Methods of breeding guayule plants

Abstract

Embodiments of the present disclosure are directed to breeding methods for guayule plants. The breeding methods disclosed herein generally include pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds. The maternal guayule plant is generally diploid and the paternal guayule plant is generally polyploid, optionally triploid. The progeny seeds are germinated to form a progeny population. Diploid progeny plants are selected from the progeny population and cultivated to produce a plant product.

Description

D&S Ref. BDGP24003WG-P24003WG011METHODS OF BREEDING GUAYULE PLANTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present specification claims the benefit of U.S. Provisional Application Serial No. 63 / 708,523 filed October 17, 2024 and entitled “Methods of Breeding Guayule Plants,” the entirety of which is incorporated by reference herein.TECHNICAL FIELD

[0002] Embodiments of the present disclosure are generally related to methods of breeding guayule plants and are specifically related to methods of breeding new lines of diploid guayule plants to increase genetic diversity.BACKGROUND

[0003] Guayule Parthenium argentatum) plants are a viable domestic source of solid rubber and hypoallergenic latex. However, the publicly available germplasm for guayule breeding is limited. Currently, only a limited number of diploid accessions and polyploid accessions are available. Further, the publicly available germplasms are relatively narrow in genetic diversity. Because diploid guayule plants primarily reproduce sexually and the majority of polyploid guayule plants all came from a few related germplasms that reproduce via facultative apomixis, introduction of genetic diversity, for example to increase rubber production, is limited.

[0004] Accordingly, a continual need exists to improve breeding strategies to increase genetic diversity and genetic gain in guayule plants.SUMMARY

[0005] Embodiments of the present disclosure are directed to methods of breeding guayule plants, which result in increased genetic diversity. The methods generally include pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds.

[0006] In an embodiment of the present disclosure, the maternal guayule plant is diploid and is self-compatible, self-incompatible, or a combination thereof; and the paternal guayule plant isD&S Ref. BDGP24003WG-P24003W0012 triploid. The progeny seeds are germinated to form a progeny population. One or more diploid plants are selected from the progeny population and cultivated to produce a plant product.

[0007] In another embodiment, the maternal guayule plant is diploid and is self-incompatible; and the paternal guayule plant is polyploid, optionally triploid or tetrapioid. The progeny seeds are germinated to form a progeny population, wherein the progeny population are hybrid plants. One or more diploid plants are selected from the progeny population and cultivated to produce a plant product.

[0008] Additional features and advantages of the embodiments described herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description which follows, and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The embodiments set forth in the drawings are illustrative and exemplary in nature and not intended to limit the disclosure. The following detailed description of the illustrative embodiments can be understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:

[0010] FIG. 1 depicts a flowchart of an example method of breeding guayule plants, according to one or more embodiments shown and described herein.DETAILED DESCRIPTION

[0011] Embodiments of the present disclosure are directed to methods of breeding guayule plants to produce guayule seed. The methods generally comprise pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds. Optionally, the maternal guayule plant is diploid. In some embodiments, the paternal guayule plant is polyploid. The progeny seeds are germinated to form a progeny population. Optionally, one or more progeny plants are selected from the progeny population, the one or more progeny plants being diploid. In some embodiments, the one or more progeny plants are cultivated to produce one or more plant products.D&S Ref. BDGP24003WG-P24003W0013

[0012] The disclosure should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the subject matter to those skilled in the art.

[0013] Definitions

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the disclosure herein is for describing particular embodiments only and is not intended to be limiting.

[0015] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0016] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order, nor that with any apparatus specific orientations be required. Accordingly, where a method claim does not actually recite an order to be followed by its steps, or that any apparatus claim does not actually recite an order or orientation to individual components, or it is not otherwise specifically stated in the claims or description that the steps are to be limited to a specific order, or that a specific order or orientation to components of an apparatus is not recited, it is in no way intended that an order or orientation be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps, operational flow, order of components, or orientation of components; plain meaning derived from grammatical organization or punctuation, and; the number or type of embodiments described in the specification.

[0017] As used in the specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, for example, reference to “a” component includes aspects having two or more such components, unless the context clearly indicates otherwise.D&S Ref. BDGP24003WO-P24003W0014

[0018] As used herein, the term “apomixis” and grammatical equivalents thereof refer to a form of asexual reproduction that allows plants to produce seeds without meiotic division of chromosomes and / or fertilization by a male gamete (sperm). In this process, the seeds develop from the ovule or other maternal tissues, resulting in offspring that are genetically identical to the parent plant. Apomixis bypasses the typical sexual reproduction cycle, including meiosis and syngamy, and may occur in various forms, including apospory, diplospory, and / or adventitious embryony.

[0019] As used herein, the terms “desired characteristic,” “desired trait,” and grammatical equivalents thereof refer to any aspect of a plant altered for a “desired benefit,” such as increasing an agriculturally desirable trait, such as any qualitative and quantitative agricultural trait, including but not limited to, rubber production, latex production, productivity, crop yield, biomass, unique plant morphology (e.g., floral structure, pod characteristics, plant height, seed weight, fruit size, leaf shape, flower color, pubescence, branching patterns, etc.), resistance to pathogens, resistance to pests, resistance to environmental changes (e.g., cold tolerance, drought tolerance, etc.), combinations thereof, and the like.

[0020] As used herein, the term “hybrid” refers to offspring resulting from the cross-breeding of two genetically distinct parent plants. The parental plants may be related by pedigree, as in production of a modified single cross, or unrelated. Fl hybrid, as used herein, refers to the first- generation progeny of the cross of two genetically dissimilar plants.

[0021] As used herein, “plant product” refers to the whole plant or part thereof removed from the guayule plant, or a product produced by or from the guayule plant including, but not limited to one or more of seeds, tissues, plant parts, biomass, rubber (e.g., solid rubber), latex, resin, fatty acid triglycerides, ethanol, adhesives, terpenes, sesquiterpenes, sugars, and / or waxes.

[0022] As used herein, the term “progeny” refers to the offspring or descendants resulting from the cross-breeding or self-fertilization of parent plants. These progeny inherit genetic material from their parent plants and may be evaluated to identify desirable traits for further breeding efforts or commercial cultivation. Progeny of a particular plant include seeds formed on Fl, F2, F3, F4, F5, F6 and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants, or seeds formed on F1BC1, F1BC2, F1BC3, and subsequent generation plants. The designation Fl refers to the progeny of a cross between two parents thatD&S Ref. BDGP24003WD-P24003W0015 are genetically distinct. The designations F2, F3, F4, F5, F6, and so on refer to subsequent generations of self- or sib-pollinated progeny of an Fl plant. In some embodiments, one or more progeny plants are cultivated to produce diploid seed.

[0023] As used herein, the term “self-incompatible” refers to a genetic mechanism in guayule plants that prevents self-fertilization and encourages cross-pollination. It will be appreciated that a self-incompatible plant cannot fertilize itself with its own pollen because biochemical interactions between the pollen and the pistil of the same plant inhibit pollen germination or pollen tube growth.

[0024] As used herein, the term “self-compatible” refers to the ability of a plant to fertilize itself with its own pollen, leading to successful seed production through self-fertilization. It will be appreciated that a self-compatible plant's reproductive organs (pollen and pistil) are genetically and biochemically compatible, allowing pollen from the same plant to germinate, grow a pollen tube, and fertilize the ovules within the same flower or in other flowers on the same plant.

[0025] Diploid plants, reproducing sexually and having limited self-compatibility, increase genetic diversity of guayule species. By increasing genetic diversity, new cultivars with desired traits, such as increased production of one or more plant products or other desired characteristics, may be developed. However, diploid guayule plants only occur naturally in a small region of Mexico, further contributing to the lack of genetic diversity.

[0026] Disclosed herein are methods for breeding guayule plants which mitigate the aforementioned problems. Specifically, the methods disclosed herein comprise pollinating a maternal guayule plant with pollen from a paternal guayule plant, resulting in progeny that have a desired trait or characteristic. In some embodiments, the parental guayule lines are selected for characteristics such as increased rubber yield, increased biomass, or other advantageous characteristics, such as higher seedling vigor, increased biotic stress resistance, and / or improved abiotic stress resistance.

[0027] As described briefly above, the present disclosure generally describes methods for breeding guayule plants to produce hybrid progeny. Although guayule plants Parthenium ar genl alum) are used for exemplary purposes as described herein, it will be appreciated that the methods disclosed may be used in the production of hybrid seeds and / or progeny of anyD&S Ref. BDGP24003WO-P24003W0016Parthenium species, including interspecies crosses. Suitable Parthenium species that may be used in the methods described herein include, but are not limited to, P. alpinum, P. argentatum, P. cineraceum, P. confertum, P. fruticosum, P. hysterophorus, P. incanum, P. integrifolium, P. ligulatum, P. rollinsianum, P. schottii, and / or P. tomentosum.

[0028] In some embodiments, both parental plants (the maternal guayule plant and the paternal guayule plant) belong to the same Parthenium species, optionally P. argentatum. In some embodiments, the maternal guayule plant and the paternal guayule plant belong to different Parthenium species. For example, and without being bound by theory, in some embodiments, the maternal plant is a P. argentatum plant, while the paternal plant is a P. alpinum, P. argentatum, P. cineraceum, P. confertum, P. fruticosum, P. hysterophorus, P. incanum, P. integrifolium, P. ligulatum, P. rollinsianum, P. schottii, or P. tomentosum species.

[0029] In some embodiments, the cross is between a maternal P. argentatum and a paternal P. tomentosum. In some embodiments, the cross is between a maternal P. tomentosum and a paternal P. argentatum. In some embodiments, the cross is between a maternal P. argentatum and a paternal P. integrifolium. In some embodiments, the cross is between a maternal P. integrifolium and a paternal P. argentatum. In some embodiments, the cross is between a maternal P. argentatum and a paternal P. alpinum. In some embodiments, the cross is between a maternal P. alpinum and a paternal P. argentatum. In some embodiments, the cross is between a maternal P. argentatum and a paternal P. incanum. In some embodiments, the cross is between a maternal P. incanum and a paternal P. argentatum. In some embodiments, the cross is between a maternal P. argentatum and a paternal P. confertum. In some embodiments, the cross is between a maternal P. confertum and a paternal P. argentatum. In some embodiments, the cross is between a maternal P. argentatum and a paternal P. hysterophorus. In some embodiments, the cross is between a maternal P. hysterophorus and a paternal P. argentatum.

[0030] In some embodiments, the maternal and / or the paternal guayule plants may be genetically modified. In some embodiments, the genetically modified plant may be a non-naturally occurring plant. As used herein, a “non-naturally occurring plant” refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include plants created through genetic engineering to add or remove one or more genes. In some embodiments, the one or more genes are added to the plant. In some embodiments, the genetically -modified plant refers to an organism in which a nucleic acid fragment containing a heterologous nucleotide sequenceD&S Ref. BDGP24003WG-P24003WG017 has been introduced. Optionally, the added genes are stable and inheritable. The heterologous nucleic acid fragment may or may not be integrated into the host genome. In some embodiments, the genetically engineered plant refers to a plant whose genetic material has been altered through genetic engineering to include one or more heterologous sequences not naturally associated with the plant. In some embodiments, the genetically engineered plant refers to a genetically modified plant that contains genes from the same species or a sexually compatible donor plant, but not from unrelated organisms.

[0031] Any suitable method to genetically engineer the parental plant(s) is contemplated and possible. For example, and without being bound theory, a genetically-engineered plant may be made or defined by U.S. Patent No. 11,530,419, the contents of which are incorporated herein by reference. Exemplary methods include, but are not limited to agrobacterium-mediated transformation, biolistic transformation, CRISPR / Cas9 gene editing techniques, interspecies crossing, induced chemical and / or x-ray mutagenesis, electroporation, microinjection, transposons, and the like.

[0032] Referring now to FIG. 1, a flowchart of an exemplary method of breeding guayule plants is depicted at 100. The method 100 begins at step 101 with pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds on the maternal guayule plant.

[0033] Generally, the maternal guayule plant is a guayule plant capable of producing an embryo. In some embodiments, the maternal guayule plant is diploid. In some embodiments, the maternal guayule plant is haploid. In some embodiments, the maternal guayule plant is a polyploid guayule plant, optionally a triploid plant, a tetrapioid plant, a pentapioid plant, a hexapioid plant, a septapioid plant, or an octoploid plant. Optionally, the polyploid may be an artificial polyploid, derived from induced chromosome doubling. For example, and without being bound by theory, in some embodiments, colchicine or other mitotic inhibitors are applied to seedlings or tissue- cultured shoots to double the chromosome number, producing artificial polyploid guayule plants.

[0034] In some embodiments, such as when a maternal plant is polyploid, the maternal plant may undergo modification (e.g., physical or chemical treatments) to suppress apomixis during flowering, thereby allowing fertilization to occur. For example, and without being bound byD&S Ref. BDGP24003WG-P24003W0018 theory, apomixis may be suppressed by environmental factors, epigenetic triggering, pollination timing, combinations thereof, and the like.

[0035] It will be appreciated that the maternal guayule plant may be self-compatible, selfincompatible, or a combination thereof. In some embodiments, the maternal guayule plant is selfincompatible. In some embodiments, described in greater detail herein, the reproductive compatibility of the maternal guayule plant is determined by genotyping the maternal plants progeny and comparing that genotyping data to the genotype of the maternal plant. In some embodiments, for example when apomixis or self-compatibility of the maternal plant is a concern, embryos from putative hybrid seeds can be tested (for example, via genetic markers) to confirm hybrid status, or the seed progeny can be grown out and obvious maternal clones (based on plant morphology and / or genetic testing) are discarded.

[0036] In embodiments, the reproductive compatibility of the maternal guayule plant is determined by isolating flowers to prevent cross pollination from other plants. Optionally, the flowers of the maternal guayule plant are pollinated with pollen from the same plant. If seeds develop on the maternal guayule plant, the plant is deemed self-compatible. In some embodiments, only self-incompatible maternal plants are used. It will be appreciated that self-incompatible plants may still shed viable pollen and are capable of pollinating plants of other varieties but are incapable of pollinating themselves or other plants of the same variety.

[0037] In some embodiments, self-pollination of the maternal guayule plant is inhibited. For example, and without being bound by theory, in some embodiments the ovules of the maternal plant are prevented from being fertilized by pollen of the same plant or of any plant of the same plant cultivar or variety. It will be appreciated that this may be done by any suitable means, such as, for example, by emasculating the flowers of the female plant, (e.g., treating or manipulating the flowers so as to prevent pollen production, in order to produce an emasculated maternal guayule plant). In some embodiments, the parental guayule plants are selected to increase the genetic diversity of guayule cultivars.

[0038] In some embodiments, the methods disclosed herein comprise pollinating a diploid maternal guayule plant with pollen from a polyploid paternal guayule plant, resulting in diploid progeny. The diploid seeds produced from the presently disclosed methods increase geneticD&S Ref. BDGP24003WG-P24003W0019 diversity of guayule species and may be used in breeding programs to increase production of one or more plant products, such as latex or rubber.

[0039] As noted briefly above, the methods of the present disclosure include pollinating the maternal guayule plant with pollen from the paternal guayule plant to produce seeds on the maternal guayule plant.

[0040] Generally, the paternal guayule plant is any plant capable of producing fertile and / or viable pollen. Non-limiting examples of suitable paternal plants include a haploid guayule plant, a diploid guayule plant, a triploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant. In some embodiments, the paternal guayule plant is polyploid. In some embodiments, the paternal guayule plant is triploid. In other embodiments, the paternal guayule plant is tetrapioid. Any polyploid state is contemplated and possible including a triploid paternal guayule plant, a tetrapioid paternal guayule plant, a pentapioid paternal guayule plant, a hexapioid paternal guayule plant, a heptapioid paternal guayule plant, an octaploid paternal guayule plant, etc. In some embodiments, the paternal plant is an artificial polyploid. For example, and without being bound by theory, in some embodiments, colchicine or other mitotic inhibitors are applied to seedlings or tissue- cultured shoots to double the chromosome number, producing artificial polyploid guayule plants.

[0041] In some embodiments, a diploid maternal plant, either naturally occurring or the result of an interploidy cross as described herein, is pollinated with pollen from a paternal polyploid plant. In some embodiments, a polyploid maternal plant is pollinated with pollen from a diploid paternal plant, either naturally occurring or the result of an interploidy cross as described herein. In some embodiments, a diploid maternal plant, either naturally occurring or the result of an interploidy cross as described herein, is pollinated with pollen from a diploid paternal plant, either naturally occurring or the result of an interploidy cross as described herein. In some embodiments, a polyploid maternal plant is pollinated with pollen from a polyploid paternal plant. Optionally, the maternal plant and the paternal plant have the same ploidy.

[0042] In some embodiments, a diploid maternal plant, either naturally occurring or the result of an interploidy cross as described herein, and belonging to P. argentatum species is pollinated with pollen from a paternal plant from a Parthenium species different from the maternal plant. In some embodiments, a maternal plant belonging to a Parthenium species different from the paternalD&S Ref. BDGP24003WO-P24003W00110 plant is pollinated with pollen from a diploid paternal plant, either naturally occurring or the result of an interploidy cross as described herein, and belonging to P. argentatum species.

[0043] In some embodiments, a diploid maternal plant, either naturally occurring or the result of an interploidy cross as described herein, is pollinated with pollen from an artificial polyploid paternal plant. In some embodiments, an artificial polyploid maternal plant is pollinated with pollen from a diploid paternal plant, either naturally occurring or the result of an interploidy cross as described herein. In some embodiments, an artificial polyploid maternal plant is pollinated with pollen from an artificial polyploid paternal plant.

[0044] In some embodiments, a maternal guayule plant is pollinated using bulk pollination. As used herein, “bulk pollination” refers to a breeding strategy in which pollen from multiple paternal plants is manually mixed together ahead of pollinating the maternal plant, or multiple paternal plants are planted in the vicinity of the maternal plant for insect-assisted pollination.

[0045] In some embodiments, the maternal guayule plant is pollinated with pollen from the paternal guayule plant via controlled pollination. As used herein, “controlled pollination” refers to a deliberate and controlled process in which pollen from the paternal guayule plant is transferred to the stigma of the maternal guayule plant.

[0046] Optionally, the controlled pollination of the maternal guayule plant is mechanical pollination. In some embodiments, female inflorescences from the maternal plant are isolated prior to pollen shed and then mechanically pollinated with pollen from the paternal plant. As used herein, “mechanical pollination” refers to a method of pollination in which pollen is transferred from the male reproductive organs of a flower of the paternal guayule plant to the reproductive organs of the maternal guayule plant using mechanical means, such as brushes, cotton swabs, vibrating devices, or air blowers, or by simply touching the flowers of the paternal guayule plant to transfer pollen. It will be appreciated that “mechanical pollination” and “hand pollination” may be used interchangeably and refer to the same process.

[0047] In some embodiments, the controlled pollination of the maternal guayule plant involves myophily. As used herein, “myophily” or “fly-pollination” refers to a method of pollination in which flowers of the maternal guayule plant are pollinated by flies. Optionally, a maternal guayule plant and a paternal guayule plant are selected and isolated to prevent cross-pollination from otherD&S Ref. BDGP24003WG-P24003WG0111 plants. In some embodiments, flies may be attracted to the isolated plants, such as by the introduction of a food source. In other embodiments, flies are placed with or near the isolated plants. Once the flies are near the plants, the flies will transfer pollen between flowers as they feed.

[0048] Referring back to FIG. 1 , the method 100 continues at step 102 with germinating the one or more progeny seeds to form a progeny population. Generally, once the maternal guayule plant is pollinated with pollen from the paternal guayule plant, progeny seeds form on the maternal guayule plant. In such embodiments, one or more progeny seeds are germinated to form a progeny population.

[0049] In some embodiments, the method 100 includes a step 103 of analyzing the ploidy status of each member of the progeny population to determine which member(s) of the progeny population are diploid. Optionally, one or more leaves are harvested from each member of the progeny population to analyze the ploidy status. In some embodiments, young leaves are harvested from each member of the progeny population for ploidy analysis.

[0050] In some embodiments, the ploidy status of each member of the progeny population is evaluated using flow cytometry. It will be appreciated that flow cytometric methods for determining ploidy status in plants are known in the art.

[0051] For example, and without being bound by theory, in some embodiments, to analyze the ploidy status of each member of the progeny population, a cell nuclei suspension is formed from the harvested leave(s). In some embodiments, the cell nuclei suspension is supplemented with one or more imaging agents, such a fluorescent dye or stain. The suspensions are analyzed using a flow cytometer to acquire a histogram to determine the relative fluorescence values are peak positions. Ploidy status of each member of the progeny population is determined by comparing the fluorescence values of the peak positions of the suspensions to standards of known ploidy status.

[0052] In some embodiments, as described briefly above, diploid members of the progeny population may be further are analyzed to identify whether the plants arose from sexual reproduction or from self-pollination of the maternal guayule plant. In some embodiments, the diploid members of the progeny population are genomically analyzed and compared to the genomeD&S Ref. BDGP24003WG-P24003W00112 to the maternal guayule plant and / or the paternal guayule plant. Any suitable genomic analysis is contemplated and possible. Exemplary analyses include, but are not-limited to, restriction site associated DNA (RAD) sequencing, single-molecule real-time sequencing, ion semiconductor sequencing, pyrosequencing, synthesis sequencing, combinatorial probe anchor synthesis (cP AS), polony sequencing, massively parallel signature sequencing, nanopore sequencing, nanoball sequencing, microfluidic systems, sanger sequencing, and the like. In some embodiments, analyzing the genome of the maternal guayule plant and / or the progeny plants includes using molecular markers, such as microsatellite markers and RAD-Seq markers. For example, and without being bound by theory, exemplary markers may be found as described in U.S. Patent Publication Serial No. US20190256933 Al, which is incorporated by reference herein in its entirety.

[0053] In some embodiments, diploid members of the progeny population arise from sexual reproduction when the genome differs from the maternal guayule plant by approximately 10% to 30% including 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% and 30%, including any subrange defined by any two of the aforementioned values. In some embodiment the diploid members of the progeny population arise from sexual reproduction when genomic analysis confirms introgression of paternal germplasm into the members of the progeny population. When members of the progeny population arise from sexual reproduction, they are considered hybrid plants.

[0054] Referring back to FIG. 1, the method 100 continues at step 104 with selecting one or more progeny plants from the progeny population, the one or more progeny plants being diploid. In some embodiments, selecting progeny plants from the progeny population involves selecting members of the progeny population that arise from sexual reproduction. In some embodiments, selecting progeny plants from the progeny population involves selecting members of the progeny population that are diploid. In some embodiments, selecting progeny plants from the progeny population involves selecting members of the progeny population that are diploid and arise from sexual reproduction.

[0055] In some embodiments, the progeny plants are evaluated for desired traits as described herein. In some embodiments, diploid progeny plants are subjected to greenhouse selection cycles using genomic selection and marker-based trait predictions. For example, tissue samples fromD&S Ref. BDGP24003WO-P24003W00113 seedlings may be collected and individual plant genotype data may be obtained. As another example, a trait prediction model may be used to select progeny plants with desired trait values.

[0056] Optionally, polyploid progeny are evaluated using a tiered screening system. Tier 1 is a single plant evaluation. Tier 2 is a plot level evaluation. Tier 3 is a field scale evaluation. Polyploid progeny that have one or more desired characteristics may be used in further breeding strategies according to the methods disclosed herein. For example, and without being bound by theory, the polyploid progeny at Tier 2 and / or Tier 3 may be recycled and used as maternal and / or paternal plants in the breeding methods and strategies described herein.

[0057] Referring back to FIG. 1, the method 100 continues at block 105 with cultivating the one or more progeny plants to produce a plant product. As described herein, the diploid progeny plants may be cultivated and developed for one or more desired traits, such as increased rubber content, increased latex content, disease and / or pest resistance, drought tolerance, cold tolerance, biomass production, harvesting time, processing time, growth rate, seedling vigor, unique morphotype, adaptability to soil or other growing conditions, seed viability, germination rate, reduced lignin content, and the like, though any desired trait in guayule plants is contemplated and possible.

[0058] In some embodiments, cultivating the one or more progeny plants to produce diploid seed comprises cross-pollinating two Fl progeny plants to form F2 generation seeds. In some embodiments, cultivating the one or more progeny plants to produce diploid seed comprises selfpollinating an Fl progeny plants to form F2 generation seeds. Optionally, the F2 generation seeds are harvested. In some embodiments, the F2 generation seeds are germinated, analyzed, and / or cultivated as described. It will be appreciated that the methods described herein may be repeated with respect to F2 and any subsequent generations.

[0059] In some embodiments, cultivating the one or more progeny plants to produce diploid seed comprises pollinating the progeny plants with pollen from a polyploid guayule plant to produce one or more seeds. Optionally, the polyploid guayule plant is triploid. Optionally, the polyploid guayule plant is tetrapioid. It will be appreciated that the methods described herein may be repeated with respect to identifying and selecting diploid offspring.D&S Ref. BDGP24003WG-P24003W00114

[0060] In some embodiments, the progeny plants are cultivated and one or more of plant products, including but not limited to biomass, rubber (e.g., solid rubber), latex, resin, fatty acid triglycerides, ethanol, adhesives, terpenes, sesquiterpenes, sugars, and / or waxes are produced.

[0061] Referring back to FIG. 1, the method 100 continues at step 106, with harvesting the plant product. In some embodiments, the plant product is harvested after the progeny plant reaches maturity. In some embodiments, the guayule plant reaches maturity from about 1 year to about 4 years, including about 1 year, about 2 years, about 3 years, and about 4 years, and including any range with endpoints defined by any two of the aforementioned values. In some embodiments, the plant product can be harvested from the progeny plants multiple times. In some embodiments, the plant product can be harvested monthly, bi-monthly, quarterly, biennially or annually. In some embodiments, a plurality of plant products can be harvested from the progeny plants. It will be appreciated that the timing and frequency of harvesting the plant product may depend on the climate, growing conditions, growth cycles, and / or environmental conditions, among other factors.

[0062] As well be demonstrated in the working examples, unlike the conventional teachings, the methods described herein demonstrate the production of hybrid diploid offspring from crossing diploid plants with polyploid plants, thereby increasing genetic diversity and providing new cultivars of guayule to develop.Examples:

[0063] The following examples are intended to illustrate specific features and aspects of the instant-disclosure and should not be construed as limiting the scope thereof.Example 1: Self-Compatibility

[0064] Conventionally, diploid plants are considered to be self-incompatible. However, in various crosses, diploid females produced a large number of self-progeny, confirmed by molecular marker data.

[0065] Self-fertilization experiments were conducted on 33 diploid plants by allowing them to self-pollinate. To assess the impact of self-compatibility on genetic diversity, the heterozygosity levels between the maternal plants and their respective progeny were compared. HeterozygosityD&S Ref. BDGP24003WG-P24003W00115 was measured using molecular markers (e.g., microsatellites or SNPs) to quantify the genetic variation.

[0066] The self-compatibility rate was determined based on the observed reduction in heterozygosity between the maternal plants and their progeny. A self-fertilization rate of 0% would indicate complete outcrossing, suggesting self-incompatibility in the plant, while a selffertilization rate of 100% would indicate full self-fertilization, indicating self-compatibility.

[0067] Across the 178 progeny analyzed from the 33 diploid maternal plants, the estimated self-fertilization rate ranged from 0% to 100%, with a mean self-fertilization rate of 29%. This suggests that, on average, the population exhibits a moderate level of self-compatibility, with a substantial variation in self-fertilization rates among individual plants.

[0068] When attempting to pollinate diploid maternal plants with pollen from a triploid paternal guayule plant, 27-60% of the progeny resulted from self-fertilization.

[0069] When attempting to pollinate diploid maternal plants with pollen from a tetrapioid paternal guayule plant, up to 56% of the progeny resulted from self-fertilization.

[0070] Accordingly, the degree of self-incompatibility of diploid guayule plants is germplasm dependent.Example 2: Hybrid Crosses

[0071] As noted above, unlike the conventional teachings, the present disclosure demonstrates the production of hybrid diploid offspring from crossing diploid plants with triploid and tetrapioid plants.

[0072] Diploid maternal guayule plants were crossed with polyploid paternal guayule plants. The progeny seeds were planted in trays and germinated. Young leaves were harvested from the offspring for ploidy analysis.

[0073] Plant tissue samples (approximately 1 sq. cm) from both a known control and the test sample were processed for analysis.D&S Ref. BDGP24003WG-P24003W00116

[0074] The tissue was homogenized in a 200 pl of extraction buffer (CyStain UV Precise P nuclei extraction buffer with polyvinylpyrrolidone (PVP) and DL-dithiothreitol) using a tissue lyser.

[0075] From the homogenate, approximately 60 pL of the solution was pipetted into a mesh fdter plate. The plate was centrifuged to pass the solution through the fdter. After centrifugation, the fdter was removed, and 160 pL of staining buffer (CyStain UV Precise P staining buffer) was added to the fdtrate.

[0076] The ploidy status of the offspring was analyzed using flow cytometry.

[0077] For the diploid X triploid crosses, 55.3 percent of 1,308 progeny were diploid hybrid plants.

[0078] For the diploid X tetrapioid crosses, 5.5 percent of the 3,343 progeny were diploid hybrid crosses.

[0079] Molecular marker data confirmed the introgression of paternal germplasm in the diploid offspring, confirming that the offspring were hybrid plants.

[0080] To evaluate the extent of introgression between diploid and tetrapioid plants, two genotyping methods were employed. Whole genome sequencing was performed on seven progeny from crosses between diploid plants and a tetrapioid (AZ-2 germplasm). The sequencing data revealed that 10-30% of the alleles in these progenies were derived from the tetrapioid parent.

[0081] In a separate analysis, reduced representation genotyping was used to assess 69 progeny derived from four different diploid parents. The genotyping results showed an introgression rate of 16-22%, indicating that the progeny's genome contained alleles from external sources, reflecting successful introgression of genetic material from other diploid or polyploid individuals.

[0082] In both cases, successful introgression was confirmed by identifying recombinant loci, where alleles from the tetrapioid or non-maternal parent were present in the progeny. These recombinant loci were distinguished from purely maternal loci, which would be expected in the absence of introgression. The presence of recombinant loci provides direct evidence of genetic exchange between the diploid and tetrapioid genomes, supporting the conclusion thatD&S Ref. BDGP24003WG-P24003W00117 introgression occurred in both the whole genome sequencing and reduced representation genotyping analyses.

[0083] Aspects Listing:

[0084] In a first aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, the method comprising: pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds, whereimthe maternal guayule plant is diploid and is self-compatible, selfincompatible, or a combination thereof; and the paternal guayule plant is triploid; germinating the one or more progeny seeds to form a progeny population; selecting one or more progeny plants from the progeny population, the one or more progeny plants being diploid; cultivating the one or more progeny plants to produce a plant product; and harvesting the plant product.

[0085] In a second aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is self-incompatible.

[0086] In a third aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, further comprising analyzing a ploidy status of each member of the progeny population.

[0087] In a fourth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the analyzing the ploidy status comprises harvesting one or more leaves from the progeny population.

[0088] In a fifth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the analyzing the ploidy status comprises performing flow cytometry.

[0089] In a sixth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, further comprising analyzing the one or more progeny plants to identify whether the progeny plants arose from sexual reproduction or from self-pollination of the maternal guayule plant.D&S Ref. BDGP24003WG-P24003W00118

[0090] In a seventh aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the selecting the one or more progeny plants further comprises selecting progeny plants that arose from sexual reproduction.

[0091] In an eighth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the pollinating the maternal guayule plant comprises controlled pollination.

[0092] In a ninth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the pollinating the maternal guayule plant comprises mechanical pollination.

[0093] In a tenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the pollinating the maternal guayule plant comprises myophily.

[0094] In an eleventh aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the plant product is a diploid seed.

[0095] In a twelfth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the cultivating the one or more progeny plants to produce the diploid seed comprises cross-pollinating two progeny plants to form F2 generation seeds.

[0096] In a thirteenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, further comprising germinating the diploid seed into a diploid plant.

[0097] In a fourteenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, further comprising pollinating the diploid plant with pollen from a polyploid guayule plant to produce one or more seeds.D&S Ref. BDGP24003WG-P24003W00119

[0098] In a fifteenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the polyploid guayule plant is triploid.

[0099] In a sixteenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the polyploid guayule plant is tetrapioid.

[0100] In a seventeenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the plant product is selected from rubber, biomass, latex, resin, fatty acid triglycerides, ethanol, adhesives, terpenes, sesquiterpenes, or waxes.

[0101] In an eighteenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, the method comprising: pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds, wherein: the maternal guayule plant is diploid and is self-incompatible; and the paternal guayule plant is polyploid; germinating the one or more progeny seeds to form a progeny population, wherein the progeny population are hybrid plants; selecting one or more progeny plants from the progeny population, the one or more progeny plants being diploid; cultivating the one or more progeny plants to produce a plant product; and harvesting the plant product.

[0102] In a nineteenth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is tetrapioid.

[0103] In a twentieth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is triploid.

[0104] In a twenty first aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, the method comprising pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one orD&S Ref. BDGP24003WG-P24003W00120 more progeny seeds, and germinating the one or more progeny seeds to form a progeny population, wherein the progeny population are hybrid plants.

[0105] In a twenty second aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein said maternal guayule plant is a haploid guayule plant, a diploid guayule plant, a triploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0106] In a twenty third aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein said paternal guayule plant is a haploid guayule plant, a diploid guayule plant, a triploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0107] In a twenty fourth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant and the paternal guayule plant have different ploidy.

[0108] In a twenty fifth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant and the paternal guayule plant have the same ploidy.

[0109] In a twenty sixth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant and / or the paternal guayule plant are genetically engineered.

[0110] In a twenty seventh aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant and / or the paternal guayule plant are non-naturally occurring.

[0111] In a twenty eighth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is a diploid guayule plant.D&S Ref. BDGP24003WG-P24003W00121

[0112] In a twenty eighth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is a haploid guayule plant, a triploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0113] In a twenty ninth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is a triploid guayule plant.

[0114] In a thirtieth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is a haploid guayule plant, a diploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0115] In a thirty first aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is a tetrapioid guayule plant, either naturally occurring or artificially induced.

[0116] In a thirty second aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is a haploid guayule plant, a diploid guayule plant, a triploid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0117] In a thirty third aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is a diploid guayule plant.

[0118] In a thirty fourth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is a haploid guayule plant, a triploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.D&S Ref. BDGP24003WG-P24003W00122

[0119] In a thirty fifth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is a triploid guayule plant.

[0120] In a thirty sixth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is a haploid guayule plant, a diploid guayule plant, a tetrapioid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0121] In a thirty seventh aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the paternal guayule plant is a tetraploid guayule plant, either naturally occurring or artificially induced..

[0122] In a thirty eighth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant is a haploid guayule plant, a diploid guayule plant, a triploid guayule plant, a pentapioid guayule plant, a hexapioid guayule plant, a septapioid guayule plant, or an octaploid guayule plant.

[0123] In a thirty eighth aspect, alone or in combination with any other aspect described herein, the present disclosure relates to a method of breeding guayule plants, wherein the maternal guayule plant and / or the paternal guayule plant is an artificial polyploid.

[0124] It will be apparent that modifications and variations are possible without departing from the scope of the disclosure defined in the appended claims. More specifically, although some aspects of the present disclosure are identified herein as preferred or particularly advantageous, it is contemplated that the present disclosure is not necessarily limited to these aspects.

Claims

D&S Ref. BDGP24003WO-P24003W00123CLAIMS1. A method of breeding guayule plants, the method comprising: pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds, wherein: the maternal guayule plant is diploid and is self-compatible, selfincompatible, or a combination thereof; and the paternal guayule plant is triploid; germinating the one or more progeny seeds to form a progeny population; selecting one or more progeny plants from the progeny population, the one or more progeny plants being diploid; cultivating the one or more progeny plants to produce a plant product; and harvesting the plant product.

2. The method of claim 1, wherein the maternal guayule plant is self-incompatible.

3. The method of claim 1, further comprising analyzing a ploidy status of each member of the progeny population.

4. The method of claim 3, wherein the analyzing the ploidy status comprises harvesting one or more leaves from the progeny population.

5. The method of claim 3, wherein the analyzing the ploidy status comprises performing flow cytometry.

6. The method of claim 1, further comprising analyzing the one or more progeny plants to identify whether the progeny plants arose from sexual reproduction or from self-pollination of the maternal guayule plant.

7. The method of claim 6, wherein the selecting the one or more progeny plants further comprises selecting progeny plants that arose from sexual reproduction.D&S Ref. BDGP24003WO-P24003W001248. The method of claim 1, wherein the pollinating the maternal guayule plant comprises controlled pollination.

9. The method of claim 1, wherein the pollinating the maternal guayule plant comprises mechanical pollination.

10. The method of claim 1, wherein the pollinating the maternal guayule plant comprises myophily.

11. The method of claim 1 , wherein the plant product is a diploid seed.

12. The method of claim 11 , wherein the cultivating the one or more progeny plants to produce the diploid seed comprises cross-pollinating two progeny plants to form F2 generation seeds.

13. The method of claim 11, further comprising germinating the diploid seed into a diploid plant.

14. The method of claim 13, further comprising pollinating the diploid plant with pollen from a polyploid guayule plant to produce one or more seeds.

15. The method of claim 14, wherein the polyploid guayule plant is triploid.

16. The method of claim 14, wherein the polyploid guayule plant is tetrapioid.

17. The method of claim 1, wherein the plant product is selected from rubber, biomass, latex, resin, fatty acid triglycerides, ethanol, adhesives, terpenes, sesquiterpenes, or waxes.

18. A method of breeding guayule plants, the method comprising: pollinating a maternal guayule plant with pollen from a paternal guayule plant to produce one or more progeny seeds, wherein: the maternal guayule plant is diploid and is self-incompatible; and the paternal guayule plant is polyploid;D&S Ref. BDGP24003WO-P24003W00125 germinating the one or more progeny seeds to form a progeny population, wherein the progeny population are hybrid plants; selecting one or more progeny plants from the progeny population, the one or more progeny plants being diploid; cultivating the one or more progeny plants to produce a plant product; and harvesting the plant product.

19. The method of claim 18, wherein the paternal guayule plant is tetrapioid.

20. The method of claim 18, wherein the paternal guayule plant is triploid.

21. A method of breeding interspecies plants from the genus Parthenium, the method comprising: pollinating a maternal Parthenium plant with pollen from a paternal Parthenium plant to produce one or more progeny seeds, wherein: the maternal plant is diploid and is self-incompatible; and the paternal plant is polyploid; and wherein the maternal plant and the paternal plant are from different Parthenium species; germinating the one or more progeny seeds to form a progeny population, wherein the progeny population are hybrid plants; selecting one or more progeny plants from the progeny population, the one or more progeny plants being diploid; cultivating the one or more progeny plants to produce a plant product; and harvesting the plant product.

22. The method of claim 21, wherein the maternal plant is P. argentatum and the paternal plant is selected from P. tomentosum, P. integrifolium. P. alpinum, P. incanum. P. confertum, or P. hysterophorus.

23. The method of claim 21, wherein the paternal plant is P. argentatum and the maternal plant is selected from P. tomentosum, P. integrifolium. P. alpinum, P. incanum. P. confertum, or P. hysterophorus.

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