Compositions and methods for generating genomic structural variation in plants

Abstract

Provided herein are methods for generating genomic structural variation in plants, e.g., using a topoisomerase II inhibitor, such as etoposide. Also provided herein are combinations and kits for generating genomic structural variation in plants. The combinations and kits generally comprise a plant cell or plant tissue that is capable of being propagated, and a topoisomerase II inhibitor.

Description

COMPOSITIONS AND METHODS FOR GENERATING GENOMIC STRUCTURAL VARIATION IN PLANTSRELATED APPLICATION S)

[0001] This application claims the benefit of U.S. Application No. 63 / 535,740, filed on August 31, 2023. The entire contents of the above application(s) are incorporated herein by reference.BACKGROUND

[0002] Structural variation is a valuable source of phenotypic variation. Genomic structural variation can include a change in the copy number of chromosomal regions (duplications and deletions leading to an increase or decrease in the number of gene copies) or the order of genes on a chromosome (inversions or translocations). Changes in the order and / or the copy number of genes can lead to novel traits that can then be selected in nature or by plant breeders. Historically, irradiation has been used to generate structural changes.

[0003] Structural variations can be artificially created in one of two ways. First, they can be created by using sources of radiation - usually research nuclear reactors or regulated radiation sources. Second, they can be created by the use of transgenic lines that harbor proteins to destabilize chromosomes. However, access to radiation sources is not readily available to everyone, and transgenic plants cannot be created cheaply or easily for most species. Accordingly, there exists a need for an alternative means to induce structural variation in the genome.SUMMARY

[0004] The present invention is based, in part, on the discovery of a new means of introducing genomic structural variation in plants using chemical agents that function as topoisomerase II inhibitors.

[0005] Accordingly, provided herein are methods of introducing genetic variation in a plant, comprising the steps of a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growthmedium to form a plant. In some embodiments, the topoisomerase II inhibitor comprises etoposide or a salt thereof.

[0006] Also provided herein are combinations and kits comprising: a) a plant cell or plant tissue that is capable of being propagated; and b) a topoisomerase II inhibitor. In some embodiments, the topoisomerase II inhibitor comprises etoposide or a salt thereof.

[0007] In addition, provided herein are methods of modifying at least one trait of a plant or part thereof, comprising the steps of: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant. In some embodiments, the topoisomerase II inhibitor comprises etoposide or a salt thereof.

[0008] Further provided herein are methods of inducing a genetic mutation in a plant, comprising the steps of: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant. In some embodiments, the topoisomerase II inhibitor comprises etoposide or a salt thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0010] FIGs. 1A-1F show that etoposide induces novel phenotypes. FIG. 1A (top) shows Arabidopsis thaliana plants that were grown on media in the absence of etoposide (0 pM), 80 pM, 160 pM, or 320 pM etoposide for two weeks, before transfer to soil. Inhibition of root and shoot growth is observed in the presence of etoposide. Arabidopsis seeds germinated on increasing concentrations of etoposide show a dose-responsive growth defect with roots particularly impacted. Seedlings grown with 320 pM etoposide exhibited no true leaves. This generation is referred to as the Ml . FIG. 1 A (bottom) shows Arabidopsis thaliana plants that were grown on media in the absence of etoposide (0 pM), 80 pM or 160 pM etoposide fortwo weeks, and then transferred to soil. The difference in the size of the transplanted seedlings can be observed even two weeks later. FIG. IB (top) illustrates that Ml was selffertilized to give rise to M2 plants. FIG. IB (bottom) shows images from a visual examination of the M2 generation that helped identify many mutant plants including plants showing the virescent phenotype with yellow-colored leaves (lower left), a brassinosteroid response-like dwarf phenotype (lower center), and a fertile dwarf with shorter internodes (lower right). FIGs. 1C-1E describe and show back-crosses to wild-type and self-fertilizing to identify genotype-phenotype relationships and the number of loci contributing to a phenotype. FIG. 1C illustrates that the virescent phenotype is recessive and caused by a single locus. FIG. ID shows that the BR-like dwarf phenotype is incompletely dominant and displays three phenotypic classes. Segregation from self-fertilized crosses suggests that this phenotype is caused by a mutation at a single locus. FIG. IE illustrates that the shortinternode t / vrar / phenotype is recessive and segregation data suggests this phenotype is caused by two loci. FIG. IF shows pigeon pea (Cajanus cajari) plants grown hydroponically in the absence (0 pM) or presence (320 pM) of etoposide. Inhibition of root growth is observed in the presence of etoposide.

[0011] FIGs. 2A-2E show representative phenotypes seen in the progeny of etoposide- treated plants. FIG. 2A shows an untreated plant with only one organ per axil (represented by *) and normal inflorescence (represented by an arrow). FIGs. 2B-2E show progeny of etoposide-treated plants. FIG. 2B shows multiple organs arising at the axil. FIG. 2C shows that the inflorescence was deformed. FIG. 2D shows dwarf plant variety in comparison to a normal looking sibling. FIG. 2E shows leaves lack chlorophyll.

[0012] FIG. 3 shows copy number variation detected by short-read sequencing of progeny of etoposide-treated plants. Each graph represents a different mutated line. Gray represents progeny of an etoposide-treated line. Black represents a control line. A y-axis value of 2 represents normal copy number in untreated plants, 3 represents a gain of one copy, and 1 represents a loss of one copy of that chromosomal region. MB stands for Megabase or position on chromosome.

[0013] FIGs. 4A-E show that whole-genome sequencing identifies structural variation in progeny of etoposide-treated plants. FIG. 4A shows duplications in etoposide-treated lines. Dosage across the indicated chromosome is shown. Chromosomal segments with one extra copy are indicated by an asterisk. FIG. 4B is a diagram of location of structural variants identified via long-read sequencing. Structural variants present in DMSO-treated (control)and Col-0 (wild-type) samples were excluded. Structural variants marked with an asterisk were verified via PCR. FIG. 4C is a JBrowse2 snapshot showing an example of a 184 bp insertion. Read alignments to reference are shown, with inserted sequence in dark grey. This insertion was present in the short internode dwarf line, including non-dwarf siblings. FIG. 4D is a JBrowse2 snapshot showing an example of an 81 bp deletion. Read alignments to reference are shown, with deleted sequence in dark grey. Coverage is shown above read alignments. This deletion was present in the variegated line. FIG. 4E shows an example of a 1.52 Mb inversion. Top, ribbon plot depicting long-read alignments at both inversion breakends on chromosome 2. Bottom, view of alignment of a single read demonstrating inverted alignment to right breakend. This inversion was present in brassinosteroid-like (BR- like) dwarf plants but not in siblings that lacked the dwarf phenotype.

[0014] FIGs. 5 A-J show RNA sequencing results for M3 plants of BR-like dwarf, shortinternode dwarf, variegated, and virescent phenotypes. FIG. 5A is a volcano plot summarizing differential expression analysis of BR-like dwarf vs. non-dwarf plants. Significantly differentially expressed genes (log2(fold change) > 1 and adjusted p-value < .01) are shown in dark grey. Key genes are labeled. FIG. 5B is a diagram depicting disruption of genes at breakends of the 1.52 Mb inversion on chromosome 2 in BR-like dwarf plants. FIG. 5C shows the average log2fold change (log2FC) of BR-like dwarf vs. non-dwarf across chromosome 2. Log2FC is plotted for individual sliding windows (30 kb windows with 10 kb overlap between windows); line represents locally estimated scatterplot smoothing to aid pattern visualization. The 1.52 Mb inversion on chromosome 2 in BR-like dwarf plants is shaded in light grey. FIG. 5D is a volcano plot summarizing differential expression analysis of short internode dwarf vs. non-dwarf plants. Significantly differentially expressed genes (log2FC > 1 and adjusted p-value < .01) are highlighted in dark grey. Select genes with small p-values are labeled. FIG. 5E shows average log2fold change (short internode dwarf vs. non- dwarf) across chromosome 4. Log2FC is plotted for individual sliding windows (30 kb windows with 10 kb overlap between windows); line represents locally estimated scatterplot smoothing to aid pattern visualization. The 3.54 Mb inversion on chromosome 4 in short internode dwarf plants is shaded in the vertical grey bar. FIG. 5F is a volcano plot summarizing differential expression analysis virescent vs. non-virescent plants. Significantly differentially expressed genes (log2FC > 1 and adjusted p value < .01) are shown in the upper left and upper right regions bounded by dashed lines. Select genes with small p-values are labeled. FIG. 5G shows average log2fold change (virescent vs. non-virescent) acrosschromosome 2. Log2FC is plotted for individual sliding windows (30 kb windows with 10 kb overlap between windows); line represents locally estimated scatterplot smoothing to aid pattern visualization. The approximate location of the nuclear insertion of the mitochondrial genome (NuMt) on chromosome 2 is shaded in the vertical grey bar. FIG. 5H is a heatmap of genes known to be associated with variegated phenotypes. IMMUTANS (AT4G22260) is marked with an asterisk. FIG. 51 is a diagram of IMMUTANS, showing 37 bp deletion in an exon. JBrowse2 snapshot shows long-read alignments to reference, with deleted sequence shown at positions 14,760,866-14,760,903. Coverage is shown above read alignments. The deletion was heterozygous in this particular plant, which was green and produced -25% variegated progeny. FIG. 5 J shows normalized counts of IMMUTANS transcripts.

[0015] FIGs. 6A-H show additional phenotyping of progeny of etoposide-treated lines. Visual observation of M2 plants arising from different etoposide-treated Ml progenitors identified multiple lines with mutant phenotypes, including early flowering (FIG. 6A), sterility (FIG. 6B), dwarfism (FIG. 6C), variegation (FIG. 6D), and altered leaf shape (FIGs. 6E-6G). FIG. 6H shows that short internode mutants show the same number of nodes as wildtype but lower distance between nodes.DETAILED DESCRIPTION

[0016] A description of example embodiments follows.

[0017] Several aspects of the disclosure are described below, with reference to examples for illustrative purposes only. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the disclosure. One having ordinary skill in the relevant art, however, will readily recognize that the disclosure can be practiced without one or more of the specific details or practiced with other methods, protocols, reagents, cell lines, and animals. The present disclosure is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts, steps, or events are required to implement a methodology in accordance with the present disclosure. Many of the techniques and procedures described, or referenced herein, are well understood and commonly employed using conventional methodology by those skilled in the art.

[0018] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonlyunderstood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0020] As used herein, the articles “a,” “an,” and “the” should be understood to include plural reference unless the context clearly indicates otherwise.

[0021] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term “comprising” can be substituted with the term “containing” or “including.”

[0022] As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the disclosure, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of’ to vary the scope of the disclosure.

[0023] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or.”

[0024] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C ”Methods of the Disclosure

[0025] Provided herein are methods of introducing genetic variation in a plant, comprising the steps of: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant.

[0026] Also provided herein are methods of modifying at least one trait of a plant or part thereof, comprising the steps of: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant.

[0027] Further provided herein are methods of inducing a genetic mutation in a plant, comprising the steps of: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant.

[0028] Non-limiting examples of topoisomerase II inhibitors that are suitable for use in the methods disclosed herein include etoposide, doxorubicin and related molecules (daunorubicin, epirubicin, idarubicin, pirarubicin), teniposide, acridine-4-carboxamide, amsacrine, ellipticine, ICRF-193, and mitoxantrone and its derivatives (e.g., pixantrone), or a salt (e.g., a pharmaceutically acceptable salt) of any of the foregoing. In some embodiments, the topoisomerase II inhibitor comprises etoposide. In some embodiments, the topoisomerase II inhibitor is a salt of etoposide (e.g., a pharmaceutically acceptable salt of etoposide), such as, e.g., etoposide phosphate.

[0029] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is pharmaceutically acceptable as defined herein and that has the desired pharmacological activity of the parent. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Non-limiting examples of pharmaceutically acceptable salts include those derived from inorganic acids, non-limiting examples of which include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and those derived from organic acids, non-limiting examples of which include acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, nicotinic acid, oxalic acid, stearic acid, malic acid, maleic acid, malonic acid, salicylic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, p- toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, and lactic acid. Either the mono-, di- or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form.

[0030] Additional non-limiting examples of pharmaceutically acceptable salts include those formed when an acidic proton in a parent compound is replaced by a metal ion, nonlimiting examples of which include an alkali metal ion and an alkaline earth metal ion, and those formed when an acidic proton present in a parent compound is replaced by an ammonium ion, a primary ammonium ion, a secondary ammonium ion, a tertiary ammonium ion, or a quaternary ammonium ion. Non-limiting examples of alkali metals and alkaline earth metals include sodium, potassium, lithium, calcium, aluminum, magnesium, copper, zinc, iron, and manganese. Additional non-limiting examples of pharmaceutically acceptable salts include those comprising one or more counterions and zwitterions.

[0031] The topoisomerase II inhibitor(s) can be provided in a composition (e.g., a pharmaceutical composition), comprising one or more pharmaceutically acceptable carriers, or diluents. “Pharmaceutically acceptable carrier” refers to a non-toxic carrier that does not destroy the pharmacological activity of the agent with which it is formulated and is nontoxic when administered in doses sufficient to deliver an effective amount (e.g., a mutagenically effective amount) of the agent to a plant. The pharmaceutically acceptable carrier may be an excipient, where “excipient” as used herein refers to a pharmaceutically acceptable material chosen from a solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, polymer, peptide,protein, cell, hyaluronidase, and mixtures thereof. Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. In some embodiments, the solvent is an aqueous solvent. Pharmaceutically acceptable carriers and excipients are well-known to persons having ordinary skill in the art and are described in, as a non-limiting example, Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2013) and any other editions, which are hereby incorporated by reference.

[0032] Compositions comprising a topoisomerase II inhibitor can further comprise one or more additional compounds, such as, for example, a compound that modulates one or more cellular processes in plants, an antimicrobial compound (e.g., an antibiotic), a plant nutrient (e.g., a mineral, a macronutrient, a micronutrient, a vitamin), an herbicide, an inhibitor of DNA methylation or histone methylation, mutagens such as alkylating agents, base analogs, DNA intercalating agents, heavy metals, or a plant hormone.

[0033] The amount or concentration of topoisomerase II inhibitor that is included in a composition can be determined empirically by a person of ordinary skill in the art to which the invention pertains (e.g., using methods described in the Exemplification herein). For example, a composition suitable for use in the methods disclosed herein can comprise at least about 80 pM topoisomerase II inhibitor (e.g., at least about 80 pM etoposide or a salt thereof), or from about 80 pM to about 380 pM topoisomerase II inhibitor (e.g., from about 80 pM to about 380 pM etoposide or a salt thereof), such as about 160 pM to 380 pM topoisomerase II inhibitor (e.g., about 160 pM to about 380 pM etoposide or a salt thereof). Thus, a composition suitable for use in the methods disclosed herein can comprise about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, about 300 pM, about 310 pM, about 320pM, about 330 pM, about 340 pM, about 350 pM, about 360 pM, about 370 pM, or about 380 pM topoisomerase II inhibitor (e.g., etoposide or a salt thereof). In some embodiments, a composition suitable for use in the methods disclosed herein comprises less than about 80 pM topoisomerase II inhibitor (e.g., less than about 80 pM etoposide or a salt thereof). In some embodiments, a composition suitable for use in the methods disclosed herein comprises greater than about 380 pM topoisomerase II inhibitor (e.g., greater than about 380 pM etoposide or a salt thereof).

[0034] The methods disclosed herein can be practiced using any plant cell or plant tissue that is capable of being propagated. Examples of plant cells and tissues that are capable of being propagated include, but are not limited to, a cultured plant cell, plant callus tissue, an embryo, a seed, a stem, a root, a root tip, a root hair, a leaf, pollen, a cotyledon, a hypocotyl, a seedling, a pistil, an anther, a flower, a meristematic cell, and a whole plant. In some embodiments, the plant cell or plant tissue that is capable of being propagated is a seed or a seedling.

[0035] The plant cell or tissue can be present in or removed from any plant that is capable of being propagated and for which an increase in genetic variation is being sought. Nonlimiting examples of suitable plants for use in the methods disclosed herein include Arabidopsis thaliana, cotton, tobacco, tomato, potato, sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, legumes, alfalfa, carrot, strawberry, poplars, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium, cactus, pigeon pea, com, rice, canola, rapeseed, false flax, pennycress, cannabis, hops, herbs, wheat, oat, apple, banana, peach, nectarine, melon, clover, orchid, and soy. The compositions and methods disclosed herein can be applied to any plant cultivated by man, e.g., for human or animal food or as a source of raw material for manufacture of other products or for ornamental, medicinal, landscape, or conservation purposes.

[0036] The methods disclosed herein include a step of contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor. An “effective amount” is an amount effective, at dosages and for periods of time necessary, to achieve a desired result (e.g., an increase in genetic variation). In some embodiments, the step of contacting the plant cell or tissue can be over a period of time that is less than one day, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days, or more. In some embodiments, the step of contacting the tissue can be, for example, over a period of time that is up to about 7 days, up to about 14 days, up to about 21 days, or up to about 28 days. In some embodiments, the step of contacting the tissue can be, for example, over a period of time that is between 1 day and 7 days, between 7 days and 28 days, or between 14 days and 21 days.

[0037] The contacting can occur, for example, by adding the topoisomerase II inhibitor to a plant growth medium (e.g., soil, agar) in which the plant, plant cell, or plant tissue is being maintained (e.g., media in which a plant seed is germinated). Alternatively, or in addition, the plant, plant cell, or plant tissue can be contacted with a solution comprising the topoisomerase II inhibitor (e.g., by spraying (for example, mixed with a surfactant), bathing and / or soaking the plant, plant cell, or plant tissue in a solution comprising the topoisomerase II inhibitor).

[0038] The methods disclosed herein further include the step of transferring (e.g., transplanting) the plant cell or plant tissue that was contacted with the topoisomerase II inhibitor to a plant growth medium (e.g., a plant growth medium lacking a topoisomerase II inhibitor). The plant growth medium can be a solid, liquid, or semi-solid designed to support the growth of a population of plants, plant parts, and / or plant cells. Suitable plant growth media for use in the methods disclosed herein include various types of soil (e.g., nutrient- supplemented soil, naturally occurring soil), plant growth media comprising agar (e.g., Murashige and Skoog (MSO or MS0) medium, with or without supplemented sucrose), and various plant cell culture media.

[0039] Non-limiting examples of genetic variation that can be generated using the methods disclosed herein include structural variations and point mutations. Structural variations include, for example, copy number changes (e.g., increases in copy number, decreases in copy number) such as duplications and deletions, and chromosomal rearrangements, such as inversions and translocations, or a combination of these events. In some embodiments, the genetic variation is a heritable structural variation.

[0040] Structural variations can be detected in plants using suitable genetic and molecular techniques known to persons of ordinary skill in the art, including those described in the Exemplification herein.

[0041] In some embodiments, the methods disclosed herein further comprise the step of screening the propagated tissue for one or more phenotypes (e.g., by visual inspection, by computational analysis of plant images, by metabolic constitution). Non-limiting examples of plant phenotypes that can be screened include yield, altered nutrient requirements, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, and photosynthetic capacity, or any combination thereof.

[0042] In some embodiments, a plant or portion thereof is screened for tolerance to an environmental stressor. In some embodiments, a plant or portion thereof is screened for changes in one or more of shoot architecture, flowering time, floral morphology, fertility, seed abortion, or root length, or any combination thereof. In some embodiments, a plant or portion thereof is screened for absence of, or a decrease in, or an increase in chlorophyll. In some embodiments, a plant or portion thereof is screened for an abiotic stress response, a biotic stress response, or a combination thereof. Non-limiting examples of abiotic stresses include low temperature, high temperature, drought, flooding, nutrient and mineral scarcity, high salinity, heavy metals, ultraviolet radiation, high light intensity, and low light intensity. Non-limiting examples of biotic stresses include a microbial infection or disease (e.g., fungal disease, viral disease, bacterial disease), a parasitic infection (e.g., by a unicellular eukaryote), an insect infestation, and a nematode infestation.

[0043] In some embodiments, the methods disclosed herein further comprise reproducing (e.g., propagating, breeding) the propagated plant having the genetic variation (e.g., to generate a population of plants comprising the genetic variation). The reproducing can be asexual or sexual reproduction.Combinations and Kits of the Disclosure

[0044] Also provided herein are combinations and kits comprising: a) a plant cell or plant tissue that is capable of being propagated; and b) a topoisomerase II inhibitor. In some embodiments, a combination comprising: a) a plant cell or plant tissue that is capable of being propagated; and b) a topoisomerase II inhibitor, is provided. In some embodiments, a kit comprising: a) a plant cell or plant tissue that is capable of being propagated; and b) a topoisomerase II inhibitor, is provided. The plant cell or plant tissue that is capable of being propagated and the topoisomerase II inhibitor can be packaged together in the same or separate containers, or can be packaged separately. Furthermore, the topoisomerase IIinhibitor can be provided in a unit dosage form. In some embodiments, the combinations and kits disclosed herein further comprise instructions for use.

[0045] Non-limiting examples of plant cells and tissues that are capable of being propagated include a cultured plant cell, plant callus tissue, an embryo, a seed, a stem, a root, a root tip, a root hair, a leaf, pollen, a cotyledon, a hypocotyl, a seedling, a pistil, an anther, a flower, a meristematic cell, and a whole plant, or any combination thereof. In some embodiments, the plant cell or plant tissue that is capable of being propagated is a seed.

[0046] The plant cell or tissue can be from any plant that is capable of being propagated and for which an increase in genetic variation is being sought. Non-limiting examples of suitable plants for use in the methods disclosed herein include Arabidopsis thaliana. cotton, tobacco, tomato, potato, sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, legumes, alfalfa, carrot, strawberry, poplars, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium, cactus, pigeon pea, corn, rice, canola, rapeseed, false flax, pennycress, cannabis, hops, herbs, wheat, and soy.

[0047] Non-limiting examples of topoisomerase II inhibitors that are suitable for inclusion in the combinations and kits disclosed herein include etoposide, doxorubicin and related molecules (daunorubicin, epirubicin, idarubicin, pirarubicin), teniposide, acridine-4- carboxamide, amsacrine, ellipticine, ICRF-193, and mitoxantrone and its derivatives (e.g., pixantrone) or a salt of any of the foregoing. In some embodiments, the topoisomerase II inhibitor comprises etoposide. In some embodiments, the topoisomerase II inhibitor is a salt of etoposide (e.g., a pharmaceutically acceptable salt of etoposide), such as, e.g., etoposide phosphate.

[0048] The topoisomerase II inhibitor(s) can be provided in a composition (e.g., a pharmaceutical composition) comprising one or more pharmaceutically acceptable carriers, or diluents. Numerous examples of pharmaceutically acceptable carriers are known in the art.

[0049] Compositions comprising a topoisomerase II inhibitor can further comprise one or more additional compounds, such as, for example, a compound that modulates one or more cellular processes in plants, an antimicrobial compound (e.g., an antibiotic), a plant nutrient (e.g., a mineral, a macronutrient, a micronutrient, a vitamin), an herbicide, an inhibitor of DNA methylation or histone methylation, mutagens such as alkylating agents, base analogs, DNA intercalating agents, heavy metals, or a plant hormone.

[0050] The amount or concentration of topoisomerase II inhibitor that is included in a composition can be determined empirically by a person of ordinary skill in the art to which the invention pertains (e.g., using methods described in the Examples herein). For example, a composition suitable for use in the methods disclosed herein can comprise at least about 80 pM topoisomerase II inhibitor (e.g., at least about 80 pM etoposide or a salt thereof), or from about 80 pM to about 380 pM topoisomerase II inhibitor (e.g., from about 80 pM to about 380 etoposide or a salt thereof), such as about 160 pM to 380 pM topoisomerase II inhibitor (e.g., about 160 pM to about 380 etoposide or a salt thereof). Thus, a composition suitable for use in the methods disclosed herein can comprise about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, about 300 pM, about 310 pM, about 320 pM, about 330 pM, about 340 pM, about 350 pM, about 360 pM, about 370 pM, or about 380 pM topoisomerase II inhibitor (e.g., etoposide or a salt thereof). In some embodiments, a composition suitable for use in the methods disclosed herein comprises less than about 80 pM topoisomerase II inhibitor (e.g., less than about 80 pM etoposide or a salt thereof). In some embodiments, a composition suitable for use in the methods disclosed herein comprises greater than about 380 pM topoisomerase II inhibitor (e.g., greater than about 380 pM etoposide or a salt thereof).

[0051] In some embodiments, the combinations and kits disclosed herein further comprise a plant growth medium. The plant growth medium can be a solid, liquid, or semisolid designed to support the growth of a population of plants, plant parts, and / or plant cells. Suitable plant growth media for inclusion in the combinations and kits disclosed herein include various types of soil (e.g., nutrient-supplemented soil, naturally occurring soil), plant growth media comprising agar (e.g., Murashige and Skoog (MSO or MS0) medium), and various plant cell culture media.Exemplification

[0052] Genomic structural variation (SV) insertions, deletions, duplications, translocations, and inversions are an important source of genetic and phenotypic novelty. Structural variants can create genetic novelty by fusing the coding regions of two genes or by altering cis-regulatory environments and gene expression patterns. In addition, deletions andduplications can alter gene or chromatin dosage (Murphy and Berger 2023, Brown et al.2020, Henikoff 1996). Per mutation, novel structural variants can have larger phenotypic effects than single nucleotide substitutions (Chiang et al. 2017) and have been selected for traits beneficial to the organism or for desirability to agriculturists (Zhou et al. 2019, Alonge et al. 2020).

[0053] Recent pan-genomic sequencing of multiple plant species has underscored the versatility and significance of structural variation to phenotypic variation, modem crop traits, and crop improvement. Lower seed sets in watermelons and grapes, important for the development of popular “seedless” varieties, have been linked to large inversions, which can cause meiotic defects (Tian et al. 2021, Wang et al. 2024). SVs have been linked to the domestication of broomcorn millet (Chen et al. 2023) and sorghum (Tao et al. 2021). In pearl millets, gain of heat tolerance has been linked to SVs at multiple loci (Yan et al. 2023). SVs are also associated with variation in immunity and plant defense responses (Hufford et al.2021, Gbktay et al. 2021, Mizuno et al. 2020). In Arabidopsis. a multi-enzyme pathway synthesizes the glucosinolate family of defense compounds. SVs that either delete or fuse enzyme-encoding paralogs modify enzymatic pathways and are responsible for ecotypespecific variation in glucosinolates (Katz et al. 2021). Studies of 0. sativa provide numerous examples of how SVs influence phenotypes. Changes in copy number of VIL1 is associated with changes to flowering time and grain number variation (Qin et al. 2021). In addition, duplications of the KALA4 gene — a regulator of the anthocyanin biosynthesis pathway — created a novel cis-regulatory region that promoted ectopic expression of anthocyanins and caused a “black pericarp” phenotype (Qin et al. 2021). In peaches, fruit flesh color around the stone and fruit shape are dependent on a deletion and a mega-base scale inversion (Guo et al. 2020). Modern varieties of sweet corn were created by selecting for an inversion that creates a loss-of-function mutation in the shrunken-2 gene, which encodes for the first enzyme of the starch biosynthesis pathway (Hu et al. 2021). In tomatoes, a duplication of a cytochrome P450 gene was linked to increased fruit weight (Alonge et al. 2020).

[0054] Extant crop genetic diversity is unlikely to provide all possible SV diversity that could impact crop improvement. Induced structural variation can, therefore, provide novel germplasm for breeding programs, and information that can be used for CRISPR-mediated targeted restructuring of crop genomes. Two methods are currently employed to induce random structural variation. The most common is exposure to ionizing radiation including X- rays, gamma irradiation, and heavy ions (Ishii et al. 2024, Du et al. 2017, Jankowicz-Cieslaket al. 2022, Henry et al. 2015, Ichida et al. 2019, Ma et al. 2021). Starting in the 1960’s, irradiation has been used to induce structural variation in numerous crops, including more recent examples in A napus (Havlickova et al. 2024), wheat (Xiong et al. 2023), cotton (Zhao et al. 2022), rice (Li et al. 2019, Ichida et al. 2019), poplar (Henry et al. 2015, Guo et al. 2021), soybean (Bolon et al. 2014, Feng et al. 2023), and buckwheat (Yasui et al. 2012). Populations carrying such structural variation have been used for the isolation of mutations that abolish hybrid sterility in rice (Koide et al. 2018) and the creation of seeds differing in oil compositions (Havlickova et al. 2024, Serson et al. 2024).

[0055] While radiation-induced structural variation remains an important element of modern plant breeding, it is logistically challenging. Radiation sources are heavily regulated and access to nuclear reactors, particle accelerators, and other sources of radiation is a bottleneck in the creation of structural variant libraries (Ma et al. 2021). One alternative is the Extended TAQing approach, in which a transgene expresses a restriction enzyme to induce conditional double-stranded DNA breaks and shuffle the plant genome (Tanaka et al. 2020). This approach can be challenging as the commercial varieties of many crops are not easily transformed.

[0056] To surmount the current challenges of creating SVs in plants, a simple, inexpensive, drug-based system that can be used in labs with minimal resources to induce SVs in plants is disclosed herein. The disclosure provided herein provides proof-of-principle that the chemotherapeutic drug etoposide can efficiently generate novel genomic structural variation in plants.Materials and MethodsPreparation of Drugs

[0057] A 0.5 M stock of HydroxyUrea was made by dissolving an appropriate weight of hydroxyurea in water and adding it to 0.5x Murashige and Skoog / Phytagar medium (MS media) supplemented with 2% sucrose to a range of concentrations between 0 and 16 mM. Hydroxyurea was also administered hydroponically at 0, 0.5, and 1 mM concentrations. A 5 mM stock solution of Aphidicolin was made by dissolving 1 mg of Aphidicolin (EMD Millipore Catalog No. 178273) in 590 pL of dimethyl sulfoxide (DMSO). This solution was added to MS media plates to a final concentration of 0, 0.1, 0.4, 0.8, and 1.2 pM. 100 mg of etoposide (Abeam Catalog No. AB 120227), an inhibitor of Topoisomerase II, was dissolvedin 1.62 mL of DMSO to obtain a 100 mM stock solution that was stored at -20 °C. Etoposide (Abeam Catalog No. AB 120227) was dissolved in DMSO to obtain a 100 mM solution that was mixed with MS media to final concentrations of 0, 20, 40, 80, 160, 320, and 640 pM. For topical application of etoposide, etoposide was diluted in an aqueous solution of 0.2% Silwet to a final concentration of 100, 300, or 600 pM. Etoposide had decreased solubility in water at concentrations beyond 300 pM.Application of Etoposide to Seeds

[0058] The application of etoposide to seeds varied by species. Seeds for different species were germinated on appropriate growth media supplemented with different concentrations of etoposide. The lowest concentration triggering a growth defect was then used to create a mutagenized population of parent plants.

[0059] Wild-type Arabidopsis thaliana seeds (Col-0) were sterilized in plant protection media (Plant Cell Technology) for 4-5 days at 4 °C and plated on 0.5x Murashige and Skoog / 1% sucrose / 0.8% bactoagar media supplemented with DMSO alone (control), or with etoposide dissolved in DMSO to a final concentration of 40, 80, or 160 pM etoposide. Seeds were germinated and grown on this media for two weeks, and seedlings were examined for signs of stunted root or shoot growth. Comparisons with control plates showed that while seeds germinated normally in the presence of etoposide, a root growth defect was discernible in two-week-old seedlings grown on 160 pM etoposide. Two to three-week old seedlings, including several showing stunted growth, were transplanted to soil, grown in a Conviron growth chamber under 16 hours of light at approximately 22 °C, and allowed to set seed. Control seedlings that had not been exposed to etoposide were planted as controls. These transplants were together labeled the M0 generation. Seeds were collected from the mature M0 plants. Seeds collected from mutagenized plants were labeled Ml. 24-100 seeds each from 24 M0 etoposide-exposed plants and five controls were planted on soil giving rise to Ml generation plants that were screened for altered appearance or phenotype. It was found that at least 40% of M0 lines exposed to etoposide produced progeny with an altered phenotype. Inheritance of the phenotype was tracked over multiple generations.

[0060] Pigeon pea (Cajanus cajari) seeds were germinated and grown under 12 hours of light at 25 °C for two to three weeks on hydroponic media (General Hydroponics HGC718281 MaxiBloom Plant Food For Flowers) that was supplemented with 160 pM or320 pM etoposide. At 320 pM etoposide, pigeon pea seedlings exhibited a stunted root formation.Phenotyping

[0061] Up to 75 seeds from each Ml line were planted over three phases. M2 plants were obtained and plants with visible phenotypes were manually identified. In cases where two or more M2 plants from an Ml progenitor showed potential leaf shape or size differences, M2 leaves were detached at 3 weeks after germination and attached with double-sided tape on white A4 sheets. These sheets were scanned using an Epson V600 scanner. Leaf circularity, blade length, perimeter and area, as well as petiole length were measured.Sequencing to establish the presence of structural variation

[0062] Based on etoposide’s ability to inhibit topoisomerase II and induce doublestranded breaks, it was speculated that the altered phenotypes that were observed in Ml plants were caused by genomic structural variation. This variation was represented by chromosomal rearrangements, inversions, deletions and duplications. To test this hypothesis, whole genome sequencing of individual plants was carried out. These included: a) One control plant descended from a DMSO-treated Ml . b) A positive control plant that was phenotypically different from a wild-type diploid plant and was derived from a pool of self-fertilized triploids and thus expected to have structural variation. c) M2 and M3 plants from 12 Ml founders, five of which had a mutant phenotype. To establish the stability of transmission of a variant, multiple M2 siblings were sequenced for two Ml lines. To establish technical replication, two leaves each were sequenced for an M2 line with a mutant phenotype. d) M4 plants.

[0063] Genomic DNA was isolated from the rosette leaves of the plants using the cetyltrimethylammonium bromide (CTAB) method. Briefly, a leaf fragment for each plant being tested was collected from three- to six-week-old plants and was flash-frozen in liquid nitrogen. The frozen leaves were then homogenized on a Qiagen TissueLyser II with the settings of 30 beats / second for 10 seconds. 200 pl of 2X CTAB buffer (2% CTAB, 100 mM Tris (pH 8.0), 20 mM ethylenediaminetetraacetic acid (EDTA), 1.4 M NaCl, 1% polyvinylpyrrolidone (PVP; molecular weight = 40,000) was added to each tube. The pulverized leaf tissue was resuspended in CTAB buffer and heated to 65 °C for 30 minutes.200 pl of chloroform was added to each tube, vortexed, and then centrifuged for ten minutes. The aqueous phase was then transferred to a tube containing 500 pl of ethanol and mixed. These tubes were then stored at -20 °C for 20 minutes and then centrifuged at maximum speed for 15 minutes. The supernatant was discarded and the DNA pellet was washed twice with 70% ethanol. Tubes were spun down and the residual ethanol was removed with a pipette. The DNA pellet was air-dried for 10 minutes at 37 °C and resuspended in nuclease- free water overnight at room temperature. DNA concentration was measured by Qubit and up to 15 ng of genomic DNA was used by the BioMicroCenter at MIT to construct DNA sequencing libraries using the Nextera Flex protocol. Each of these libraries was sequenced in the Illumina 150 bp paired-end format to a depth of about 20 million reads using an Illumina NovaSeq S4. DNA sequence for each library was aligned to the “telomere-to-telomere” A. thaliana assembly (Naish et al. 2021) using BWA (Burrows-Wheeler Aligner) or to the Col-0 Centromere assembly using Bwa mem set to default options.

[0064] Structural variants were identified by two separate methods. Larger deletions and duplications were identified using a method proposed by the Comai Lab (Henry et al. 2015). In brief, aligned reads output by BWA was used to calculate read depth over non-overlapping 100 kb windows. Number of reads per million (rpm) that mapped to 100 kb windows across the genome, or fraction read depth, was calculated by the formula “reads in a 100 kb window / total number of reads in the library”. Then, the median read depth for that window across all sequenced individuals was calculated. As most lines were not expected to share a structural variant, this median read depth was considered to be the wild-type genome dosage and was set to a ploidy of two. The true ploidy of a 100 kb window was determined by the formula “(fraction read depth in that individual for window ‘a’ / median read depth for that window ‘a’ across all individuals) x 2” or “(rpm of a line / median rpm for all lines) x 2”. High confidence duplications or deletions were defined as a 100 kb window having a dosage greater than or equal to 3 or less than or equal to 1. Deletions or duplications smaller than 100 kb have not been investigated.

[0065] Inversions and structural variation were identified using LUMPY Express using a previously benchmarked pipeline (Gbktay et al. 2021). Briefly, Samtools was used to identify discordant and split reads from the SAM file output by BWA. Files containing discordant reads, split reads, and all reads were used by LUMPY Express to identify putative structural variants. Due to mapping artifacts, LUMPY Express and other variant callers often identify false-positive structural variant candidates at repetitive regions. Bedtools multiinter wasutilized to identify and remove structural variants occurring in three or more independent M2 lines. In addition, all structural variants that were also detected in an M2 line derived from a control Ml plant treated only with DMSO were removed.Nanopore sequencing and identification of structural variants

[0066] Two mature leaves from 32-day-old plants were flash frozen in liquid nitrogen. Frozen tissue was ground into a powder with a TissueLyser II (QIAGEN; 30 beats / second for 1 minute). High molecular weight DNA was extracted using a Wizard HMW DNA Extraction Kit (Promega). DNA was sheared by passing through a 23G needle twice. Contaminating RNA and proteins were removed using RNase A (New England Biolabs) and Proteinase K (New England Biolabs). DNA was further cleaned and small fragments removed using a modified solid-phase reversible immobilization (SPRI) bead mixture in which the buffer of Agencourt AMPure XP beads was exchanged with a custom buffer of 10 mM Tris-HCl, 1 mM EDTA pH 8, 1.6 M NaCl, 11% w / v polyethylene glycol (PEG) 8000 (Schalamun et al. 2018). To perform the size selection, in brief, 0.7x volume of modified bead solution was added to each DNA sample, then incubated on a nutator for 10 minutes. Samples were placed on a magnetic stand to bind the beads to the side of the tube, then washed twice with 70% ethanol. Beads were not allowed to dry before adding nuclease-free water to elute the DNA. To increase yield and remove residual ethanol, samples were incubated at 50 °C for 2 minutes and then at room temperature for two hours with the lids open.

[0067] All library preparation and sequencing was performed at the MIT BioMicro Center. For two samples showing the BR-like dwarf phenotype, high molecular weight DNA extractions had low yield. For these samples and corresponding controls (individual from sibling line without dwarf phenotype and individual from a line treated with DMSO), library preparation was performed utilizing a Rapid Barcoding Kit VI 0 (Oxford Nanopore Technologies Catalog No. SQK-RBK110-96). These four samples were multiplexed and sequenced on a single PromethlON R9.4.1 flow cell (Oxford Nanopore Technologies Catalog No. FLO-PRO002). For all other samples, library preparation was performed with a Native Barcoding Kit V14 (Oxford Nanopore Technologies Catalog No. SQK-NBD114-24). These samples were multiplexed into groups of three and each group was sequenced on its own PromethlON R10.4.1 flow cell (Oxford Nanopore Technologies Catalog No. FLO- P1RO14M). Both runs utilized the following software versions for sequencing and basecalling: MinKNOW 23.04.6, Bream 7.5.10, Configuration 5.5.14, Guppy 6.5.7, and MinKNOW Core 5.5.5. On average, the former group yielded 4.26 Gbp per sample with an N50 of 8,026 bp and the latter group yielded 24.02 Gbp per sample with an N50 of 10,586 bp.

[0068] Nanopore reads were filtered to select the best reads in terms of length and quality, up to a max coverage of 50x, utilizing Filtlong —min length 1000 —minywindow q 40 —trim —split 1500 —target bases 6605000000), Filtered reads were mapped to the Col- CEN vl.2 genome using Vulcan dual-mode alignment pipeline (Fu et al. 2021), which leverages both minimap2 (Li 2018) and NGMLR (Sedlazeck et a. 2018), using default settings for ONT reads. Structural variants in each sample were detected with cuteSV (— max cluster bias INS 100 —diff ratio merging INS 0.3 —max cluster bias DEL 100 — diff ratio merging DEL 0.3 —min size 30 —max size -1 -genotype; Jiang et al. 2020, Jiang et al. 2023). SVs identified in all samples were merged with SURVIVOR (merge 1000 1 1 1 0 30; Jeffares et al. 2017), then samples were genotyped with cuteSV for all SVs identified (- -min mapq 20 —max cluster bias INS 100 —diff ratio merging INS 0.3 — max cluster bias DEL 100 —diff ratio merging DEL 0.3 —min size 30 —max size -7). Structural variants present in the DMSO and / or Col-0 controls, present in more than one phenotypic line, or with a quality less than 20 were removed. Data was visualized in R using ggplot2 (Wickham 2016).RNA sequencing and analysis

[0069] RNA sequencing was conducted on M3 generation plants. Six plants of each BR- like dwarf short-internode dwarf and virescent phenotypes, as well as four non-phenotypic individuals corresponding to sibling lines of each phenotype were sequenced. For the fraction variegated line, thirteen green plants (three of which produced -25% variegated progeny) and one white plant were sampled. Nine M3 progeny of DMSO-treated plants were included as controls.

[0070] Mature leaf tissue from 32-day-old plants was flash frozen in liquid nitrogen. Frozen tissue was ground into a powder with a TissueLyser II (QIAGEN; 30 beats / second for 30 seconds). RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN). Library preparation was performed at the MIT BioMicro Center using an NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Samples were multiplexed on one lane of an Illumina NovaSeq 600 S4 flow cell and sequenced at the MIT BioMicro Center on an Illumina NovaSeq 6000 with 150 bp paired end reads.

[0071] Adapters and low-quality ends were removed from reads with Trim Galore (— paired —retain unpaired -q 25). Salmon (Patro et al. 2017) was used to quantify the expression of transcripts, utilizing the Araport 11 transcriptome as the reference and the Col- CEN vl.2 genome (Naish et al. 2021) as decoy sequences. Six samples were identified as outliers and removed from the dataset. DESeq2 (Love et al. 2014) was used to quantify differential expression; for a gene to be considered it must have at least 10 normalized counts in four or more samples. Each phenotype of interest was analyzed independently; DESeq2 design (~ treatment + phenotype) incorporated both treatment (etoposide, DMSO, or none) and phenotype (present or absent). Data was visualized utilizing R and packages ggplot2 (Wickham 2016), Enhanced Volcano (Blighe et al. 2018), and pheatmap.Results

[0072] To create genomic structural variants, the impact of three different drugs (etoposide, aphidicolin, and hydroxyurea) on the model organism A. thaliana was examined. Treatment with 10 pM aphidicolin did not lead to any observable growth defect or any detectable novel traits / phenotypes in a preliminary observation of several hundred plants. Although treatment with hydroxyurea did inhibit growth of the parent plants, starting at 10 mM, examination of several hundred progeny showed no evidence of plants with novel traits. Etoposide on the other hand inhibited growth of parents and there was a high frequency of visible mutants that were identified among the progeny. Etoposide is a well characterized, non-proprietary, anti-cancer drug that inhibits the activity of the topoisomerase II class of enzymes. Topoisomerase II inhibition leads to chromosomal breakage, which when repaired by the cell, can result in heritable structural variation.

[0073] Accordingly, to induce structural variation, wild-type Col-0 seeds were germinated and grown on MS media supplemented with sucrose and differing concentrations of etoposide (0 pM or DMSO only, 40 pM, 80 pM, and 160 pM) for up to two weeks. Plants grown on DMSO alone showed no observable growth defects. Two-week-old seedlings grown on 40 pM of etoposide exhibited gnarled leaves but no differences in size. Seedlings growing on 80 pM etoposide showed marginally reduced growth. Of the seedlings grown on 160 pM of etoposide, less than half (75 / 184) exhibited true leaves. In addition, these plants showed stunted root development (FIG. 1 A). Seedlings grown on DMSO only, 80 pM and 160 pM etoposide (referred to as Ml plants) were transplanted to soil and compared atmaturity. Plants exposed to 160 pM of etoposide exhibited significantly more abnormal phenotypes than DMSO only or 80 pM etoposide, including loss of apical dominance, gnarled leaves, reduced plant size, seed abortion and lower seed number at maturity. For thaliana, it was found that a 160 pM concentration of etoposide lead to a reduction in root length and seedling growth (FIG. 1 A) while for pigeon pea, this concentration was found to be 320 pM (FIG. IF. These results suggest that the exact concentration of etoposide to be used may vary by species.

[0074] Seedlings grown on etoposide for 2 weeks were transferred onto soil and seeds were collected for each individual founder plant. 24 to 100 progeny of each etoposide-treated parent were screened either in optimal conditions or under heat stress, for visible phenotypes that impacted shoot architecture, flowering time, floral morphology, fertility and seed abortion. Thus far, 10 out of 25 screened lines showed a visible phenotype (FIGs. 2A-E) suggesting that this technology was efficient at generating genetic variation. It was also found that the variation generated by this technique can be stably transmitted to future plant generations.

[0075] To confirm that plants with visible phenotypes had a structural variation, DNA was extracted from plants with visible phenotypes and sequenced and regions with structural variation were identified by computational analyses. Some lines without an abnormal phenotype were also sequenced to assess how many plants without visible phenotypes also have structural variation. Through sequencing, 18 putative structural variants (duplications, deletions, inversions) among the progeny of 13 etoposide-treated parents were detected, underlining the efficacy of this technology (see FIG. 3 for examples of increased copy number after etoposide treatment).

[0076] To test if these abnormal plants produced progeny with mutant phenotypes, the progeny (M2) of plants grown at 160 pM etoposide were examined. 24 / 37 M2 progeny derived from different Ml parents exhibited at least one visible phenotype (Table 1).

[0077] Table 1 : List of phenotypes observed in progeny of etoposide treated (Ml) plants.

[0078] These phenotypes included variegated or albino plants, plants with altered flowering time, leaf shape and size (FIGs. 6A-H), plants exhibiting sterility or seed abortion,and dwarf plants. The large proportion of plants showing visible phenotypes suggested that etoposide was an excellent mutagen for efficiently creating large-effect mutations.

[0079] To assess the heritability of the phenotypes and to establish genotype-phenotype relationships, four M2 lines with visible phenotypes were closely examined. These included: a line with a variegated phenotype with varying proportions of green and white sectors; a line with delayed greening, or virescent (Line 13B); a dwarf line reminiscent of mutations in the brassinosteroid response pathway (Line 1 A), referred to as brassinosteriod-like dwarf, and a dwarf plant with short internodes, or short internode dwarf (Line 34C). The virescent line exhibited variegation in chlorophyll pigmentation in juvenile plants that vanished in adult plants (FIG. IB, lower left). The BR-like dwarf (FIG. IB, lower center) exhibited short, thick stems that bear fleshy leaves and was semi-sterile. In contrast, the short internode dwarf mutant, while short in stature, was fertile, had the same number of siliques as wild-type plants, and lacked the fleshy leaf phenotype observed in the BR dwarf (FIG. IB, lower right).

[0080] These phenotypes were transmitted from self-fertilized parents to offspring for at least two additional monitored generations and suggest that mutations are true-breeding and the underlying mutant genotype was stable. To assess whether phenotypes were dominant or recessive, the mutant lines, except for variegated, were crossed to wild-type. Fl progeny from the virescent line and the short-internode dwarf lines exhibited no phenotype, indicating that these phenotypes are recessive (FIGs. 1C, IE). The Fl progeny from the BR-like dwarf exhibited intermediate dwarfing, suggesting that the phenotype was incompletely dominant (FIG. ID).

[0081] To estimate the number of loci contributing to each phenotype, F2 progeny obtained by self-fertilizing the Fl plants were examined. 20.9% of the progeny generated by self-fertilizing the virescent line displayed a mutant phenotype (FIG. 1C). This was consistent with the phenotype being cause by a mutation at a single locus (%2= 1.699, two-tailed p-value = 0.1924). F2 progeny of the BR-like dwarf generated 26.5% plants with a wild-type phenotype, 58% with an intermediate phenotype, and 15.5% with a severe mutant phenotype (FIG. ID). This result suggests that a single incompletely dominant locus causes the BR-like t / nw phenotype (%2= 3.030, two-tailed p-value = 0.08). The short-internode dwarf phenotype was observed in 4.5% of progeny obtained by self-fertilizing F 1 plants, which was consistent with a phenotype arising when two mutant loci are present (%2= 1.699, two-tailed p-value = 0.9393,' FIG. IE). These observations suggest that etoposide mutagenesis cancreate novel phenotypes by efficiently generating a broad spectrum of large-effect single and double mutants.

[0082] To characterize the molecular nature of etoposide-induced mutations, Illumina short-read technology was employed to sequence M2 lines derived from four control Ml lines treated only with DMSO and 33 M2 and M3 progeny of etoposide-treated lines with and without visible phenotypes. As a control, one triploid plant obtained from crossing a diploid and a tetrapioid was also sequenced. Triploid plants often exhibit structural variation and aneuploidy. The sequenced, etoposide-mutagenized lines included siblings of plants exhibiting virescent, BR-like dwarf, and short-internode dwarf phenotypes. The sequencing of siblings with and without visible phenotypes allowed for accurately identifying mutations that potentially cause the phenotypes. To identify novel structural variants (SVs) in these data, LUMPY Express (Gbktay et al. 2021) was employed. This pipeline identified nearly 2,224 variants that included inversion, deletions, duplications, and breakpoints. The predicted SVs included a high proportion mapping to Nucleolar Organizer Regions, plastid genomes, and a small subset of other genomic loci. These high frequency SVs were found across control and multiple mutant lines suggesting that they are likely to be false positives or are non-reference SVs found in lab stocks of wild-type Col-0. SVs that were found in control lines and in more than four M2 / M3 lines that were descended from different MO lines were therefore filtered out. After filtering out potential false positives, 232 SVs across 32 lines were identified. LUMPY and other short read-based SV callers may not detect segmental duplications or deletions. Therefore, a computational approach that uses genome-wide coverage to identify segmental deletions or duplications was adapted. Using this technique, 6 duplications and 1 deletion in the genome were identified (FIG. 4A).

[0083] Long-read sequencing can identify structural variants that are not easily resolved or cannot be identified by short-read sequencing. Using Nanopore technology, the genomes of two M3 plants with the BR-like dwarf short-internode dwarf and virescent phenotypes, one albino plant from the variegated line, and a plant from each corresponding sibling line that lacked the phenotype, as well as a single plant from the variegated line that was green but produced a proportion of variegated progeny were sequenced (FIGs. 4B). To control for structural variation already present in wild-type stocks compared to the reference genome, two M3 plants that were the progeny of Ml plants grown on DMSO only and an untreated Col-0 plant were also sequenced. In total, 12 SVs were identified: seven deletions (ranging from 37 bp to 176 bp long), two insertions (71 bp and 184 bp), two inversions (1.52 Mb and3.54 Mb), and one translocation (FIGs. 4A-E). A third of these SVs were also confirmed via PCR, including the 1.52 Mb inversion (FIG. 4B).

[0084] To quantify the changes in gene expression associated with each phenotype, RNA sequencing was performed for M3 plants of BR-like dwarf, short-internode dwarf, variegated, and virescent phenotypes. Non-phenotypic individuals corresponding to sibling lines of each phenotype and M3 progeny of DMSO-treated plants were included as controls. Plants that exhibited the BR-like dwarf phenotype showed misregulation of 309 genes (114 downregulated and 195 upregulated; FIG. 5 A). Notably, the differentially expressed genes included AT2G37630 (ASYMMETRIC LEAVES 7; AST), which was located at the right breakend of the 1.52 Mb inversion on chromosome 2 that was present in BR-like dwarf plants. The inversion resulted in the first 42 bp of the 5’UTR of AS1 to be separated from the rest of the gene (FIG. 5B). Previously-identified mutations in AS1 share a similar phenotype to the BR-like dwarf suggesting that the misregulation of AS1 induced by the inversion is causal for the phenotype. Short-internode dwarf plants showed considerably less expression changes in leaves: 18 genes were downregulated and 26 were upregulated compared to nondwarf plants (FIG. 5D). Genes in windows surrounding the large 3.54 Mb inversion on chromosome 4 which was present in short-internode dwarf plants did not show increases or decreases in average fold change (FIG. 53E). Virescent plants had more misregulated genes than either dwarf phenotype, with a total of 1273 differentially expressed genes (FIG. 5F). While no structural variants were identified via long read sequencing in virescent plants, genes in windows near the nuclear insertion of the mitochondrial genome (NuMt) on chromosome 2 had lower average fold changes than elsewhere on the chromosome (FIG.5G). Differential expression analysis could not reliably be conducted for the variegated line, due to few variegated plants recovered. As an alternative strategy, expression of 18 genes known to be associated with variegation was examined (FIG. 5H). In the one white plant recovered, AT4G22260 (I M I NANS,' IM) stood out as a downregulated gene compared to all other green plants (FIGs. 5H, 5J). Long-read sequencing identified a 37 bp deletion in the third exon of IM,' a frame-shift mutation resulting in a premature stop codon (FIG. 51), suggesting that the deletion in IM underlies the mutant phenotype.References

[0085] Alonge, M., Wang, X., Benoit, M., Soyk, S., Pereira, L., Zhang, L., Suresh, H., Ramakrishnan, S., Maumus, F., Ciren, D., Levy, Y., Harel, T.H., Shalev-Schlosser, G.,Amsellem, Z., Razifard, H., Caicedo, A.L., Tieman, D.M., Klee, H., Kirsche, M., Aganezov, S., Ranallo-Benavidez, T.R., Lemmon, Z.H., Kim, J., Robitaille, G., Kramer, M., Goodwin, S., McCombie, W.R., Hutton, S., Eck, J.V., Gillis, J., Eshed, Y., Sedlazeck, F.J., Knaap, E. van der, Schatz, M.C., Lippman, Z.B., 2020. Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato. Cell 182, 145-161. e23. https: / / doi.Org / 10.1016 / j.cell.2020.05.021

[0086] Blighe, K., Rana, S., Lewis, M., 2018. EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling.

[0087] Bolon, Y.-T., Stec, A.O., Michno, J.-M., Roessler, J., Bhaskar, P.B., Ries, L., Dobbels, A.A., Campbell, B.W., Young, N.P., Anderson, J.E., Grant, D.M., Orf, J.H., Naeve, S.L., Muehlbauer, G.J., Vance, C.P., Stupar, R.M., 2014. Genome resilience and prevalence of segmental duplications following fast neutron irradiation of soybean. Genetics 198, 967- 981. https: / / doi.org / 10.1534 / genetics.114.170340

[0088] Brown, E.J., Nguyen, A.H., Bachtrog, D., 2020. The Drosophila Y Chromosome Affects Heterochromatin Integrity Genome-Wide. Molecular Biology and Evolution 37, 2808-2824. https: / / doi.org / 10.1093 / molbev / msaa082

[0089] Chen, I, Liu, Y., Liu, M., Guo, W., Wang, Y., He, Q., Chen, W., Liao, Y., Zhang, W., Gao, Y., Dong, K., Ren, R., Yang, T., Zhang, L., Qi, M., Li, Z., Zhao, M., Wang, Haigang, Wang, J., Qiao, Z., Li, Haiquan, Jiang, Y., Liu, G., Song, X., Deng, Y., Li, Hai, Yan, F., Dong, Y., Li, Q., Li, T., Yang, W., Cui, J., Wang, Hongru, Zhou, Y., Zhang, X., Jia, G., Lu, P., Zhi, H., Tang, S., Diao, X., 2023. Pangenome analysis reveals genomic variations associated with domestication traits in broomcorn millet. Nat Genet 55, 2243-2254. https: / / doi.org / 10.1038 / s41588-023-01571-z

[0090] Chiang, C., Scott, A.J., Davis, J.R., Tsang, E.K., Li, X., Kim, Y., Hadzic, T., Damani, F.N., Ganel, L., Montgomery, S.B., Battle, A., Conrad, D.F., Hall, I.M., 2017. The impact of structural variation on human gene expression. Nat Genet 49, 692-699. https: / / doi.org / 10.1038 / ng.3834

[0091] Du, Y., Luo, S., Li, X., Yang, J., Cui, T., Li, W., Yu, L., Feng, H., Chen, Y., Mu, J., Chen, X., Shu, Q., Guo, T., Luo, W., Zhou, L., 2017. Identification of Substitutions and Small Insertion-Deletions Induced by Carbon-Ion Beam Irradiation in Arabidopsis thaliana. Front. Plant Sci. 8. https: / / doi.org / 10.3389 / fpls.2017.01851

[0092] Feng, Z., Du, Y., Chen, J., Chen, X., Ren, W., Wang, L., Zhou, L., 2023. Comparison and Characterization of Phenotypic and Genomic Mutations Induced by aCarbon-Ion Beam and Gamma-ray Irradiation in Soybean (Glycine max (L.) Merr.). International Journal of Molecular Sciences 24, 8825. https: / / doi.org / 10.3390 / ijms24108825

[0093] Fu, Y., Mahmoud, M., Muraliraman, V.V., Sedlazeck, F.J., Treangen, T.J., 2021. Vulcan: Improved long-read mapping and structural variant calling via dual-mode alignment.GigaScience 10, giab063. https: / / doi.org / 10.1093 / gigascience / giab063

[0094] Gbktay, M., Fulgione, A., Hancock, A.M., 2021. A New Catalog of Structural Variants in 1,301 A. thaliana Lines from Africa, Eurasia, and North America Reveals a Signature of Balancing Selection at Defense Response Genes. Molecular Biology and Evolution 38, 1498-1511. https: / / doi.org / 10.1093 / molbev / msaa309

[0095] Guo, J., Cao, K., Deng, C., Li, Y., Zhu, G., Fang, W., Chen, C., Wang, X , Wu, I, Guan, L., Wu, S., Guo, W., Yao, J.-L., Fei, Z., Wang, L., 2020. An integrated peach genome structural variation map uncovers genes associated with fruit traits. Genome Biol 21, 258. https: / / doi.org / 10.1186 / sl3059-020-02169-y

[0096] Havlickova, L., He, Z., Berger, M., Wang, L., Sandmann, G., Chew, Y.P., Yoshikawa, G.V., Lu, G., Hu, Q., Banga, S.S., Beaudoin, F., Bancroft, I., 2024. Genomics of predictive radiation mutagenesis in oilseed rape: modifying seed oil composition. Plant Biotechnol J 22, 738-750. https: / / doi.org / 10.l l l l / pbi.14220

[0097] Henikoff, S., 1996. Dosage-dependent modification of position-effect variegation in Drosophila. BioEssays 18, 401-409. https: / / doi.org / 10.1002 / bies.950180510

[0098] Henry, I.M., Zinkgraf, M.S., Groover, A.T., Comai, L., 2015. A System for Dosage-Based Functional Genomics in Poplar. The Plant Cell27, 2370-2383. https: / / doi.org / 10.1105 / tpc.15.00349

[0099] Hu, Y., Colantonio, V., Muller, B.S.F., Leach, K.A., Nanni, A., Finegan, C., Wang, B., Baseggio, M., Newton, C.J., Juhl, E.M., Hislop, L., Gonzalez, J.M., Rios, E.F., Hannah, L.C., Swarts, K., Gore, M.A., Hennen-Bierwagen, T.A., Myers, A.M., Settles, A.M., Tracy, W.F., Resende, M.F.R., 2021. Genome assembly and population genomic analysis provide insights into the evolution of modem sweet corn. Nat Commun 12, 1227. https: / / doi .org / 10.1038 / s41467-021-21380-4

[0100] Hufford, M.B., Seetharam, A.S., Woodhouse, M.R., Chougule, K.M., Ou, S., Liu, J., Ricci, W.A., Guo, T., Olson, A., Qiu, Y., Della Coletta, R., Tittes, S., Hudson, A.I., Marand, A.P., Wei, S., Lu, Z., Wang, B., Tello-Ruiz, M.K., Piri, R.D., Wang, N., Kim, D. won, Zeng, Y., O’Connor, C.H., Li, X., Gilbert, A.M., Baggs, E., Krasileva, K.V., Portwood, J.L., Cannon, E.K.S., Andorf, C.M., Manchanda, N., Snodgrass, S.J., Hufnagel, D.E., Jiang,Q., Pedersen, S., Syring, M.L., Kudma, D.A., Llaca, V., Fengler, K., Schmitz, R.J., Ross- Ibarra, J., Yu, J., Gent, J.I., Hirsch, C.N., Ware, D., Dawe, R.K., 2021. De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science 373, 655-662. https: / / doi.org / 10.1126 / science.abg5289

[0101] Ichida, H., Morita, R., Shirakawa, Y., Hayashi, Y., Abe, T., 2019. Targeted exome sequencing of unselected heavy-ion beam-irradiated populations reveals less-biased mutation characteristics in the rice genome. The Plant Journal 98, 301-314. https: / / doi.org / 10.l l l l / tpj.14213

[0102] Ishii, K., Kazama, Y., Hirano, T., Fawcett, J.A., Sato, M., Hirai, M.Y., Sakai, F., Shirakawa, Y., Ohbu, S., Abe, T., 2024. Genomic view of heavy-ion-induced deletions associated with distribution of essential genes in Arabidopsis thaliana. Front. Plant Sci. 15. https: / / doi.org / 10.3389 / fpls.2024.1352564

[0103] Jankowicz-Cieslak, J., Hofinger, B.J., Jarc, L., Junttila, S., Galik, B., Gyenesei, A., Ingelbrecht, I.L., Till, B.J., 2022. Spectrum and Density of Gamma and X-ray Induced Mutations in a Non-Model Rice Cultivar. Plants (Basel) 11, 3232. https: / / doi.org / 10.3390 / plantsl l233232

[0104] Jeffares, D.C., Jolly, C., Hoti, M., Speed, D., Shaw, L., Rallis, C., Balloux, F., Dessimoz, C., Bahler, J., Sedlazeck, F.J., 2017. Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Nat Commun8, 14061. https: / / doi.org / 10.1038 / ncommsl4061

[0105] Jiang, T., Cao, S., Liu, Y., Liu, S., Liu, B., Wang, G., Wang, Y., 2022.Regenotyping structural variants through an accurate force-calling metho. https: / / doi.org / 10.1101 / 2022.08.29.505534

[0106] Jiang T, Liu Y, Jiang Y, Li J, Gao Y, Cui Z, Liu Y, Liu B, Wang Y. Long-readbased human genomic structural variation detection with cuteSV. Genome Biol. 2020 Aug 3;21(1): 189. doi: 10.1186 / sl3059-020-02107-y.

[0107] Katz, E., Li, J.-J., Jaegle, B., Ashkenazy, H., Abrahams, S.R., Bagaza, C., Holden, S., Pires, C.J., Angelovici, R., Kliebenstein, D.J., 2021. Genetic variation, environment and demography intersect to shape Arabidopsis defense metabolite variation across Europe. eLife 10, e67784. https: / / doi.org / 10.7554 / eLife.67784

[0108] Koide, Y., Ogino, A., Yoshikawa, T., Kitashima, Y., Saito, N., Kanaoka, Y., Onishi, K., Yoshitake, Y., Tsukiyama, T., Saito, H., Teraishi, M., Yamagata, Y., Uemura, A., Takagi, H., Hayashi, Y., Abe, T., Fukuta, Y., Okumoto, Y., Kanazawa, A., 2018. Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice. Proceedings of the National Academy of Sciences 115, E1955-E1962. https: / / doi.org / 10.1073 / pnas.1711656115

[0109] Li, F., Shimizu, A., Nishio, T., Tsutsumi, N., Kato, H., 2019. Comparison and Characterization of Mutations Induced by Gamma-Ray and Carbon-Ion Irradiation in Rice (Oryza sativa L.) Using Whole-Genome Resequencing. G3 Genes|Genomes|Genetics 9, 3743-3751. https: / / doi.org / 10.1534 / g3.119.400555

[0110] Li H, Durbin R. Fast and accurate short read alignment with Burrows- Wheel er transform. Bioinformatics. 2009 Jul 15;25(14): 1754-60. doi: 10.1093 / bioinformatics / btp324.

[0111] Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018 Sep 15;34(18):3094-3100. doi: 10.1093 / bioinformatics / btyl91.

[0112] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DES eq2. Genome Biol. 2014;15(12):550. doi: 10.1186 / S13059-014-0550-8. PMID: 25516281; PMCID: PMC4302049.

[0113] Ma, L., Kong, F., Sun, K., Wang, T., Guo, T., 2021. From Classical Radiation to Modem Radiation: Past, Present, and Future of Radiation Mutation Breeding. Front Public Health 9, 768071. https: / / doi.org / 10.3389 / fpubh.2021.768071

[0114] Mizuno, H., Katagiri, S., Kanamori, H., Mukai, Y., Sasaki, T., Matsumoto, T., Wu, J., 2020. Evolutionary dynamics and impacts of chromosome regions carrying R-gene clusters in rice. Sci Rep 10, 872. https: / / doi.org / 10.1038 / s41598-020-57729-w

[0115] Murphy, P.J., Berger, F., 2023. The chromatin source-sink hypothesis: a shared mode of chromatin-mediated regulations. Development 150, dev201989. https: / / doi.org / 10.1242 / dev.201989

[0116] Naish, M., Alonge, M., Wlodzimierz, P., Tock, A.J., Abramson, B.W., Schmucker, A., Mandakova, T., Jamge, B., Lambing, C., Kuo, P., Yelina, N., Hartwick, N., Colt, K., Smith, L.M., Ton, J., Kakutani, T., Martienssen, R.A., Schneeberger, K., Lysak, M.A., Berger, F., Bousios, A., Michael, T.P., Schatz, M.C., Henderson, I.R., 2021. The genetic and epigenetic landscape of the Arabidopsis centromeres. Science374, eabi7489. https: / / doi.org / 10.1126 / science.abi7489

[0117] Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017 Apr;14(4):417-419. doi: 10.1038 / nmeth.4197. Epub 2017 Mar 6. PMID: 28263959; PMCID: PMC5600148.

[0118] Qin, P., Lu, H., Du, H., Wang, H., Chen, W., Chen, Z., He, Q., Ou, S., Zhang, H., Li, Xuanzhao, Li, Xiuxiu, Li, Y., Liao, Y., Gao, Q., Tu, B., Yuan, H., Ma, B., Wang, Y., Qian, Y., Fan, S., Li, W., Wang, J., He, M., Yin, J., Li, T., Jiang, N., Chen, X., Liang, C., Li, S., 2021. Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations. Cell 184, 3542-3558. el6. https: / / doi.Org / 10.1016 / j.cell.2021.04.046

[0119] Schalamun, M., Nagar, R., Kainer, D., Beavan, E., Eccles, D., Rathjen, J.P., Lanfear, R., Schwessinger, B., 2019. Harnessing the MinlON: An example of how to establish long-read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora. Molecular Ecology Resources 19, 77-89. https: / / doi.org / 10.l l l l / 1755- 0998.12938

[0120] Sedlazeck, F.J., Rescheneder, P., Smolka, M., Fang, H., Nattestad, M., von Haeseler, A., Schatz, M.C., 2018. Accurate detection of complex structural variations using single-molecule sequencing. Nat Methodsl5, 461-468. https: / / doi.org / 10.1038 / s41592-018- 0001-7

[0121] Serson, W.R., Gishini, M.F.S., Stupar, R.M., Stec, A.O., Armstrong, P.R., Hildebrand, D., 2024. Identification and Candidate Gene Evaluation of a Large Fast Neutron- Induced Deletion Associated with a High-Oil Phenotype in Soybean Seeds. Genes (Basel) 15, 892. https: / / doi.org / 10.3390 / genesl5070892

[0122] Tanaka, H., Muramoto, N., Sugimoto, H., Oda, A.H., Ohta, K., 2020. Extended TAQing system for large-scale plant genome reorganization. The Plant Journal 103, 2139- 2150. https: / / doi.org / 10.l l l l / tpj.14888

[0123] Tao, Y., Luo, H., Xu, J., Cruickshank, A., Zhao, X., Teng, F., Hathorn, A., Wu, X., Liu, Y., Shatte, T., Jordan, D., Jing, H., Mace, E., 2021. Extensive variation within the pan-genome of cultivated and wild sorghum. Nat. Plants 7, 766-773. https: / / d0i.0rg / l 0.1038 / s41477-021 -00925-x

[0124] Tian, S., Ge, J., Ai, G., Jiang, J., Liu, Q., Chen, X., Liu, M., Yang, J., Zhang, X., Yuan, L., 2021. A 2.09 Mb fragment translocation on chromosome 6 causes abnormalities during meiosis and leads to less seed watermelon. Horticulture Research 8, 256. https: / / doi .org / 10.1038 / s41438-021 -00687-9

[0125] Wang, X., Liu, Zhongjie, Zhang, Fan, Xiao, H., Cao, S., Xue, H., Liu, W., Su, Y., Liu, Zhenya, Zhong, H., Zhang, Fuchun, Ahmad, B., Long, Q., Zhang, Y., Liu, Y., Gan, Y., Hou, T., Jin, Z., Wu, X., Liu, G., Wang, Y., Peng, Y., Zhou, Y., 2024. Integrative genomicsreveals the polygenic basis of seedlessness in grapevine. Current Biology 34, 3763-3777. e5. https: / / doi.Org / 10.1016 / j.cub.2024.07.022

[0126] Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.

[0127] Xiong, H., Guo, H., Fu, M., Xie, Y., Zhao, L., Gu, I, Zhao, S., Ding, Y., Du, Q., Zhang, J., Qiu, L., Xie, X., Zhou, L., Chen, Z., Liu, L., 2023. A large-scale whole-exome sequencing mutant resource for functional genomics in wheat. Plant Biotechnol J 21, 2047- 2056. https: / / doi.Org / 10.l 111 / pbi.14111

[0128] Yan, H., Sun, M., Zhang, Z., Jin, Y., Zhang, A., Lin, C., Wu, B., He, M., Xu, B., Wang, Jing, Qin, P., Mendieta, J.P., Nie, G., Wang, Jianping, Jones, C.S., Feng, G., Srivastava, R.K., Zhang, X., Bombarely, A., Luo, D., Jin, L., Peng, Y., Wang, X., Ji, Y., Tian, S., Huang, L., 2023. Pangenomic analysis identifies structural variation associated with heat tolerance in pearl millet. Nat Genet 55, 507-518. https: / / doi.org / 10.1038 / s41588-023- 01302-4

[0129] Yasui, Y., Mori, M., Aii, J., Abe, T., Matsumoto, D., Sato, S., Hayashi, Y., Ohnishi, O., Ota, T., 2012. S-LOCUS EARLY FLOWERING 3 Is Exclusively Present in the Genomes of Short-Styled Buckwheat Plants that Exhibit Heteromorphic Self-Incompatibility. PLOS ONE 7, e31264. https: / / doi.org / 10.1371 / journal.pone.0031264

[0130] Zhao, Z., Liu, Z., Zhou, Y., Wang, J., Zhang, Y., Yu, X., Wu, R., Guo, C., Qin, A., Bawa, G., Sun, X., 2022. Creation of cotton mutant library based on linear electron accelerator radiation mutation. Biochem Biophys Rep 30, 101228. https: / / doi.Org / 10.1016 / j.bbrep.2022.101228

[0131] Zhou, Y., Minio, A., Massonnet, M., Solares, E., Lv, Y., Beridze, T., Cantu, D., Gaut, B.S., 2019. The population genetics of structural variants in grapevine domestication. Nat. Plants 5, 965-979. https: / / doi.org / 10.1038 / s41477-019-0507-8

[0132] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0133] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of introducing genetic variation in a plant, comprising: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant, thereby producing a plant having genetic variation.

2. The method of claim 1, wherein the topoisomerase II inhibitor comprises etoposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, teniposide, acridine-4- carboxamide, amsacrine, ellipticine, ICRF-193, mitoxantrone, pixantrone, or a salt of any of the foregoing.

3. The method of claim 1 or 2, wherein the plant cell or plant tissue that is capable of being propagated includes a cultured plant cell, plant callus tissue, an embryo, a seed, a stem, a root, a root tip, a root hair, a leaf, pollen, a cotyledon, a hypocotyl, a seedling, a pistil, an anther, a flower, a meristematic cell, or a whole plant, or any combination thereof.

4. The method of claim 1, 2 or 3, wherein the growth medium comprises a soil, a plant growth medium comprising agar, Murashige and Skoog medium with supplemented sucrose, Murashige and Skoog medium without supplemented sucrose, or a plant cell culture medium.

5. The method of any one of the preceding claims, wherein the genetic variation comprises a structural variation.

6. The method of claim 5, wherein the structural variation comprises a change in copy number of at least one chromosomal region.

7. The method of claim 6, wherein the change in copy number of at least one chromosomal region comprises at least one duplication event.

8. The method of claim 7, wherein the duplication event is characterized by an increase in at least one gene copy number.

9. The method of claim 6, wherein the change in copy number of at least one chromosomal region comprises at least one deletion event.

10. The method of claim 9, wherein the deletion event is characterized by a decrease in at least one gene copy number.

11. The method of claim 5, wherein the structural variation comprises a chromosomal rearrangement.

12. The method of claim 11, wherein the chromosomal rearrangement comprises an inversion, translocation, or combination thereof.

13. The method of claim 11 or 12, wherein the chromosomal rearrangement involves one or more chromosomes.

14. The method of any one of claims 1-13, wherein the composition comprises from about 80 pM to about 380 pM etoposide or a salt thereof.

15. The method of claim 14, wherein the composition comprises from about 160 pM to about 320 pM etoposide or a salt thereof.

16. The method of any one of claims 1-15, wherein the composition comprising a topoisomerase II inhibitor further comprises a pharmaceutically acceptable carrier, an antimicrobial compound, a plant nutrient, an herbicide, an inhibitor of DNA methylation, an inhibitor of histone methylation, a mutagen, a nucleotide base analog, a DNA intercalating agent, a heavy metal, or a plant hormone, or any combination thereof.

17. The method of any one of claims 1-16, wherein the step of contacting the tissue is over a period of time of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at leastabout 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days.

18. The method of any one of claims 1-17, wherein the plant comprises Arabidopsis thaliana, cotton, tobacco, tomato, potato, sugar beet, broccoli, cassava, sweet potato, pepper, poinsettia, legumes, alfalfa, carrot, strawberry, poplars, lettuce, oak, maple, walnut, rose, mint, squash, daisy, geranium, cactus, pigeon pea, com, rice, canola, rapeseed, false flax, pennycress, cannabis, hops, herbs, wheat, and soy.

19. The method of any one of claims 1-18, further comprising: c) screening the propagated tissue for one or more phenotypes.

20. The method of claim 19, wherein the screening is for yield, altered nutrient requirements, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, or photosynthetic capacity, or any combination thereof.

21. The method of claim 19 or 20, wherein the screening is for tolerance to an environmental stressor, shoot architecture, flowering time, floral morphology, fertility, seed abortion, or root length, or any combination thereof.

22. The method of any one of claims 19-21, wherein the phenotype comprises absence of, or a decrease in, chlorophyll.

23. The method of any one of claims 19-22, wherein the screening includes screening for an abiotic stress response or a biotic stress response, or a combination thereof.

24. The method of claim 23, wherein the abiotic stress comprises low temperature, high temperature, drought, flooding, high salinity, nutrient and mineral scarcity, heavy metals, ultraviolet radiation, high light intensity, low light intensity, or any combination thereof.

25. The method of claim 23, wherein the biotic stress comprises a fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, or any combination thereof.

26. The method of any one of claims 1-25, wherein the genetic variation is a heritable structural variation.

27. The method of any one of claims 1-26, further comprising breeding the propagated plant.

28. A combination or kit comprising: a) a plant cell or plant tissue that is capable of being propagated; and b) a topoisomerase II inhibitor.

29. The combination or kit of claim 28, wherein the topoisomerase II inhibitor comprises etoposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, teniposide, acridine-4-carboxamide, amsacrine, ellipticine, ICRF-193, mitoxantrone, pixantrone, or a salt of any of the foregoing.

30. The combination or kit of claim 28 or 29, wherein the plant cell or plant tissue that is capable of being propagated includes a cultured plant cell, plant callus tissue, an embryo, a seed, a stem, a root, a root tip, a root hair, a leaf, pollen, a cotyledon, a hypocotyl, a seedling, a pistil, an anther, a flower, a meristematic cell, or a whole plant, or any combination thereof.

31. The combination or kit of any one of claims 28-30, further comprising a plant growth medium.

32. The combination or kit of any one of claims 28-31, further comprising instructions for use.

33. A method of modifying at least one trait of a plant or part thereof, comprising: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant or part thereof, thereby producing a plant or part thereof having at least one modified trait.

34. The method of claim 33, wherein the topoisomerase II inhibitor comprises etoposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, teniposide, acridine-4-carboxamide, amsacrine, ellipticine, ICRF-193, mitoxantrone, pixantrone, or a salt of any of the foregoing.

35. The method of claim 33 or 34, wherein the composition comprises from about 80 pM to about 380 pM etoposide or a salt thereof.

36. A method of inducing a genetic mutation in a plant, comprising: a) contacting a plant cell or plant tissue that is capable of being propagated with an effective amount of a composition comprising a topoisomerase II inhibitor; and b) transferring the plant cell or plant tissue that was contacted with the inhibitor to a plant growth medium, wherein the plant cell or plant tissue propagates in the growth medium to form a plant or part thereof, thereby inducing a genetic mutation in the plant.

37. The method of claim 36, wherein the gene mutation is in a shoot architecture gene, a flowering time gene, a floral morphology gene, a fertility gene, a seed abortion gene, a root length gene, or any combination thereof.

38. The method of claim 36 or 37, wherein the mutagen comprises etoposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, teniposide, acridine-4- carboxamide, amsacrine, ellipticine, ICRF-193, mitoxantrone, pixantrone, or a salt of any of the foregoing.

39. The method of claim 36, 37, or 38, wherein the tissue capable of being propagated from a plant is contacted with a composition comprising etoposide, or a salt thereof, in an amount from about 80 pM to about 380 pM.

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