herbicide, but widespread and continual use has resulted in the evolution of resistance. Kochia scoparia (kochia) has evolved resistance via tandem gene amplification of glyphosate's target, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and resistant populations have been reported from the Canadian Prairies and the Northern Great Plains. Here, we evaluated the fitness costs of EPSPS amplification in kochia by comparing susceptible and resistant full siblings from segregating F2 populations generated from within six populations. Kochia was expected to be highly diverse because of strong gene flow; however, six of the seven field-collected parents with higher EPSPS copy number were homozygous. Under competitive greenhouse conditions, the EPSPS type of the line's maternal parent showed persistent effects: delayed emergence, delayed flowering, and reductions in viable seed count and weight overall. High EPSPS copy number individuals had reduced seed count and weight, reduced competitive ability, and reduced final height in mixed stands, but better germination of the F3. However, all characteristics were highly variable and fitness costs were not constant across genetic backgrounds. In the absence of selection from glyphosate, kochia with increased EPSPS copy number will be at a competitive disadvantage in some genetic backgrounds.

Genome size variation in the genus Avena.

Genome size is an indicator of evolutionary distance and a metric for genome characterization. Here, we report accurate estimates of genome size in 99 accessions from 26 species of Avena. We demonstrate that the average genome size of C genome diploid species (2C = 10.26 pg) is 15% larger than that of A genome species (2C = 8.95 pg), and that this difference likely accounts for a progression of size among tetraploid species, where AB < AC < CC (average 2C = 16.76, 18.60, and 21.78 pg, respectively). All accessions from three hexaploid species with the ACD genome configuration had similar genome sizes (average 2C = 25.74 pg). Genome size was mostly consistent within species and in general agreement with current information about evolutionary distance among species. Results also suggest that most of the polyploid species in Avena have experienced genome downsizing in relation to their diploid progenitors. Genome size measurements could provide additional quality control for species identification in germplasm collections, especially in cases where diploid and polyploid species have similar morphology.

Bidirectional but asymmetrical sexual hybridization between Brassica carinata and Sinapis arvensis (Brassicaceae).

With transgenic crop development it is important to evaluate the potential for transgenes to escape into populations of wild, weedy relatives. Ethiopian mustard (Brassica carinata, BBCC) is easily transformed and is being investigated for uses from biodiesel fuels to biopharmaceuticals. However, little work has been done evaluating its ability to cross with relatives such as wild mustard (Sinapsis arvensis, SrSr), an abundant, cosmopolitan weedy relative. Here we conducted bidirectional crosses with Ethiopian mustard as a maternal parent in 997 crosses and paternal parent in 1,109 crosses. Hybrids were confirmed using flow cytometry and species-specific ITS molecular markers and indicate a high hybridization rate of 6.43 % between Ethiopian mustard (♀) and wild mustard (♂) and a lower, but not insignificant, hybridization rate of 0.01 % in the reverse direction. The majority of the hybrids were homoploid (BCSr) with less than 1 % of pollen production of their parents and low seed production (0.26 seeds/pollination) in crosses and backcrosses indicating a potential for advanced generation hybrids. The accession used had a significant effect on hybrid seed production with different accessions of Ethopian mustard varying in their production of hybrid offspring from 2.69 to 16.34 % and one accession of wild mustard siring almost twice as many hybrid offspring per flower as the other. One pentaploid (BBCCSr) and one hexaploid (BBCCSrSr) hybrid were produced and had higher pollen viability, though no and low seed production, respectively. As wild mustard is self-incompatible and the outcrossing rate of Ethiopian mustard has been estimated as 30 % potential for hybrid production in the wild appears to be high, though the hybridization rate found here represents a worst case scenario as it does not incorporate pre-pollination barriers. Hybridization in the wild needs to be directly evaluated as does the propensity of Ethiopian mustard to volunteer.

Synoptic weather typing applied to air pollution mortality among the elderly in 10 Canadian cities.

BACKGROUND: Synoptic circulation patterns (large-scale weather systems) affect ambient levels of air pollution, as well as the relationship between air pollution and human health. OBJECTIVE: To investigate the air pollution-mortality relationship within weather types and seasons, and to determine which combination of atmospheric conditions may pose increased health threats in the elderly age categories. METHODS: The relative risk of mortality (RR) due to air pollution was examined using Poisson generalized linear models (GLMs) within specific weather types. Analysis was completed by weather type and age group (all ages, ≤64, 65-74, 75-84, ≥85 years) in ten Canadian cities from 1981 to 1999. RESULTS: There was significant modification of RR by weather type and age. When examining the entire population, weather type was shown to have the greatest modifying effect on the risk of dying due to ozone (O3). This effect was highest on average for the dry tropical (DT) weather type, with the all-age RR of mortality at a population weighted mean (PWM) found to be 1.055 (95% CI 1.026-1.085). All-weather type risk estimates increased with age due to exposure to carbon monoxide (CO), nitrogen dioxide (NO2), and sulphur dioxide (SO2). On average, RR increased by 2.6, 3.8 and 1.5% for the respective pollutants between the ≤64 and ≥85 age categories. Conversely, mean ozone estimates remained relatively consistent with age. Elevated levels of air pollution were found to be detrimental to the health of elderly individuals for all weather types. However, the entire population was negatively effected by air pollution on the hot dry (DT) and hot humid (MT) days. CONCLUSIONS: We identified a significant modification of RR for mortality due to air pollution by age, which is enhanced under specific weather types. Efforts should be targeted at minimizing pollutant exposure to the elderly and/or all age groups with respect to weather type in question.

Adaptation of diploid and tetraploid chamerion angustifolium to elevation but not local environment.

Polyploid organisms often have different geographic ranges than their diploid relatives. However, it is unclear whether this divergence is maintained by adaptation or results from historical differences in colonization. Here, we conducted a reciprocal transplant experiment with diploid and autotetraploid Chamerion angustifolium to test for adaptation at the ploidy and population level. In the Rocky Mountains, pure diploid populations occur at high elevations and pure autotetraploid populations occur at low elevations with mixed ploidy populations between. We planted 3134 seedlings in 2004 and 3890 juveniles (bolting) in 2005 among nine plots, three in each of the diploid, mixed ploidy, and tetraploid zones, and monitored survival until 2008. For both seedlings and juvenile plants, elevation significantly influenced survival. The juvenile plants also showed a significant ploidy by elevation interaction, indicating that diploids and tetraploids survived best at their native elevations. In contrast, we found no evidence of local adaptation to plot within elevation. This suggests that the current distribution of diploids and tetraploids across elevations is the result of adaptation and that genome duplication may have facilitated the invasion of lower elevation habitats by limiting the movement of maladapted alleles from diploid populations at higher elevations.

Whole genome duplication affects evolvability of flowering time in an autotetraploid plant.

Whole genome duplications have occurred recurrently throughout the evolutionary history of eukaryotes. The resulting genetic and phenotypic changes can influence physiological and ecological responses to the environment; however, the impact of genome copy number on evolvability has rarely been examined experimentally. Here, we evaluate the effect of genome duplication on the ability to respond to selection for early flowering time in lines drawn from naturally occurring diploid and autotetraploid populations of the plant Chamerion angustifolium (fireweed). We contrast this with the result of four generations of selection on synthesized neoautotetraploids, whose genic variability is similar to diploids but genome copy number is similar to autotetraploids. In addition, we examine correlated responses to selection in all three groups. Diploid and both extant tetraploid and neoautotetraploid lines responded to selection with significant reductions in time to flowering. Evolvability, measured as realized heritability, was significantly lower in extant tetraploids (^b(T) =  0.31) than diploids (^b(T) =  0.40). Neotetraploids exhibited the highest evolutionary response (^b(T)  =  0.55). The rapid shift in flowering time in neotetraploids was associated with an increase in phenotypic variability across generations, but not with change in genome size or phenotypic correlations among traits. Our results suggest that whole genome duplications, without hybridization, may initially alter evolutionary rate, and that the dynamic nature of neoautopolyploids may contribute to the prevalence of polyploidy throughout eukaryotes.

Design, fabrication and demonstration of a magnetophoresis chamber with 25 output fractions.

Our goal is to develop an instrument for parallel and multiplexed bioassay using magnetic labels. Toward this end we are developing a multi-outlet magnetophoresis instrument incorporating a fluidic flow chamber placed inside a magnetic field gradient. Magnetic microparticles are sorted by their magnetic moment for eventual use as biological labels based on magnetic signature.In this paper we concentrate on developments in our flow chamber fabrication methods that have allowed us to scale the number of sorting channels from 8 to 25. We present data for instrument performance and reproducibility of sorting.