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The adaptation of weedy plants to herbicide is both a significant problem in agriculture and a model for the study of rapid adaptation under regimes of strong selection. Despite recent advances in our understanding of simple genetic changes that lead to resistance, a significant gap remains in our knowledge of resistance controlled by many loci and the evolutionary factors that influence the maintenance of resistance over time. Here, we perform a multi-level analysis involving whole genome sequencing and assembly, resequencing and gene expression analysis to both uncover putative loci involved in nontarget herbicide resistance and to examine evolutionary forces underlying the maintenance of resistance in natural populations. We found loci involved in herbicide detoxification, stress sensing, and alterations in the shikimate acid pathway to be under selection, and confirmed that detoxification is responsible for glyphosate resistance using a functional assay. Furthermore, we found interchromosomal linkage disequilibrium (ILD), most likely associated with epistatic selection, to influence NTSR loci found on separate chromosomes thus potentially mediating resistance through generations. Additionally, by combining the selection screen, differential expression and LD analysis, we identified fitness cost loci that are strongly linked to resistance alleles, indicating the role of genetic hitchhiking in maintaining the cost. Overall, our work strongly suggests that NTSR glyphosate resistance in I. purpurea is conferred by multiple genes which are maintained through generations via ILD, and that the fitness cost associated with resistance in this species is a by-product of genetic-hitchhiking.

The Pacific crabapple (Malus fusca) is a wild relative of the commercial apple (Malus×domestica). With a range extending from Alaska to Northern California,M. fuscais extremely hardy and disease resistant. The species represents an untapped genetic resource for development of new apple cultivars with enhanced stress resistance. However, gene discovery and utilization ofM. fuscahas been hampered by the lack of genomic resources. Here, we present a high-quality, haplotype-resolved, chromosome-scale genome assembly and annotation forM. fusca. The genome was assembled using high-fidelity long-reads and scaffolded using genetic maps and high-throughput chromatin conformation capture sequencing, resulting in one of the most contiguous apple genomes to date. We annotated the genome using public transcriptomic data from the same species taken from diverse plant structures and developmental stages. Using this assembly, we explored haplotypic structural variation within the genome ofM. fusca,identifying thousands of large variants. We further showed high sequence co-linearity with other domesticated and wildMalusspecies. Finally, we resolve a known quantitative trait locus associated with resistance to fire blight (Erwinia amylovora). Insights gained from the assembly of a reference-quality genome of this hardy wild apple relative will be invaluable as a tool to facilitate DNA-informed introgression breeding.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants. Saponins, a type of plant-derived glycoside, are useful in formulations including soaps, medicines, and root beer foam. Reed et al. studied how the Chilean soapbark tree (Quillaja saponaria) makes its saponins, which lend immunostimulatory activity to vaccines against shingles, malaria, and COVID-19 (see the Perspective by Chubatsu Nunes and Dang). The saponins are currently extracted from the tree?s bark. The identification of enzymes in the biosynthetic pathway opens doors to the more sustainable production of these useful adjuvants and opportunities to engineer new adjuvants. ?PJH Genome-based pathway discovery reveals the biosynthetic route to vaccine adjuvants produced by the soapbark tree.

Triterpenes with complex scaffold modifications are widespread in plants yet little is known regarding biosynthesis. Limonoids are an exemplary family that includes the bitter taste in citrus (e.g., limonin) and the active constituents in neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite limonoid commercial value, a complete biosynthetic route has not been described. Here, we report the discovery of 22 enzymes that catalyze 12 unique reactions including the 4-carbon scission and furan installation that are a signature of the limonoid family and a pair of sterol isomerases previously unknown in plants. This gene set is then used for the total biosynthesis of kihadalactone A and azadirone in a heterologous plant. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

Euphorbia peplus(petty spurge) is a small, fast-growing plant that is native to Eurasia and has become a naturalized weed in North America and Australia.E. peplusis not only medicinally valuable, serving as a source for the skin cancer drug ingenol mebutate, but also has great potential as a model for latex production owing to its small size, ease of manipulation in the laboratory, and rapid reproductive cycle. To help establishE. peplusas a new model, we generated a 267.2 Mb HiC-anchored PacBio HiFi nuclear genome assembly with an embryophyta BUSCO score of 98.5%, a genome annotation based on RNA-seq data from six tissues, and publicly accessible tools including a genome browser and an interactive organ-specific expression atlas. Chromosome number is highly variable acrossEuphorbiaspecies. Using a comparative analysis of our newly sequencedE. peplusgenome with other Euphorbiaceae genomes, we show that variation inEuphorbiachromosome number is likely due to fragmentation and rearrangement rather than aneuploidy. Moreover, we found that theE. peplusgenome is relatively compact compared to related members of the genus in part due to restricted expansion of the Ty3 transposon family. Finally, we identify a large gene cluster that contains many previously identified enzymes in the putative ingenol mebutate biosynthesis pathway, along with additional gene candidates for this biosynthetic pathway. The genomic resources we have created forE. pepluswill help advance research on latex production and ingenol mebutate biosynthesis in the commercially important Euphorbiaceae family.Significance statementEuphorbiais one of the five largest genera in the plant kingdom. Despite an impressive phenotypic and metabolic diversity in this genus, only oneEuphorbiagenome has been sequenced so far, restricting insights intoEuphorbiabiology.Euphorbia peplushas excellent potential as a model species due to its latex production, fast growth rate and production of the anticancer drug ingenol mebutate. Here, we present a chromosome-levelE. peplusgenome assembly and publicly accessible resources to support molecular research for this unique species and the broader genus. We also provide an explanation of one reason the genome is so small, and identify more candidate genes for the anticancer drug and related compounds.

The mustard family (Brassicaceae) is a scientifically and economically important family, containing the model plant Arabidopsis thaliana and numerous crop species that feed billions worldwide. Despite its relevance, most published family phylogenies are incompletely sampled, generally contain massive polytomies, and/or show incongruent topologies between datasets. Here, we present the most complete Brassicaceae genus-level family phylogenies to date (Brassicaceae Tree of Life, or BrassiToL) based on nuclear (>1,000 genes, almost all 349 genera and 53 tribes) and plastome (60 genes, 79% of the genera, all tribes) data. We found cytonuclear discordance between nuclear and plastome-derived phylogenies, which is likely a result of rampant hybridisation among closely and more distantly related species, and highlight rogue taxa. To evaluate the impact of this rampant hybridisation on the nuclear phylogeny reconstruction, we performed four different sampling routines that increasingly removed variable data and likely paralogs. Our resulting cleaned subset of 297 nuclear genes revealed high support for the tribes, while support for the main lineages remained relatively low. Calibration based on the 20 most clock-like nuclear genes suggests a late Eocene to late Oligocene ‘icehouse origin’ of the family. Finally, we propose five new or re-established tribes, including the recognition of Arabidopsideae, a monotypic tribe to accommodate Arabidopsis. With a worldwide community of thousands of researchers working on this family, our new, densely sampled family phylogeny will be an indispensable tool to further highlight Brassicaceae as an excellent model family for studies on biodiversity and plant biology.

Plastic phenotypic responses to environmental change are common, yet we lack a clear understanding of the fitness consequences of these plastic responses. Here, we use the evolution of herbicide resistance in the common morning glory (Ipomoea purpurea) as a model for understanding the relative importance of adaptive and maladaptive gene expression responses to herbicide. Specifically, we compare leaf gene expression changes caused by herbicide to the expression changes that evolve in response to artificial selection for herbicide resistance. We identify a number of genes that show plastic and evolved responses to herbicide and find that for the majority of genes with both plastic and evolved responses, plastic responses appear to be adaptive. We also find that selection for herbicide response increases gene expression plasticity. Overall, these results show the importance of adaptive plasticity for herbicide resistance in a common weed and that expression changes in response to strong environmental change can be adaptive., Impact Statement Predicting whether and how organisms will adapt to environmental change is a crucial goal. However, this goal can be complicated because environmental change can alter traits, in a process called plasticity. The extent and fitness consequences of plasticity will have important effects on the adaptive process. In this study, we use adaptation to herbicide in the agricultural weed, the common morning glory, as a model for understanding the extent and fitness consequences of plasticity in gene expression. We find evidence that gene expression plasticity is adaptive in the presence of herbicide, suggesting that understanding plasticity is crucial for understanding how organisms adapt to new environments.

A goosegrass [Eleusine indica (L.) Gaertn.] population uncontrolled by paraquat (R) in a vegetable production field in St. Clair County, AL, was collected in summer 2019. Research was conducted to assess the level of resistance of the suspected resistant population compared with three populations with no suspected paraquat resistance (S1, S2, and S3). Visual injury at all rating dates and biomass reduction at 28 d after treatment (DAT) of S populations occurred exponentially to increasing paraquat rates. S biotypes were injured more than R at 3 DAT, with biomass recovery at 28 DAT only occurring at rates −1. Plant death or biomass reduction did not occur for any rate at any date for R. Paraquat rates that induced 50% or 90% injury or reduced biomass 50% or 90% compared with the non-treated (I50 or I90, respectively) ranged from 10 to 124 times higher I50 for R compared with S and 54 to 116 times higher I90 for R compared with S biotypes. These data confirm a paraquat-resistant E. indica biotype in Alabama, providing additional germplasm for study of resistance to photosystem I electron-diverting (PSI-ED) resistance mechanisms.

Premise One evolutionary path from hermaphroditism to dioecy is via a gynodioecious intermediate. The evolution of dioecy may also coincide with the formation of sex chromosomes that possess sex-determining loci that are physically linked in a region of suppressed recombination. Dioecious papaya (Carica papaya) has an XY chromosome system, where the presence of a Y chromosome determines maleness. However, in cultivation, papaya is gynodioecious, due to the conversion of the male Y chromosome to a hermaphroditic Yh chromosome during its domestication. Methods We investigated gene expression linked to the X, Y, and Yh chromosomes at different floral developmental stages to identify differentially expressed genes that may be involved in the sexual transition of males to hermaphrodites. Results We identified 309 sex-biased genes found on the sex chromosomes, most of which are found in the pseudoautosomal regions. Female (XX) expression in the sex-determining region was almost double that of X-linked expression in males (XY) and hermaphrodites (XYh ), which rules out dosage compensation for most sex-linked genes; although, an analysis of hemizygous X-linked loci found evidence of partial dosage compensation. Furthermore, we identified a candidate gene associated with sex determination and the transition to hermaphroditism, a homolog of the MADS-box protein SHORT VEGETATIVE PHASE. Conclusions We identified a pattern of partial dosage compensation for hemizygous genes located in the papaya sex-determining region. Furthermore, we propose that loss-of-expression of the Y-linked SHORT VEGETATIVE PHASE homolog facilitated the transition from males to hermaphrodites in papaya.

Sex chromosomes have evolved hundreds of independent times across eukaryotes. As genome sequencing, assembly, and scaffolding techniques rapidly improve, it is now feasible to build fully phased sex chromosome assemblies. Despite technological advances enabling phased assembly of whole chromosomes, there are currently no standards for representing sex chromosomes when publicly releasing a genome. Furthermore, most computational analysis tools are unable to efficiently investigate their unique biology relative to autosomes. We discuss a diversity of sex chromosome systems and consider the challenges of representing sex chromosome pairs in genome assemblies. By addressing these issues now as technologies for full phasing of chromosomal assemblies are maturing, we can collectively ensure that future genome analysis toolkits can be broadly applied to all eukaryotes with diverse types of sex chromosome systems. Here we provide best practice guidelines for presenting a genome assembly that contains sex chromosomes. These guidelines can also be applied to other non-recombining genomic regions, such as S-loci in plants and mating-type loci in fungi and algae.

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. Here, we studied the genome and proteome of Spirodela polyrhiza, or greater duckweed, which has the largest body plan yet the smallest genome size in the family (1C=150 Mb). Using Oxford Nanopore sequencing combined with Hi-C scaffolding, we generated a highly contiguous, chromosome-scale assembly of S. polyrhiza line Sp7498 (Sp7498_HiC). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations relative to a recent PacBio assembly of Sp7498, highlighting the need for orthogonal long-range scaffolding techniques such as Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of ~2250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

The early 1900s delivered many foundational discoveries in genetics, including re-discovery of Mendel's research and the chromosomal theory of inheritance. Following these insights, many focused their research on whether the development of separate sexes had a chromosomal basis or if instead it was caused by environmental factors. It is Dr Nettie M. Stevens'Studies in spermatogenesis(1905) that provided the unequivocal evidence that the inheritance of the Y chromosome initiated male development in mealworms. This result established that sex is indeed a Mendelian trait with a genetic basis and that the sex chromosomes play a critical role. In Part II ofStudies in spermatogenesis(1906), an XY pair was identified in dozens of additional species, further validating the function of sex chromosomes. Since this formative work, a wealth of studies in animals and plants have examined the genetic basis of sex. The goal of this review is to shine a light again on Stevens’Studies in spermatogenesisand the lasting impact of this work. We additionally focus on key findings in plant systems over the last century and open questions that are best answered, as in Stevens' work, by synthesizing across many systems.This article is part of the theme issue ‘Sex determination and sex chromosome evolution in land plants’.

The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes.

The large size and complexity of most fern genomes have hampered efforts to elucidate fundamental aspects of fern biology and land plant evolution through genome-enabled research. Here we present a chromosomal genome assembly and associated methylome, transcriptome and metabolome analyses for the model fern species Ceratopteris richardii. The assembly reveals a history of remarkably dynamic genome evolution including rapid changes in genome content and structure following the most recent whole-genome duplication approximately 60 million years ago. These changes include massive gene loss, rampant tandem duplications and multiple horizontal gene transfers from bacteria, contributing to the diversification of defence-related gene families. The insertion of transposable elements into introns has led to the large size of the Ceratopteris genome and to exceptionally long genes relative to other plants. Gene family analyses indicate that genes directing seed development were co-opted from those controlling the development of fern sporangia, providing insights into seed plant evolution. Our findings and annotated genome assembly extend the utility of Ceratopteris as a model for investigating and teaching plant biology.

The rapid development of sequencing technologies has led to a deeper understanding of horticultural plant genomes. However, experimental evidence connecting genes to important agronomic traits is still lacking in most non-model organisms. For instance, the genetic mechanisms underlying plant architecture are poorly understood in pome fruit trees, creating a major hurdle in developing new cultivars with desirable architecture, such as dwarfing rootstocks in European pear (Pyrus communis). Further, the quality and content of genomes vary widely. Therefore, it can be challenging to curate a list of genes with high-confidence gene models across reference genomes. This is often an important first step towards identifying key genetic factors for important traits. Here we present a draft genome of P. communis ‘d’Anjou’ and an improved assembly of the latest P. communis ‘Bartlett’ genome. To study gene families involved in tree architecture in European pear and other rosaceous species, we developed a workflow using a collection of bioinformatic tools towards curation of gene families of interest across genomes. This lays the groundwork for future functional studies in pear tree architecture. Importantly, our workflow can be easily adopted for other plant genomes and gene families of interest.

Duckweeds are some of the smallest angiosperms, possessing a simple body architecture and high rates of biomass accumulation. They can grow near-exponentially via clonal propagation. Understanding their reproductive biology, growth, and development is essential to unlock their potential for phytoremediation, carbon capture, and nutrition. However, there is a lack of non-laborious and convenient methods for spatially and temporally imaging an array of duckweed plants and growth conditions in the same experiment. We developed an automated microscopy approach to record time-lapse images of duckweed plants growing in 12-well cell culture plates. As a proof-of-concept experiment, we grew duckweed on semi-solid media with and without sucrose and monitored its effect on their growth over 3 days. Using the PlantCV toolkit, we quantified the thallus area of individual plantlets over time, and showed that L. minor grown on sucrose had an average growth rate four times higher than without sucrose. This method will serve as a blueprint to perform automated high-throughput growth assays for studying the development patterns of duckweeds from different species, genotypes, and conditions.

James “Jim” A. Birchler, Curators’ Distinguished Professor at the University of Missouri (MU or Mizzou), is perhaps the closest thing that the 21st century gets to a “jack of all trades.” He’s known by the fly world as the scientist who discovered cosuppression in Drosophila , by the

For centuries, scientists have been intrigued by the origin of dioecy in plants, characterizing sex-specific development, uncovering cytological differences between the sexes, and developing theoretical models. Through the invention and continued improvements in genomic technologies, we have truly begun to unlock the genetic basis of dioecy in many species. Here we broadly review the advances in research on dioecy and sex chromosomes. We start by first discussing the early works that built the foundation for current studies and the advances in genome sequencing that have facilitated more-recent findings. We next discuss the analyses of sex chromosomes and sex-determination genes uncovered by genome sequencing. We synthesize these results to find some patterns are emerging, such as the role of duplications, the involvement of hormones in sex-determination, and support for the two-locus model for the origin of dioecy. Though across systems, there are also many novel insights into how sex chromosomes evolve, including different sex-determining genes and routes to suppressed recombination. We propose the future of research in plant sex chromosomes should involve interdisciplinary approaches, combining cutting-edge technologies with the classics to unravel the patterns that can be found across the hundreds of independent origins.

Genome sizes of plants have long piqued the interest of researchers due to the vast differences among organisms. However, the mechanisms that drive size differences have yet to be fully understood. Two important contributing factors to genome size are expansions of repetitive elements, such as transposable elements (TEs), and whole-genome duplications (WGD). Although studies have found correlations between genome size and both TE abundance and polyploidy, these studies typically test for these patterns within a genus or species. The plant order Brassicales provides an excellent system to further test if genome size evolution patterns are consistent across larger time scales, as there are numerous WGDs. This order is also home to one of the smallest plant genomes, Arabidopsis thaliana—chosen as the model plant system for this reason—as well as to species with very large genomes. With new methods that allow for TE characterization from low-coverage genome shotgun data and 71 taxa across the Brassicales, we confirm the correlation between genome size and TE content, however, we are unable to reconstruct phylogenetic relationships and do not detect any shift in TE abundance associated with WGD.

International audience; The genetic basis and evolution of sex determination in dioecious plants is emerging as an active area of research with exciting advances in genome sequencing and analysis technologies. As the sole species within the sister lineage to all other extant flowering plants, Amborella trichopoda is an important model for understanding the evolution and development of flowers. Plants typically produce only male or female flowers, but sex determination mechanisms are unknown for the species. Sequence data derived from plants of natural origin and an F1 mapping population were used to identify sex-linked genes and the nonrecombining region. Amborella trichopoda has a ZW sex determination system. Analysis of genes in a 4 Mb nonrecombining sex-determination region reveals recent divergence of Z and W gametologs, and few Z- and W-specific genes. The sex chromosomes of A. trichopoda evolved less than 16.5 Myr ago, long after the divergence of the extant angiosperms.

Sex determination is poorly understood in plants. Amborella trichopoda is a well-known plant model for evo-devo studies, which is also dioecious (has male and female individuals), with an unknown sex determination mechanism. A. trichopoda is a “sex switcher”, which points to possible environmental factors that act on sex, but populations grown from seed under greenhouse conditions exhibit a 50:50 sex ratio, which indicates the operation of genetic factors. Here, we use a new method (SDpop) to identify sex-linked genes from genotyping data of male and female individuals sampled in the field, and find that A. trichopoda has a ZW sex-chromosome system. The sex-linked genes map to a 4 Mb sex-determining region on chromosome 9. The low extent of ZW divergence suggests these sex chromosomes are of recent origin, which is consistent with dioecy being derived character in the A. trichopoda lineage. Our work has uncovered clearly formed sex chromosomes in a species in which both genetic and environmental factors can influence sex.One Sentence SummaryAmborella trichopoda, a dioecious species in which both genetics and the environment influence sex, possesses a pair of quite recently evolved ZW chromosomes.

5-methylcytosine (5mC) is a modified base often described as necessary for the proper regulation of genes and transposons and for the maintenance of genome integrity in plants. However, the extent of this dogma is limited by the current phylogenetic sampling of land plant species diversity. Here, we report that a monocot plant, Spirodela polyrhiza, has lost CG gene body methylation, genome-wide CHH methylation, and the presence or expression of several genes in the highly conserved RNA-directed DNA methylation (RdDM) pathway. It has also lost the CHH methyltransferase CHROMOMETHYLASE 2. Consequently, the transcriptome is depleted of 24-nucleotide, heterochromatic, small interfering RNAs that act as guides for the deposition of 5mC to RdDM-targeted loci in all other currently sampled angiosperm genomes. Although the genome displays low levels of genome-wide 5mC primarily at LTR retrotransposons, CG maintenance methylation is still functional. In contrast, CHG methylation is weakly maintained even though H3K9me2 is present at loci dispersed throughout the euchromatin and highly enriched at regions likely demarcating pericentromeric regions. Collectively, these results illustrate that S. polyrhiza is maintaining CG and CHG methylation mostly at repeats in the absence of small RNAs. S. polyrhiza reproduces rapidly through clonal propagation in aquatic environments, which we hypothesize may enable low levels of maintenance methylation to persist in large populations.Significance StatementDNA methylation is a widespread chromatin modification that is regularly found in all plant species. By examining one aquatic duckweed species, Spirodela polyrhiza, we find that it has lost highly conserved genes involved in methylation of DNA at sites often associated with repetitive DNA, and within genes, however DNA methylation and heterochromatin is maintained during cell division at other sites. Consequently, small RNAs that normally guide methylation to silence repetitive DNA like retrotransposons are diminished. Despite the loss of a highly conserved methylation pathway, and the reduction of small RNAs that normally target repetitive DNA, transposons have not proliferated in the genome, perhaps due in part to the rapid, clonal growth lifestyle of duckweeds.

Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH spots that reflect the copy number of target RNA.

One evolutionary path from hermaphroditism to dioecy is via a gynodioecious intermediate. The evolution of dioecy may also coincide with the formation of sex chromosomes that possess sex-determining loci that are physically linked in a region of suppressed recombination. Dioecious papaya (Carica papaya) has an XY chromosome system, where the presence of a Y chromosome determines males. However, in cultivation, papaya is gynodioecious, due to the conversion of the male Y chromosome to a hermaphroditic Yh chromosome during its domestication. We investigated gene expression linked to the X, Y, and Yh chromosomes at different floral developmental stages in order to identify differentially expressed genes (DEGs) that may be involved in the sexual reversion of males to hermaphrodites. We identified 309 sex-biased genes found on the sex chromosomes, most of which are found in the pseudoautosomal regions (PARs). Female (XX) expression in the sex determining region (SDR) was almost double that of X-linked expression in males (XY) and hermaphrodites (XYh), which rules out dosage compensation for most sex-linked gene; although, an analysis of hemizygous X-linked loci found evidence of partial dosage compensation. Furthermore, we identified a potential candidate gene associated with both sex determination and the transition to hermaphroditism, a homolog of the MADS-box protein SHORT VEGETATIVE PHASE (SVG).

Genome size of plants has long piqued the interest of researchers due to the vast differences among organisms. However, the mechanisms that drive size differences have yet to be fully understood. Two important contributing factors to genome size are expansions of repetitive elements, such as transposable elements (TEs), and whole-genome duplications (WGD). Although studies have found correlations between genome size and both TE abundance and polyploidy, these studies typically test for these patterns within a genus or species. The plant order Brassicales provides an excellent system to test if genome size evolution patterns are consistent across larger time scales, as there are numerous WGDs. This order is also home to one of the smallest plant genomes, Arabidopsis thaliana - chosen as the model plant system for this reason - as well as to species with very large genomes. With new methods that allow for TE characterization from low-coverage genome shotgun data and 71 taxa across the Brassicales, we find no correlation between genome size and TE content, and more surprisingly we identify no significant changes to TE landscape following WGD.

In monocots other than maize (Zea mays) and rice (Oryza sativa), the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNA) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, small interfering RNAs (siRNAs) and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of transfer RNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th, and 3′-end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nucleotide (nt) reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5, important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of sRNA diversification in flowering plants.

A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

The origin and early evolution of sex chromosomes have been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis) is an ideal species to investigate this hypothesis, as the X and Y chromosomes are cytologically homomorphic and evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with single-molecule fluorescence in situ hybridization directly implicate Y-specific genes that respectively suppress female (pistil) development and are necessary for male (anther) development. Comparison of contiguous X and Y chromosome assemblies shows that hemizygosity underlies the loss of recombination between the genes suppressing female organogenesis (SUPPRESSOR OF FEMALE FUNCTION) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION1 [aspTDF1]). We also experimentally demonstrate the function of aspTDF1. These findings provide direct evidence that sex chromosomes can function through linkage of two sex determination genes.

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Spirodela polyrhiza, the Greater Duckweed, has the largest body plan yet the smallest genome size in the family (1C = 150 Mb). Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. We generated a highly contiguous, chromosome-scale assembly of Spirodela polyrhiza line Sp7498 using Oxford Nanopore plus Hi-C scaffolding (Sp7498_HiC) which is highly syntenic with a related line (Sp9509). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations in a recent PacBio assembly of Sp7498, highlighting the necessity of orthogonal long-range scaffolding techniques like Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of ∼2,250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

Single-molecule FISH (smFISH) has been widely used in animal tissue to localize and quantify RNAs with high specificity. This protocol describes an smFISH method optimized for highly autofluorescent plant tissue. It provides details on fixation buffers and protocols to protect the integrity of plant samples. We also provide smFISH hybridization conditions to detect plant RNA with ~50 fluorescently labeled DNA oligonucleotides. In addition, this protocol provides instructions on linear spectral unmixing of smFISH signal from background autofluorescence by confocal microscopy and a method to quantify the smFISH spots that reflect the copy number of target RNA.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These “phasiRNAs” are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115–117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.

Regulated gene expression is key to the orchestrated progression of the cell cycle. Many genes are expressed at specific points in the cell cycle, including important cell cycle regulators, plus factors involved in signal transduction, hormonal regulation, and metabolic control. We demonstrate that post-embryonic depletion of Arabidopsis (Arabidopsis thaliana) ARGONAUTE1 (AGO1), the main effector of plant microRNAs (miRNAs), impairs cell division in the root meristem. We utilized the highly synchronizable tobacco (Nicotiana tabacum) Bright yellow 2 (BY2) cell suspension to analyze mRNA, small RNAs, and mRNA cleavage products of synchronized BY2 cells at S, G2, M, and G1 phases of the cell cycle. This revealed that in plants, only a few miRNAs show differential accumulation during the cell cycle, and miRNA-target pairs were only identified for a small proportion of the more than 13,000 differentially expressed genes during the cell cycle. However, this unique set of miRNA-target pairs could be key to attenuate the expression of several transcription factors and disease resistance genes. We also demonstrate that AGO1 binds to a set of 19-nucleotide, tRNA-derived fragments during the cell cycle progression.

The early maize (Zea mays) seed undergoes several developmental stages after double fertilization to become fully differentiated within a short period of time, but the genetic control of this highly dynamic and complex developmental process remains largely unknown. Here, we report a high temporal-resolution investigation of transcriptomes using 31 samples collected at an interval of 4 or 6 h within the first six days of seed development. These time-course transcriptomes were clearly separated into four distinct groups corresponding to the stages of double fertilization, coenocyte formation, cellularization, and differentiation. A total of 22,790 expressed genes including 1415 transcription factors (TFs) were detected in early stages of maize seed development. In particular, 1093 genes including 110 TFs were specifically expressed in the seed and displayed high temporal specificity by expressing only in particular period of early seed development. There were 160, 22, 112, and 569 seed-specific genes predominantly expressed in the first 16 h after pollination, coenocyte formation, cellularization, and differentiation stage, respectively. In addition, network analysis predicted 31,256 interactions among 1317 TFs and 14,540 genes. The high temporal-resolution transcriptome atlas reported here provides an important resource for future functional study to unravel the genetic control of seed development.

PREMISE OF THE STUDY: Previous phylogenetic studies employing molecular markers have yielded various insights into the evolutionary history across Brassicales, but many relationships between families remain poorly supported or unresolved. A recent phylotranscriptomic approach utilizing 1155 nuclear markers obtained robust estimates for relationships among 14 of 17 families. Here we report a complete family-level phylogeny estimated using the plastid genome. METHODS: We conducted phylogenetic analyses on a concatenated data set comprising 44,926 bp from 72 plastid genes for species distributed across all 17 families. Our analysis includes three additional families, Tovariaceae, Salvadoraceae, and Setchellanthaceae, that were omitted in the previous phylotranscriptomic study. KEY RESULTS: Our phylogenetic analyses obtained fully resolved and strongly supported estimates for all nodes across Brassicales. Importantly, these findings are congruent with the topology reported in the phylotranscriptomic study. This consistency suggests that future studies could utilize plastid genomes as markers for resolving relationships within some notoriously difficult clades across Brassicales. We used this new phylogenetic framework to verify the placement of the At-α event near the origin of Brassicaceae, with median date estimates of 31.8 to 42.8 million years ago and restrict the At-β event to one of two nodes with median date estimates between 85 to 92.2 million years ago. These events ultimately gave rise to novel chemical defenses and are associated with subsequent shifts in net diversification rates. CONCLUSIONS: We anticipate that these findings will aid future comparative evolutionary studies across Brassicales, including selecting candidates for whole-genome sequencing projects.

Plants constantly face the threat of attack from many directions. Organisms like bacteria, viruses, and fungi must be blocked from entering a plant’s cells or quickly targeted for destruction once inside. Within the plant genome itself, transposable elements lie in wait for reactivation. In

Resurrection plants have the unique ability to survive extreme dehydration (desiccation), lying dormant for months or sometimes years until rehydration is possible. This formidable survival strategy has evolved independently several times across the land plant phylogeny, and several phylogenetically

Sex chromosomes have evolved independently in phylogenetically diverse flowering plant lineages. The genes governing sex determination in dioecious species remain unknown, but theory predicts that the linkage of genes influencing male and female function will spur the origin and early evolution of sex chromosomes. For example, in an XY system, the origin of an active Y may be spurred by the linkage of female suppressing and male promoting genes. Garden asparagus (Asparagus officinalis) serves as a model for plant sex chromosome evolution, given that it has recently evolved an XX/XY sex chromosome system. In order to elucidate the molecular basis of gender differences and sex determination, we used RNA-sequencing (RNA-Seq) to identify differentially expressed genes between female (XX), male (XY) and supermale (YY) individuals. We identified 570 differentially expressed genes, and showed that significantly more genes exhibited male-biased than female-biased expression in garden asparagus. In the context of anther development, we identified genes involved in pollen microspore and tapetum development that were specifically expressed in males and supermales. Comparative analysis of genes in the Arabidopsis thaliana, Zea mays and Oryza sativa anther development pathways shows that anther sterility in females probably occurs through interruption of tapetum development before microspore meiosis.

With the rapid development of sequencing technology and the plummeting cost, assembling whole genomes from non-model plants will soon become routine for plant systematists and evolutionary biologists. Here we summarize and compare several of the latest genome sequencing and assembly approaches, offering a practical guide on how to approach a genome project. We also highlight certain precautions that need to be taken before investing time and money into a genome project.

Summary Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver ( Kindr ) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.