Soybean gene edits for increased seeds per pod

Abstract

The disclosure relates to novel soybean plants, plant parts, and nucleotide sequences in soybean plants comprising an amorphic allele of a RPF1 gene, along with methods of using and making the same.

Description

TITLE: SOYBEAN GENE EDITS FOR INCREASED SEEDS PER PODCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional application U.S. Serial No. 63 / 711,978 filed October 25, 2024, which is hereby incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The present application includes a Sequence Listing in electronic format as an XML file entitled “P14990WO00_SequenceListing.xml” which was created on October 21, 2025, which has a size of 38,508 bytes (measured in MS-Windows), and which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0003] Disclosed herein are novel plants, plant parts, and nucleotide sequences in soybean varieties comprising an amorphic allele of a RPF1 gene, along with methods of making the same by growing a soybean plant or lot, and methods of using the same.BACKGROUND

[0004] Agriculture is an essential industry for the global economy and the United States in particular. Soybean (Glycine max) is an important legume crop worldwide due to its ability to fix atmospheric nitrogen. Soybeans serve as a major source of animal feed protein and soybean oil has uses in a wide variety of industries, including the food and beverage, biodiesel, and other industries.

[0005] Soybean sustainability is a priority for farmers worldwide. Farming practices such as water and nutrient management help farmers improve efficiencies, boost crop productivity, conserve water, enrich soil quality, improve nutrient efficiencies of the soil, and produce sustainable soybean crops. The benefits of bioengineering for soybean farmers include increased yields and extreme weather hardiness.SUMMARY

[0006] Disclosed herein are soybean plant cells comprising an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1, SEQ ID NO: 14, or an allelic variant thereof. Soybean plant parts, including a stem, root, leaf, flower, pod, or seed, and soybean plants comprising the aforementioned soybean plant cells are also provided.

[0007] Also disclosed are plants or plant parts of elite soybean plants, cultivars, or varieties with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield comprising: (a) an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1, SEQ ID NO: 14, or an allelic variant thereof, wherein the at least one amorphic allele comprises at least one mutation in the RPF1 gene; or (b) (i) an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1 or an allelic variant thereof; (ii) a hypom orphic or amorphic allele of at least one of an AlPlOa gene, AlPlOb gene, AML4 gene, CRN gene, RIC1 gene, RIC2 gene, JAG1 gene, JAG2 gene, TCP5-L gene, FTla gene, BS1 gene, BS2 gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene; wherein the increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of the soybean plant or a soybean plant grown from the plant part is in comparison to a wild-type or control soybean plant lacking the amorphic allele of (a) or lacking the amorphic and hypomorphic alleles of (b).

[0008] Also disclosed are soybean plant cells, soybean plant parts, or soybean plants obtainable by the process comprising: (i) directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof; and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising at least one mutation in the RPF1 gene of SEQ ID NO. 1 or an allelic variant thereof, optionally wherein the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6, are provided.

[0009] Methods of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising the aforementioned soybean plants; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot are provided.

[0010] Methods for generating the aforementioned soybean plant cells, soybean plant parts, or soybean plants comprising introducing at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0011] Methods for increasing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in a soybean plant, comprising: introducing at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof are provided.

[0012] Methods for generating the aforementioned soybean plant cells, soybean plant parts, or soybean plants comprising: (i) screening a population of soybean plant cells, parts, or plants for the presence of the at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or anallelic variant thereof; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0013] Use of any of the aforementioned soybean plant cells, soybean plant parts, or soybean plants to produce soybean seed, grow a soybean crop, and / or obtain a soybean by-product are provided herein.

[0014] Biological samples comprising one or more nucleic acids containing: (i) an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1 or an allelic variant thereof, wherein the at least one amorphic allele comprises at least one mutation in the RPF1 gene; or (ii) an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1 or an allelic variant thereof, wherein the at least one amorphic allele comprises at least one mutation in the RPF1 gene; and a hypomorphic or amorphic allele of at least one of an AlPlOa, AlPlOb, AML4, CRN, RIC1 gene, RIC2 gene, FT la gene, JAG1 gene, JAG2 gene, TCP5-L gene, BS1 gene, BS2 gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene comprises at least one mutation in the AlPlOa, AlPlOb, AML4, CRN, RIC1 gene, RIC2 gene, JAG 1 gene, JAG2 gene, TCP5-L gene, FTla gene, BS1 gene, BS2 gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene are also provided.

[0015] Gene editing system comprising a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that has 100% sequence identity to the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof. Also provided are expression cassettes comprising: a polynucleotide encoding CRISPR-Cas effector protein comprising a cleavage domain and the aforementioned guide RNA molecule. Use of the guide RNA molecule, the gene editing system, or the expression cassette to introduce at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof are also provided.

[0016] Methods for determining whether a soybean plant cell, plant part, or plant comprises at least one mutation of the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof comprising: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof, or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof, from the plant cell, plant part, or plant, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic orbiochemical activity of the polypeptide is indicative of the presence of the at least one mutation are provided.

[0017] Methods for determining whether a soybean seed lot comprises soybean seed comprising at least one mutation of the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof comprising: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof, or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof, from a sample of the soybean seed lot, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from a sample of the soybean seed lot, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation are provided.

[0018] Soybean genomes characterized by the fact that they comprise: (a) an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1 or an allelic variant thereof, wherein the at least one amorphic allele comprises at least one mutation in the RPF1 gene; or (b) (i) an amorphic allele of the endogenous RPF1 gene comprising the DNA molecule of SEQ ID NO: 1, wherein the at least one amorphic allele comprises at least one mutation in the RPF1 gene; and (iii) a hypom orphic or amorphic allele of at least one of an AlPlOa gene, AlPlOb gene, AML4 gene, JAG1 gene, JAG2 gene, TCP5-L gene, CRN gene, RIC1 gene, RIC2 gene, FTla gene, BS1 gene, BS2 gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene; are provided.DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A, B, C, D, E and F shows the wild-type RPF1 gene of SEQ ID NO: 1 which encompasses the 5’ untranslated region (SEQ ID NO: 9), protein coding region, and 3’ untranslated regions (SEQ ID NO: 10). The guide RNA target sites for the spacer RNA molecules encoded by SEQ ID NO: 4-6 are indicated.DETAILED DESCRIPTION

[0020] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a particular gene at particular locus in a different strain, variety, or isolate of a given organism.

[0021] As used herein, the phrase “amorphic allele” refers to an allele of a gene having no gene activity in comparison to the wild-type allele of the gene. Amorphic alleles are also known as null alleles.

[0022] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] As used herein, the phrase “biological sample” refers to either intact or non-intact (e.g., milled soybean seed or soybean plant tissue, chopped soybean plant tissue, lyophilized tissue) soybean plant tissue. It may also be an extract comprising intact or non-intact seed or soybean plant tissue. The biological sample can comprise flour, meal, syrup, oil, starch, and cereals manufactured in whole or in part to contain soybean plant by-products. In certain embodiments, the biological sample is “non-regenerable” ( / .< ., incapable of being regenerated into a soybean plant or soybean plant part).

[0024] As used herein, the terms “correspond,” “corresponding,” and “equivalent,” when used in the context of an nucleotide position, mutation, and / or substitution in any given polynucleotide (e.g., an allelic variant of SEQ ID NO: 1) with respect to the reference polynucleotide sequence (e.g, SEQ ID NO: 1) all refer to the position of the nucleotide in the given sequence that has identity to the nucleotide in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g, CLUSTAL O 1.2.4 with default parameters).

[0025] As used herein, the terms “Cpfl” and “Cast 2a” are used interchangeably to refer to the same RNA dependent DNA endonuclease (RdDe).

[0026] As used herein, a “cultivar” refers to a cultivated variety. A cultivar is generally developed using crossing, selfing, and / or selection and is maintained by any suitable method of propagation, through open pollination, selfing, or the like. Details of cultivar development can be found in “Principles of Cultivar Development” by Fehr, Macmillan Publishing Company (1993), which is incorporated herein by reference in its entirety.

[0027] As used herein, the phrases “elite soybean line” or “elite soybean plant” refer to any line or plant which has undergone breeding to provide one or more trait improvements (e.g., desirable agronomic performance (typically commercial production) or superior grain quality). In some cases, an elite line can be an agronomically or otherwise superior line or variety that hasresulted from several or many cycles of breeding and selection for one or more trait improvements (e.g., superior agronomic performance or superior grain quality). Similarly, “elite germplasm” is a germplasm resulting from breeding and selection for desirable agronomic performance (typically commercial production). Such germplasm may be agronomically superior germplasm, derived from and / or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of soybean. Elite crop plant lines include plants which are an essentially homozygous, e.g., inbred or doubled haploid. Elite crop plants can include inbred lines used as is or used as pollen donors or pollen recipients in breeding (e.g., used to produce Fl plants). Elite crop plants can include inbred lines which are selfed to produce non-hybrid cultivars or varieties. Elite crop plants can include hybrid Fl progeny of a cross between two distinct elite inbred or doubled haploid plant lines.

[0028] As used herein, the phrase “endogenous gene” refers to the native form of a gene unit in its natural location in the genome of an organism.

[0029] As used herein, the term “expression” refers to the production of a functional endproduct (e.g., an mRNA, guide RNA, or a protein) in either precursor or mature form.

[0030] As used herein, the phrase “hypomorphic allele” refers to an allele of a gene with less gene activity than a wild-type allele but more gene activity than an amorphic allele.

[0031] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0032] As used herein, the term “isomorphic allele” refers to an allele of a gene having wildtype gene activity.

[0033] The term “isolated” as used herein means having been removed from its natural environment.

[0034] As used herein, the term “introduced” means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods. Thus, “introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct / expression construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., nuclear chromosome, plasmid, plastid, chloroplast, or mitochondrialDNA), converted into an autonomous replicon, or transiently expressed e.g., transfected mRNA).

[0035] As used herein, a “loss-of-function allele” can include an amorphic allele or a hypomorphic allele of a gene.

[0036] As used herein in the specification, the phrase “RPF1 gene” is understood to include: (i) a RPF1 gene alone or an amorphic allele thereof; or (ii) a RPF1 gene alone or an amorphic allele thereof wherein the RPF1 genes or amorphic alleles can in certain embodiments be combined in a soybean plant cell, plant, or part thereof such as a seed, or biological sample, or used in a related method, with additional hypomorphic or amorphic alleles of a distinct soybean gene, including an AlPlOa gene, AlPlOb gene, AML4 gene, CRN gene, RIC1 gene, RIC2 gene, JAG1 gene, JAG2 gene, TCP5-L gene, FTla gene, BS1 gene, BS2 gene, TFLlb gene, CYP76C gene, and / or NF -YC4 gene.

[0037] As used herein, a “non-natural” or “non-naturally occurring” mutation refers to a mutation in a gene which is generated via human intervention or descended from the mutation generated via human intervention. Non-limiting examples of human intervention which can be used to generate a non-naturally occurring mutation include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis), mutagenesis followed by DNA sequence-based screening and selection (TILLING), and targeted genetic modifications (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods).

[0038] As used herein, the term “plant” includes a whole soybean plant and any descendant, cell, tissue, part, or parts of the plant. The term “plant” thus includes reference to an immature or mature whole soybean plant, including a plant from which seed or grain or anthers have been removed.

[0039] The term “plant part” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); grain; stover; a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, pods; flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.

[0040] As used herein, the term “variety” refers to a group of similar plants that by one or more structural features, genetic features, and / or performance can be distinguished from other varieties within the same species. In certain embodiments, the term variety refers to the botanical taxonomic designation whereby variety is ranked below species or subspecies, as well as the legal definition whereby the term “variety” refers to a commercial plant that is protected under the terms outlined in the International Convention for the Protection of New Varieties of Plants.

[0041] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

[0042] The present disclosure provides for soybean plant cells, plant parts including seed, plants, seed lots, and biological samples comprising an amorphic allele of a RPF1 gene ( / .< ., comprising at least one mutation in the endogenous RPF1 gene). These soybean plants and parts can be utilized for human food, livestock feed, as a raw material in industry, or as breeding material for development of other soybean varieties.

[0043] The target endogenous soybean transcription factor RNA Processing Factor 1 (RPF1) gene comprises the genomic DNA of SEQ ID NO: 1 (depicted in Figure 1 A-F), SEQ ID NO: 14 (a shortened version of SEQ ID NO: 1), and allelic variants thereof located on soybean chromosome 8. The endogenous soybean RPF1 gene is located at nucleotides 8326291-8330229 of chromosome 8 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.vl; Glyma.08G108400 on the world wide web internet site “legacy.soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Allelic variants of an endogenous soybean RPF1 gene include variants which encode RPF1 proteins having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 3. Allelic variants of an endogenous soybean RPF1 gene also include variants which comprise genomic DNA having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 1, 7, or 14. RPF1 gene transcripts comprising distinct 3’ UTRs are disclosed herein (SEQ ID NO: 10) and in sequence databases (Glyma.08G108400.1 (SEQ ID NO: 11, 12, and 13) and Glyma.08G108400.2 (SEQ ID NO: 16 and 17). All RPF1 gene transcripts comprise a single exon which contains the open reading frame (ORF) encoding the RFP1 protein. Guide RNAs comprising the spacers encoded by SEQ ID NO: 4, 5, and 6 are used in conjunction with suitable Cas nucleases to introduce amorphic orhypomorphic mutations in the single exon which contains the open reading frame (ORF) encoding the RFP1 protein.

[0044] Soybean plant cells, plant parts, and plants comprising at least one mutation in the soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof are provided. In certain embodiments, the at least one mutation is a non-natural mutation. In certain embodiments, the at least one mutation comprises a loss-of-function allele of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof. Examples of mutations can include a deletion, an insertion, and / or a substitution of one or more nucleotides of the endogenous RPF1 gene. The insertion, deletion, and / or substitution can be made anywhere in the RPF1 gene including, for example, in the promoter region, an exon, an intron, and / or the untranslated regions (5’ UTR or 3’ UTR). In certain embodiments, the at least one mutation comprises, consists essentially of, or consists of a deletion, insertion, and / or substitution of at least one nucleotide (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, or 3939 nucleotides) of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments, the at least one mutation comprises, consists essentially of, or consists of a deletion, insertion, and / or substitution in the coding region (z.e., nucleotides 120-1955 of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1) of the RPF1 gene. In certain embodiments, the at least one mutation in the RPF1 gene can comprise a deletion of the entire coding region or any portion of the coding region required for biological activity. In certain embodiments, the at least one mutation in the RPF1 gene comprises, consists essentially of, or consists of a deletion, insertion, and / or substitution of one or more nucleotides of: (i) an N-terminal RPF1 protein coding region (e g., nucleotides 120-1955 of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1) or in an equivalent position of an allelic variant of SEQ ID NO: 1). In certain embodiments, the at least one mutation comprises, consists essentially of, or consists of a deletion, insertion, and / or substitution of at least one nucleotide (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, or 1836 nucleotides) corresponding to nucleotides 120-1955 (z.e., within the protein and intron coding region) of the RPF1 gene of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1. In certain embodiments, the at least one mutation comprises, consists essentially of, or consists of a deletion, insertion, and / or substitution of at least one nucleotide(e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides) corresponding to: (i) nucleotides 415-439; (ii) nucleotides 675-699; or (iii) nucleotides 770-794; all of the RPF1 gene of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1.

[0045] In certain embodiments, the at least one mutation comprises, consists essentially of, or consists of a deletion, an insertion, and / or substitution that results in a frameshift mutation and / or a nonsense mutation in the coding region of the RPF1 gene. In certain embodiments, mutations of the RPF1 gene can comprise a deletion of any number of nucleotides that are not divisible by 3 in the exon of the RPF1 gene (e.g., the exon located between nucleotides 120-1955 of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1). In certain embodiments, mutations of the RPF1 gene can comprise, consist essentially of, or consist of a deletion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163, 164, 166, 167, 169, 170, 172, 173, 175, 176, 178, 179, 181, 182, 184, 185, 187, 188, 190, 191, 193, 194, 196, 197, 199, 200, 202, 203, 205, 206, 208, 209, 211, 212, 214, 215, 217, 218, 220, 221, 223, 224, 226, 227, 229, 230, 232, 233, 235, 236, 238, 239, 241, 242, 244, 245, 247, 248, 250, 251, 253, 254, 256, 257, 259, 260, 262, 263, 265, 266, 268, 269, 271, 272, 274, 275, 277, 278, 280, 281, 283, 284, 286, 287, 289, 290, 292, 293, 295, 296, 298, 299, 301, 302, 304, 305, 307, 308, 310, 311, 313, 314, 316, 317, 319, 320, 322, 323, 325, 326, 328, 329, 331, 332, 334, 335, 337, 338, 340, 341, 343, 344, 346, 347, 349, 350, 352, 353, 355, 356, 358, 359, 361, 362, 364, 365, 367, 368, 370, 371, 373, 374, 376, 377, 379, 380, 382, 383, 385, 386, 388, 389, 391, 392, 394, 395, 397, 398, 400, 401, 403, 404, 406, 407, 409, 410, 412, 413, 415, 416, 418, 419, 421, 422, 424, 425, 427, 428, 430, 431, 433, 434, 436, 437, 439, 440, 442, 443, 445, 446, 448, 449, 451, 452, 454, 455, 457, 458, 460, 461, 463, 464, 466, 467, 469, 470, 472, 473, 475, 476, 478, 479, 481, 482, 484, 485, 487, 488, 490, 491, 493, 494, 496, 497, 499, 500, 502, 503, 505, 506, 508, 509, 511, 512, 514, 515, 517, 518, 520, 521, 523, 524, 526, 527, 529, 530, 532, 533, 535, 536, 538, 539, 541, 542, 544, 545, 547, 548, 550, 551, 553, 554, 556, 557, 559, 560, 562, 563, 565, 566, 568, 569, 571, 572, 574, 575, 577, 578, 580, 581, 583, 584, 586, 587, 589, 590, 592, 593, 595, 596, 598, 599, 601, 602, 604, 605, 607, 608, 610, 611, 613, 614, 616, 617, 619, 620, 622, 623, 625, 626, 628, 629, 631, 632, 634, 635, 637, 638, 640, 641, 643, 644, 646, 647, 649, 650, 652, 653, 655, 656, 658, 659, 661, 662, 664, 665, 667, 668, 670, 671, 673, 674, 676, 677,679, 680, 682, 683, 685, 686, 688, 689, 700, 701, 703, 704, 706, 707, 709, 710, 712, 713, 715, 716, 718, 719, 721, 722, 724, 725, 727, 728, 730, 731, 733, 734, 736, 737, 739, 740, 742, 743, 745, 746, 748, 749, 751, 752, 754, 755, 757, 758, 760, 761, 763, 764, 766, 767, 769, 770, 772, 773, 775, 776, 778, 779, 781, 782, 784, 785, 787, 788, 790, 791, 793, 794, 796, 797, 799, 800, 802, 803, 805, 806, 808, 809, 811, 812, 814, 815, 817, 818, 820, 821, 823, 824, 846, 847, 829, 830, 832, 833, 835, 836, 838, 389, 841, 842, 844, 845, 847, 848, 850, 851, 853, 854, 856, 857, 859, 860, 862, 863, 865, 866, 868, 869, 871, 872, 874, 875, 877, 878, 880, 881, 883, 884, 886, 887, 889, 890, 892, 893, 895, 896, 898, 899, 901, 902, 904, 905, 907, 908, 910, 911, 913, 914, 916, 917, 919, 920, 922, 923, 925, 926, 928, 929, 931, 932, 934, 935, 937, 938, 940, 941, 943, 944, 946, 947, 949, 950, 952, 953, 955, 956, 958, 959, 961, 962, 964, 965, 967, 968, 970, 971, 973. 974. 976. 977. 979. 980. 982. 983. 985. 986. 988. 989. 991. 992. 994. 995. 997. 998. 1000 1001, 1003, 1004, 1006, 1007, 1009, 1010, 1012, 1013, 1015, 1016, 1018, 1019, 1021, 1022, 1024, 1025, 1027, 1028, 1030, 1031, 1033, 1034, 1036, 1037, 1039, 1040, 1042, 1043, 1045, 1046, 1048, 1049, 1051, 1052, 1054, 1055, 1057, 1058, 1060, 1061, 1063, 1064, 1066, 1067, 1069, 1070, 1072, 1073, 1075, 1076, 1078, 1079, 1081, 1082, 1084, 1085, 1087, 1088, 1090, 1091, 1093, 1094, 1096, 1097, 1099, 1100, 1102, 1103, 1105, 1106, 1108, 1109, 1111, 1112, 1114, 1115, 1117, 1118, 1120, 1121, 1123, 1124, 1126, 1127, 1129, 1130, 1132, 1133, 1135, 1136, 1138, 1139, 1141, 1142, 1144, 1145, 1147, 1148, 1150, 1151, 1153, 1154, 1156, 1157, 1159, 1160, 1162, 1163, 1165, 1166, 1168, 1169, 1171, 1172, 1174, 1175, 1177, 1178, 1180, 1181, 1183, 1184, 1186, 1187, 1189, 1190, 1192, 1193, 1195, 1196, 1198, 1199, 1201, 1202, 1204, 1205, 1207, 1208, 1210, 1211, 1213, 1214, 1216, 1217, 1219, 1220, 1222, 1223, 1225, 1226, 1228, 1229, 1231, 1232, 1234, 1235, 1237, 1238, 1240, 1241, 1243, 1244, 1246, 1247, 1249, 1250, 1252, 1253, 1255, 1256, 1258, 1259, 1261, 1262, 1264, 1265, 1267, 1268, 1270, 1271, 1273, 1274, 1276, 1277, 1279, 1280, 1282, 1283, 1285, 1286, 1288, 1289, 1291, 1292, 1294, 1295, 1297, 1298, 1300, 1301, 1303, 1304, 1306, 1307, 1309, 1310, 1312, 1313, 1315, 1316, 1318, 1319, 1321, 1322, 1324, 1325, 1327, 1328, 1330, 1331, 1333, 1334, 1336, 1337, 1339, 1340, 1342, 1343, 1345, 1346, 1348, 1349, 1351, 1352, 1354, 1355, 1357, 1358, 1360, 1361, 1363, 1364, 1366, 1367, 1369, 1370, 1372, 1373, 1375, 1376, 1378, 1379, 1381, 1382, 1384, 1385, 1387, 1388, 1390, 1391, 1393, 1394, 1396, 1397, 1399, 1400, 1402, 1403, 1405, 1406, 1408, 1409, 1411, 1412, 1414, 1415, 1417, 1418, 1420, 1421, 1423, 1424, 1426, 1427, 1429, 1430, 1432, 1433, 1435, 1436, 1438, 1439, 1441, 1442, 1444, 1445, 1447, 1448, 1450, 1451, 1453, 1454, 1456, 1457, 1459, 1460, 1462, 1463, 1465, 1466, 1468, 1469, 1471, 1472, 1474, 1475, 1477, 1478, 1480, 1481, 1483, 1484, 1486, 1487, 1489, 1490, 1492, 1493, 1495, 1496, 1498, 1499, 1501, 1502 1504, 1505, 1507, 1508, 1510 1511, 1513, 1514, 1516, 15171519, 1520, 1522, 1523, 1525, 1526, 1528, 1529, 1531, 1532, 1534, 1535, 1537, 1538, 1540, 1541, 1543, 1544, 1546, 1547, 1549, 1550, 1552, 1553, 1555, 1556, 1558, 1559, 1561, 1562, 1564, 1565, 1567, 1568, 1570, 1571, 1573, 1574, 1576, 1577, 1579, 1580, 1582, 1583, 1585, 1586, 1588, 1589, 1591, 1592, 1594, 1595, 1597, 1598, 1600, 1601, 1603, 1604, 1606, 1607, 1609, 1610, 1612, 1613, 1615, 1616, 1618, 1619, 1621, 1622, 1624, 1625, 1627, 1628, 1630, 1631, 1633, 1634, 1636, 1637, 1639, 1640, 1642, 1643, 1645, 1646, 1648, 1649, 1651, 1652, 1654, 1655, 1657, 1658, 1660, 1661, 1663, 1664, 1666, 1667, 1669, 1670, 1672, 1673, 1675, 1676, 1678, 1679, 1681, 1682, 1684, 1685, 1687, 1688, 1690, 1691, 1693, 1694, 1696, 1697, 1699, 1700, 1702, 1703, 1705, 1706, 1708, 1709, 1711, 1712, 1714, 1715, 1717, 1718, 1720, 1721, 1723, 1724, 1726, 1727, 1729, 1730, 1732, 1733, 1735, 1736, 1738, 1739, 1741, 1742, 1744, 1745, 1747, 1748, 1750, 1751, 1753, 1754, 1756, 1757, 1759, 1769, 1771, 1772, 1774, 1775, 1777, 1778, 1780, 1781, 1783, 1784, 1786, 1787, 1789, 1790, 1792, 1793, 1795, 1796, 1798, 1799, 1801, 1802, 1804, 1805, 1807, 1808, 1810, 1811, 1813, 1814, 1816, 1817, 1819, 1820, 1822, 1823, 1825, 1826, 1828, 1829, 1831, 1832, 1834, or 1836 nucleotides of the exon located in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1 and result in a frameshift mutation and / or a nonsense mutation. In certain embodiments, the frameshift mutation and / or a nonsense mutation occurs at nucleotides corresponding to: (i) nucleotides 415-439; (ii) nucleotides 675-699; or (iii) nucleotides 770-794; all of the RPF1 gene of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1.

[0046] In certain embodiments, the at least one mutation comprises, consists essentially of, or consists of an internal deletion that preserves the reading frame of the encoded RPF1 protein while removing at least one, two, three codons, thus resulting in a mutant RPF1 protein lacking at least one, two, or three amino acid residues. In certain embodiments, mutations of the RPF1 gene can comprise a deletion of any number of nucleotides that are divisible by 3 in a protein coding region of an exon of the RPF1 gene. In certain embodiments, the at least one mutation of the RPF1 gene can comprise, consist essentially of, or consist of a deletion of 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 414, 417, 420, 423, 426, 429, 432, 435, 438,441, 444, 447, 450, 453, 456, 459, 462, 465, 468, 471, 474, 477, 480, 483, 486, 489, 492, 495, 498, 501, 504, 507, 510, 513, 516, 519, 522, 525, 528, 531, 534, 537, 540, 543, 546, 549, 552, 555, 558, 561, 564, 567, 570, 573, 576, 579, 582, 585, 588, 591, 594, 597, 600, 603, 606, 609, 612, 615, 618, 621, 624, 627, 630, 633, 636, 639, 642, 645, 648, 651, 654, 657, 660, 663, 666, 669, 672, 675, 678, 681, 684, 687, 690, 693, 696, 699, 702, 705, 708, 711, 714, 717, 720, 723, 726, 729, 732, 735, 738, 741, 744, 747, 750, 753, 756, 759, 762, 765, 768, 771, 774, 777, 780, 783, 786, 789, 792, 795, 798, 801, 804, 807, 810, 813, 816, 819, 822, 825, 828, 831, 834, 837, 840, 843, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 876, 879, 882, 885, 888, 891, 894, 897, 900, 903, 906, 909, 912, 915, 918, 921, 924, 927, 930, 933, 936, 939, 942, 945, 948, 951, 954, 957, 960, 963, 966, 969, 972, 975, 978, 981, 984, 987, 990, 993, 996, 999, 1002, 1005, 1008, 1011, 1014, 1017, 1020, 1023, 1026, 1029, 1032, 1035, 1038, 1041, 1044, 1047, 1050, 1053, 1056, 1059, 1062, 1065, 1068, 1071, 1074, 1077, 1080, 1083, 1086, 1089, 1092, 1095, 1098, 1101, 1104, 1107, 1110, 1113, 1116, 1119, 1122, 1125, 1128, 1131, 1134, 1137, 1140, 1143, 1146, 1149, 1152, 1155, 1158, 1161, 1164, 1167, 1170, 1173, 1176, 1179, 1182, 1185, 1188, 1191, 1194, 1197, 1200, 1203, 1206, 1209, 1212, 1215, 1218, 1221, 1224, 1227, 1230, 1233, 1236, 1239, 1242, 1245, 1248, 1251, 1254, 1257, 1260, 1263, 1266, 1269, 1272, 1275, 1278, 1281, 1284, 1287, 1290, 1293, 1296, 1299, 1302, 1305, 1308, 1311, 1314, 1317, 1320, 1323, 1326, 1329, 1332, 1335, 1338, 1341, 1344, 1347, 1350, 1353, 1356, 1359, 1362, 1365, 1368, 1371, 1374, 1377, 1380, 1383, 1386, 1389, 1392, 1395, 1398, 1401, 1404, 1407, 1410, 1413, 1416, 1419, 1422, 1425, 1428, 1431, 1434, 1437, 1440, 1443, 1446, 1449, 1452, 1455, 1458, 1461, 1464, 1467, 1470, 1473, 1476, 1479, 1482, 1485, 1488, 1491, 1494, 1497, 1500, 1503, 1506, 1509, 1512, 1515, 1518, 1521, 1524, 1527, 1530, 1533, 1536, 1539, 1542, 1545, 1548, 1551, 1554, 1557, 1560, 1563, 1566, 1569, 1572, 1575, 1578, 1581, 1584, 1587, 1590, 1593, 1596, 1599, 1602, 1605, 1608, 1611, 1614, 1617, 1620, 1623, 1626, 1629, 1632, 1635, 1638, 1641, 1644, 1647, 1650, 1653, 1656, 1659, 1662, 1665, 1668, 1671, 1674, 1677, 1680, 1683, 1686, 1689, 1692, 1695, 1698, 1701, 1704, 1707, 1710, 1713, 1716, 1719, 1722, 1725, 1728, 1731, 1734, 1737, 1740, 1743, 1746, 1749, 1752, 1755, 1758, 1761, 1764, 1767, 1770, 1773, 1776, 1779, 1782, 1785, 1788, 1791, 1794, 1797, 1800, 1803, 1806, 1809, 1812, 1815, 1818, 1821, 1824, 1827, 1830, 1833, 1836 in a protein coding region of an exon of SEQ ID NO: 1 or at an equivalent position in an allelic variant of SEQ ID NO: 1 and preserve the reading frame. In certain embodiments, the at least one mutation comprises an internal deletion of at least 3, 6, or 9 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of SEQ ID NO: 1 or an allelic variant thereof which preserves the reading frame. In certain embodiments, the at least one mutation comprises an internal deletion of at least 3 nucleotidesencoding one or more amino acids corresponding to: (i) amino acids 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and / or 110; (ii) amino acids 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, and / or 197; and / or (iii) amino acids 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, and / or 229; all of SEQ ID NO: 3 or an allelic variant thereof, wherein the deletion preserves the reading frame.

[0047] In certain embodiments, seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of the soybean plant comprising at least one amorphic allele of the endogenous soybean RPF1 gene is increased in comparison to the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean plant lacking the at least one amorphic allele of the RPF1 gene. Increased yield of the soybean plant can be measured in a number of ways, including pod count per plant, seed count per plant, total harvested seed weight per plant, or total harvested seed weight per unit area (e.g., seed weight per acre or seed weight per hectare). In certain embodiments, the leaf thickness, chlorophyll content, seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of the soybean plant comprising at least one amorphic allele of the endogenous soybean RPF1 gene is preserved or further enhanced when the at least one amorphic allele of the endogenous soybean RPF1 gene is combined with least one mutation in a distinct soybean gene (e.g., a hypom orphic or amorphic allele of a soybean AML4 gene, AlPlOa gene, AlPlOb gene, RIC1 gene, RIC2 gene, FTla gene, JAG1 gene, JAG2 gene, TCP5-L gene, BS1 gene, BS2 gene, CRN gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene).

[0048] In certain embodiments, the seeds per pod for the soybean plant comprising the at least one mutation in the RPF1 gene is increased in comparison to the seeds per pod for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the seeds per pod is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the seeds per pod from the corresponding wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the average number of seeds per pod for the soybean plant comprising the at least one mutation in the RPF1 gene is at least about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, or 3.8. In certain embodiments, the percentage of 3 seeded pods for the soybean plant comprising the at least one mutation in the RPF1 gene is increased in comparison to the percentage of 3 seeded pods for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the percentage of 3 seeded pods is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the percentage of 3 seeded pods from the corresponding wild-type orcontrol soybean plant lacking the at least one mutation. In certain embodiments, the percentage of 3 seeded pods for the soybean plant comprising the at least one mutation in the RPF1 gene is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. In certain embodiments, the percentage of 4 seeded pods for the soybean plant comprising the at least one mutation in the RPF1 gene is increased in comparison to the percentage of 4 seeded pods for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the percentage of 4 seeded pods is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% in comparison to the percentage of 4 seeded pods from the corresponding wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the percentage of 4 seeded pods for the soybean plant comprising the at least one mutation in the RPF1 gene is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%.

[0049] In certain embodiments, the pod count per soybean plant comprising the at least one mutation in the RPF1 gene is increased in comparison to the pod count per plant for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the pod count per plant is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the pod count per plant from the corresponding wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the seed count per plant comprising the at least one mutation in the RPF1 gene is increased in comparison to the seed count per plant for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the seed count per plant comprising the at least one mutation in the RPF1 gene is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the seed count per plant from the corresponding wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the total harvested seed weight per plant comprising the at least one mutation in the RPF1 gene is increased in comparison to the total harvested seed weight per plant for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the total harvested seed weight per plant is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the total harvested seed weight per plant from the corresponding wildtype or control soybean plant lacking the at least one mutation. In certain embodiments, the totalharvested seed weight per unit area for soybean plants comprising the at least one mutation in the RPF1 gene is increased in comparison to the total harvested seed weight per unit area for a wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the total harvested seed weight per unit area for soybean plants comprising the at least one mutation in the RPF1 gene is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the total harvested seed weight per unit area from the corresponding wild-type or control soybean plant lacking the at least one mutation. In certain embodiments, the average weight of 1000 seeds obtained from the soybean plant is equivalent to or essentially the same as the average weight of 1000 seeds obtained from a wild-type or control soybean plant lacking the at least one mutation.

[0050] In certain embodiment, the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for the soybean plant comprising the at least one mutation in the RPF1 gene are increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for a wild-type or control soybean plant lacking the at least one mutation when grown under stress or specific agronomic practices. Non-limiting examples of stresses include drought, cold, heat, salt, shade, nutrient deficiency (e.g., nitrogen deficiency), high planting density, and the presence of pests or pathogens. In certain embodiments, the stress comprises an abiotic stress. In certain embodiments, the abiotic stress comprises drought, cold, heat, salt stress, or nutrient deficiency (e.g., nitrogen deficiency). In these embodiments, the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area can be increased when the plant comprising the at least one mutation in the RPF1 gene is grown under abiotic stress in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for a wild-type or control soybean plant lacking the at least one mutation grown under abiotic stress.

[0051] Soybean seed lots comprising the soybean seeds comprising the at least one mutation in the RPF1 gene are provided. In certain embodiments, soybean plants comprising the mutated RPF1 gene can yield seed lots wherein the average weight of 1000 seeds in the seed lot is equivalent to or essentially the same as the average weight of 1000 seeds in a control seed lot obtained from a wild-type or control plant lacking the at least one mutation in the RPF1 gene(e.g., a wild-type soybean plant homozygous for a wild-type RPF1 gene). In certain embodiments, the average number of seeds per kilogram of seeds in the seed lot is equivalent to or essentially the same as the average number of seeds per kilogram of seeds in a control seed lot obtained from a wild-type or control soybean plant lacking the at least one mutation in the RPF1 gene. In certain embodiments, the seed lot is packaged in lots comprising about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms) of seeds.

[0052] Also provided are polynucleotides comprising any of the aforementioned mutated RPF1 genes or fragments thereof. In certain embodiments, polynucleotides comprising at least one mutation relative to the endogenous soybean RPF1 gene of SEQ ID NO: 1 are provided. In certain embodiments, the polynucleotide comprises a sequence having at least 95%, 96, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity across the entire length of SEQ ID NO: 1 with the proviso that the sequences are not identical to across their entire length to SEQ ID NO: 1. In certain embodiments, the polynucleotide is an isolated polynucleotide.

[0053] Biological samples and soybean by-products comprising any of the aforementioned polynucleotides are also provided. In certain embodiments, the by-products are processed products are made from the soybean plants of the disclosure or their seeds, including: (a) soybean seed meal (defatted or non-defatted); (b) extracted soybean proteins, oils, sugars, syrups, and starches; (c) soy fermentation products; (d) soybean based animal feed or human food products (e.g., feed and food comprising soybean seed meal (defatted or non-defatted) and other ingredients e.g., other cereal grains, other seed meal, other protein meal, other oil, other starch, other sugar, a binder, a preservative, a humectant, a vitamin, and / or mineral); (e) a pharmaceutical; (f) raw or processed biomass (e.g., cellulosic and / or lignocellulosic material; silage); and (g) various industrial products.

[0054] Methods of using the soybean plants, seeds, and seed lots of the disclosure to produce soybean by-products are also provided. Such methods will typically include at least one processing step of cleaning, cracking, flaking, crushing, macerating, pressing, extracting, expelling, and / or extruding the seed.

[0055] This disclosure is also directed to methods for producing a soybean plant having at least one mutation in the endogenous soybean RPF1 gene by crossing a first parent soybean plant with a second parent soybean plant wherein the first or second parent soybean plant comprises the at least one mutation. Further, both the first and second parent soybean plants can comprise the at least one mutation. Any such methods using a soybean plant comprising the at least one mutation are part of this disclosure: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using a soybean plant comprising the at least onemutation as a parent are within the scope of this disclosure, including plants derived from a soybean plant having the at least one mutation. Also provided are the Fi progeny soybean plants produced from the crossing of a soybean plant comprising the at least one mutation with any other soybean plant, Fi seed, and various parts of the Fi soybean plant. The following describes breeding methods that can be used with soybean plants of the disclosure in the development of further soybean plants. One such embodiment is a method for developing a progeny soybean plant in a soybean plant breeding program comprising: obtaining the soybean plant, or its parts, comprising at least one mutation in the endogenous soybean RPF1 gene and utilizing said plant or plant parts as a source of breeding material; and selecting a progeny plant having the at least one mutation. Breeding steps that can be used in the soybean plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as restriction fragment polymorphism enhanced selection, genetic marker enhanced selection (for example SNP or SSR markers), and the making of double haploids can be utilized.

[0056] Field crops are bred through techniques that take advantage of the plant’s method of pollination. A soybean plant of the disclosure can be self-pollinated, sib-pollinated, or cross pollinated to create a pedigree soybean plant. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is sib-pollinated when individuals within the same family or variety are used for pollination. A plant is cross-pollinated if the pollen comes from a flower on a different plant from a different family or variety. The terms “cross-pollination” and “out-cross” as used herein do not include self-pollination or sib-pollination. Soybean plants (Glycine max) are recognized to be naturally self-pollinated plants which, while capable of undergoing cross-pollination, rarely do so in nature. Insects are reported by some researchers to carry pollen from one soybean plant to another and it generally is estimated that less than one percent of soybean seed formed in an open planting can be traced to cross-pollination, z.e., less than one percent of soybean seed formed in an open planting is capable of producing Fi hybrid soybean plants.

[0057] Any other suitable breeding, selection, or growing methods may be used. Choice of the particular breeding or selection method will vary depending on environmental factors, population size, and the like.

[0058] In certain embodiments, soybean plant cells, plant parts e.g., seeds), and plants comprising at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof and a transgenic locus are provided. In certain embodiment, the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variantthereof is combined with one or more soybean GM events providing tolerance to any one or a combination of glyphosate-based, glufosinate-based, HPPD inhibitor-based, sulfonylurea- or imidazolinone-based, AHAS- or ALS-inhibiting and / or auxin-type (e.g., dicamba, 2,4-D) herbicides and / or an insect resistance trait. GM events that can be combined with the mutations disclosed herein include Event EE-GM3 (aka FG-072, MST-FG072-3, described in WO2011063411, USDA-APHIS Petition 09-328-01p), Event SYHTOH2 (aka 0H2, SYN-000H2-5, described in WO2012 / 082548 and 12-215-01p), Event DAS-68416-4 (aka Enlist Soybean, described in WO2011 / 066384 and WO2011 / 066360, USDA-APHIS Petition 09-349-Olp), Event DAS-44406-6 (aka Enlist E3, DAS-44406-6, described in WO2012 / 075426 and USDA-APHIS 1 l-234-01p), Event MON87708 (dicamba-tolerant event of Roundup Ready 2 Xtend Soybeans, described in WO2011 / 034704 and USDA-APHIS Petition 10- 188-0 Ip, MON-87708-9), Event MON89788 (aka Genuity Roundup Ready 2 Yield, described in W02006 / 130436 and USDA-APHIS Petition 06-178-01p), Event 40-3-2 (aka Roundup Ready, GTS 40-3-2, MON-04032-6, described in USDA-APHIS Petition 93-258-01), Event A2704-12 (aka LL27, ACS-GM005-3, described in W02006108674 and USDA-APHIS Petition 96-068-Olp), Event 127 (aka BPS-CV127-9, described in WO2010 / 080829), Event A5547-127 (aka LL55, ACS-GM006-4, described in W02006108675 and in USDA-APHIS Petition 96-068-Olp), event MON87705 (MON-87705-6, Vistive Gold, published PCT patent application W02010 / 037016, USDA-APHIS Petition 09-201-0 Ip), or event DP305423 (aka DP-305423-1, published PCT patent application W02008 / 054747, USDA-APHIS Petition 06-354-01p), or EE-GM5 is combined with a combination of the following events: Event MON98788*MON87708 (aka Roundup Ready 2 Xtend Soybeans, MON-87708-9*MON-89788-l), Event HOS*Event 40-3-2 (aka Plenish High Oleic Soybeans xRoundup Ready Soybeans), Event EE-GM3*EE-GM2 (aka FG-072xLL55, described in WO2011063413), Event MON 87701 xMON 89788 (aka Intacta RR2 Pro Soybean, MON-87701-2xMON-89788-l), DAS-81419-2xDAS-44406-6 (aka Conkesta™ Enlist E3™ Soybean, DAS-81419-2xDAS-444O6-6), Event DAS-68416-4 xEvent MON 89788 (aka Enlist™ RoundUp Ready® 2 Soybean, DAS-68416-4xMON-89788-l), Event MON-87769-7xEvent MON-89788-1 (aka Omega-3 x Genuity Roundup Ready 2 Yield Soybeans), Event MON 87705 xEvent MON 89788 (aka Vistive Gold, MON-87705-6xMON-89788-1), or Event MON87769xEvent MON89788 (aka Omega-3 x Genuity Roundup Ready 2 Yield Soybeans, MON-87769-7xMON-89788-l), where all published PCT patent applications or US national stages thereof are incorporated herein by reference in there entireties.Representative transgenic events that can be combined with the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof include those setforth in Table 1. Also provided herein are soybean plant cells, plant parts (e.g., seeds), and plants comprising at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof and a modification of any of the aforementioned transgenic events or transgenic events set forth in Table 1 below. Modifications of the transgenic events include those disclosed in: WO2022 / 026375, WO2022 / 026379, W02022 / 026390, WO2022 / 026395, W02022 / 026403; US Patent Applic. Pub. Nos. US20220030822 and US20230250441; and U.S. Patent No. 11,242,534, which are each incorporated herein by reference in their entireties.Table 1. Transgenic Soybean EventsEvent Name Patent or Patent ATCC;3NCIMB4Trait(traits)1Application Deposit Number; expressionNumber(s)2or Commercial cassette(s)SourceA5547- 127 (HT) US 20080196127 NCIMB 41660 PATRE44962DAS44406-6 US 9,540,655 PTA-11336 Aad-12,(HT)5US 10,400,250 2mepsps, PAT DAS68416-4 US 9,738,904 PTA- 10442 Aad-12, PAT(IR, HT)6PTA-12006DAS81419-2 US 8680363 PTA-12006 crylAc, crylF,(IR, HT) US 8632978 PATUS9695441US 9738904GTS 40-3-2 US 20070136836 M690GT | 0.9 RM cp4epsps(HT) Soybean7MON87701 (IR) US 8049071 PTA-8194 cry 1 AcMON87708 US 9447428 PTA-9670 DM0(HT)8MON89788 US 9944945 PTA-6708 cp4epsps(HT)MST-FG072-3 US 8592650 NCIMB 41659 hppdPF W336,(HT)92mepspsSYHTOH210US 10,184,134 PTA- 11226 cAvHPPD-031Traits: IR=Insect Resistance; HT=Herbicide Tolerance; AR= Antibiotic Resistance;2Each U S Patent or Patent Application Publication is incorporated herein by reference in itsentirety.3ATCC is the American Type Culture Collection, 10801 University Boulevard Manassas, VA20110 USA (for “PTA-XXXXX” deposits).4NCIMB is the National Collection of Industrial, Food and Marine Bacteria, FergusonBuilding, Craibstone Estate, Bucksbum, Aberdeen AB9YA, Scotland.5HT to 2,4-D; glyphosate, and glufosinate; also refered to as pDAB8264.44.06.1.6Independent IR / HT and HT events combined by breeding. IR / HT event (Cry IF, Cry 1 Acsynpro (Cry lAc), and PAT) is DAS81419-2, deposited with ATCC under PTA-12006, alsoreferred to as DAS81419-2.7Elk Mound Seed, 308 Railroad Street Elk Mound, WI, USA 54739.8HT to dicamba.9HT to both glyphosate and isoxaflutole herbicides.10HT to glufosinate and mesotrione herbicides.

[0059] In certain embodiments, soybean plant cells, plant parts (e.g., seeds), and plants comprising the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof and at least one mutation in a distinct soybean gene are provided. In certain embodiments, soybean plant cells, plant parts (e.g., seeds), and plants comprising the at least one mutation in the endogenous soybean RPF1 and at least one mutation in a distinct soybean gene are provided. Non-limiting examples of mutations in distinct soybean genes that may be combined with the at least one mutation in the endogenous soybean RPF1 gene of the disclosure include mutations in any one or a combination of the soybean AML4 gene, AlPlOa gene, AlPlOb gene, RIC1 gene, RIC2 gene, FT la gene, JAG1 gene, JAG2, gene, TCP5-L gene, BS1 gene, BS2 gene, CRN gene, TFLlb gene, CYP76C gene, and / or NF- YC4 gene, where the mutation is optionally a loss-of-function mutation e.g., a hypomorphic or amorphic allele). In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean AML4 gene located at nucleotides 23,215,077 to 23,222,035 of chromosome 8 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.vl; Glyma.08g257400 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). In certain embodiments, the at least one mutation in the AML4 gene comprises a deletion, insertion, and / or substitution in the coding region (e.g., 5’ UTR, an exon, an intron, and / or a 3’ UTR) of the AML4 gene. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean AlPlOa gene. The AlPlOa gene (Glyma.07G021400) is located at nucleotides 1,664,710 to 1,668,114 of chromosome 7 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the mutation (e.g., a deletion) in the AlPlOa gene can be in the AlPlOa gene promoter and / or 5’ UTR. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean AlPlOb gene. The AlPlOb gene (Glyma.08G220400) is located at nucleotides 17,985,027 to 17,988,197 of chromosome 8 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the mutation (e.g., a deletion) in the AlPlOb gene can be in the AlPlOb gene promoter and / or 5’ UTR. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean RIC1 gene. The RIC1 gene (Glyma.l3g292300) is located at nucleotides 38,587,351 to 38,588,133 of chromosome 13 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean RIC2 gene, where the mutation in the RIC2 gene is optionally a deletion in the promoter region of the gene. The RIC2 gene (Glyma.06g284100) is located atnucleotides 46,845,530 to 46,845,811 of chromosome 6 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean FTla gene. The FTla gene (Glyma.l8G298900) is located at nucleotides 57,922,912 to 57,928,648 of chromosome 18 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof is combined with a mutation in the soybean JAG1 gene. The JAG1 gene is located at nucleotides 35827671 to 35830107 of chromosome 20 of the Glycine max Wm82.a2.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof is combined with a mutation in the soybean JAG2 gene. The JAG2 gene is located at nucleotides 49718688 to 49722581 of chromosome 10 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof is combined with a mutation in the soybean TCP5-L gene. The TCP5-L gene is located at nucleotides 38,837,566 to 38,842,283 of chromosome 4 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof is combined with a mutation in the soybean CYP76C gene. The CYP76C gene (Glyma.l8G222900) is located at nucleotides 51,262,793 to 51,265,190 of chromosome 18 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean BS1 gene. The BS1 gene (Glyma.l0g244400) is located at nucleotides 47,330,160 to 47,335,971 of chromosome 10 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the soybean BS2 gene. The BS2 gene (Glyma.20gl 50000) is located at nucleotides 38,879,463 to 38,885,344 of chromosome 20 of the Glycine max Wm82.a4.vl genome assembly. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation in the endogenous soybean CRN gene is located at nucleotides 23,254,545 to 23,257,685 of chromosome 8 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.vl; Glyma.08g257700 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798), where the mutation in the CRN gene is optionally a loss-of-function mutation (e.g., a hypomorphic or amorphic allele). In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation (e.g., a deletion) in the TFLlb gene (Glyma.l9gl94300) which can be a promoter element deletion asdescribed in WO2023086765, which is incorporated herein by reference in its entirety. In certain embodiments, the at least one mutation in the endogenous soybean RPF1 gene is combined with a mutation (e.g., a deletion) in the NF-YC4 gene (Glyma06gl7780 and / or Glyma04g37291) which can be a promoter element deletion as described in US20230139093, which is incorporated herein by reference in its entirety.

[0060] Methods of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising an amorphic allele of a RPF1 gene to maturity; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot, wherein the soybean plants are homozygous for the amorphic allele of a RPF1 gene. In certain embodiments, the seed lot is packaged in lots comprising about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms).

[0061] Also provided herein are methods of treating the soybean seeds and seed lots of the disclosure and the resultant treated seeds and seed lots. Seeds can be treated with such fertilizers, biological agents, nematicides, insecticides, and fungicides by methods including in-furrow applications or by coating (e.g., with a drum coater, rotary coater, tumbling drum, fluidized bed, and / or spouted bed apparatus). Methods and compositions including various binders, fillers, film coats, and active ingredients such as fertilizers, surfactants, plant growth regulators, crop desiccants, fungicides, bacteriocides, bacteriostats, insecticides, and insect repellants for coating seeds that can be adapted for use with seeds provided herein are disclosed in US Patent No. 10745578, which is incorporated herein by reference in its entirety.

[0062] The disclosure also provides a method of making a soybean plant comprising an amorphic allele of a RPF1 gene. In certain embodiments, the methods can comprise making a deletion, an insertion and / or a substitution which results in an amorphic allele of a RPF1 gene. Gene editing molecules of use in methods provided herein include molecules capable of introducing a double-strand break (“DSB”) or single-strand break (“SSB”) at a specific site or sequence in a double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA. In certain embodiments, the at least one mutation results from introduction of a DSB at a target site in the RPF1 gene e.g., SEQ ID NO: 1 or an allelic variant thereof) to induce non-homologous end joining (NHEJ) at the site of the break followed by recovery of the desired mutation. In certain embodiments, the at least one mutation results from introduction of a DSB at a target site in the RPF1 gene (e.g., SEQ ID NO: 1 or an allelic variant thereof) followed by homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ to introduce a desired donor or other DNA template polynucleotide at the DSB, followed by recovery of the desired mutation. Examples of suchgene editing molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type nuclease system, a class 2 type II Cas nuclease, a Cas9, a nCas9 nickase, a class 2 type V Cas nuclease, a Cas 12a nuclease, a nCasl2a nickase, a Cas 12d (CasY), a Casl2e (CasX), a Cas 12b (C2cl), a Casl2c (C2c3), a Casl2i, a Casl2j, a Casl4, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (including introduction of a DSB or SSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for use with an RNA-guided nuclease, or a DNA encoding a gRNA for use with an RNA-guided nuclease; (d) optionally donor DNA template polynucleotides suitable for insertion at a break in genomic DNA by homology-directed repair (HDR) or microhomology-mediated end joining (MMEJ); and (e) optionally other DNA templates (e.g., dsDNA, ssDNA, or combinations thereof) suitable for insertion at a break in genomic DNA (e.g, by non-homologous end joining (NHEJ). In certain embodiments, the at least one mutation is made with a cytosine and / or adenine base editor, or by a PRIME editing system.

[0063] In certain embodiments, the mutated RPF1 gene and plant cells, parts including seeds, and plants comprising the mutated RPF1 gene are generated by CRISPR technology. CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016 / 0138008 Al and US2015 / 0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 Al. Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in International Patent Application PCT / US2015 / 018104 (published as WO 2015 / 131101 and claiming priority to US Provisional Patent Application 61 / 945,700). Methods of using CRISPR technology for genome editing in plants are disclosed in US Patent Application Publications US 2015 / 0082478 Al and US 2015 / 0059010A1 and in International Patent Application PCT / US2015 / 038767 Al (published as WO 2016 / 007347 and claiming priority to US Provi sional Patent Applicati on 62 / 023,246). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety. In certain embodiments, an RNA-guided endonuclease that leaves a blunt end following cleavage of the target site is used. Blunt-end cutting RNA-guided endonucleases include Cas9.In certain embodiments, an RNA-guided endonuclease that leaves a staggered single stranded DNA overhanging end following cleavage of the target site following cleavage of the target site is used. Staggered-end cutting RNA-guided endonucleases include Casl2a, Casl2b, Casl2d, Casl2e, and Casl2i.

[0064] Guide RNA molecules comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof are provided. In certain embodiments, the spacer RNA molecule targets a portion of the protein and intron coding region ( / .< ., nucleotides 120-1955 of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1) of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments, the spacer RNA molecule comprises the RNA encoded by SEQ ID NO: 4, 5, or 6. Guide RNAs comprising a spacer RNA molecule encoded by SEQ ID NO: 4, 5, or 6 can be used in conjunction with a type V (Casl2a or Casl2i nuclease) to generate mutated RPF1 genes which: (i) comprise, consist essentially of, or consist of a deletion, insertion, and / or substitution of at least one nucleotide (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, or 3939 nucleotides) of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof; (ii) comprise, consist essentially of, or consist of a deletion, insertion, and / or substitution of at least one nucleotide (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, or 1836 nucleotides) corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1; or (iii) comprise, consist essentially of, or consist of a deletion, insertion, and / or substitution of at least one nucleotide (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides) corresponding to: (i) nucleotides 415-439; (ii) nucleotides 675-699; and / or (iii) nucleotides 770-794; all in a RPF1 gene of SEQ ID NO: 1 or in an equivalent position of an allelic variant of SEQ ID NO: 1.

[0065] CRISPR-type genome editing can be adapted for use in the plant cells and methods provided herein in several ways. CRISPR elements, e.g., gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or guide RNAs in combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are provideddirectly to the eukaryotic cell (e.g., soybean plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, soybean plants or soybean plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpfl-type or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA or scoutRNA) to form an RNA-guided endonuclease / guide RNA complex which can specifically bind sequences in the gDNA target site that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5’-NGG are typically targeted for design of crRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), 5’-NNGRRT or 5’-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5’-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5’-TTN or 5’-TTTV, where “V” is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with Cast 2a proteins. In some instances, Casl2a can also recognize a 5’-CTA PAM motif. Other examples of potential Casl2a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 Al, which is incorporated herein by reference for its disclosure of DNA encoding Cpfl endonucleases and guide RNAs and PAM sites. Engineered endonucleases with altered or eliminated PAM recognition sites can also be used.

[0066] Identification of a suitable PAM site is only one factor in selecting an efficacious gRNA. Different guides can also have different cutting efficiencies and specificities, which depend on a number of factors. In some embodiments, identification of effective gRNA target sequences in the genome is approached through a combination of in silico selection and experimental evaluation.

[0067] In some cases, a target sequence that perfectly hybridizes with the gRNA spacer sequence occurs only once in a given plant genome. In some embodiments, the genome comprises additional sequences that imperfectly hybridize with the gRNA spacer sequence, forexample, sequences having one or more mismatches (e.g., 1, 2, 3, 4, or 5 mismatches) and / or bulges, relative to the gRNA spacer sequence. In some embodiments, the genome comprises sequences that hybridize the gRNA spacer sequence that are adjacent to a PAM sequence having at least one mismatch relative to the canonical PAM sequence. Such genomic sequences (e.g., target sequences that imperfectly hybridize the gRNA spacer sequence and / or target sequences comprising a non-canonical PAM sequences) are called off-target sites. A favorable off-target profile is typically one that minimizes or eliminates the number of off-target sites and / or the frequency of cutting at these sites.

[0068] The nuclease efficiency and occurrence of off-target activity for a given gRNA / endonuclease combination can be influenced by a number of factors including similarities and dissimilarities between the target site and various off-target sites, as well as the particular endonuclease used. For example, the ability of a given gRNA to promote cleavage at a target sequence in a genomic DNA molecule may relate to the accessibility of the target sequence, which depends on one or more factors that include the chromatin structure of the genomic DNA molecule and / or proximity to transcription factor binding sites. For example, target sequences located within a region of the genomic DNA molecule having a high condensed chromatin structure are less accessible than target sequences located within a region of the genomic DNA molecule having an open chromatin structure. As a further example, target sequences proximal to a region of the genomic DNA molecule bound by a transcription factor or other regulatory protein may be less accessible than target sequences proximal a region of the genomic DNA molecule that is unbound by regulatory proteins. Moreover, the cell state and type of cell may influence the accessibility of target sequences, for example, by influencing the chromatin structure of genomic DNA.

[0069] In certain embodiments, the mutated RPF1 gene and plant cells, parts including seeds, and plants comprising the mutated RPF1 gene are generated by use of zinc finger nucleases or zinc finger nickases. Zinc-finger nucleases are site-specific endonucleases comprising two protein domains: a DNA-binding domain, comprising a plurality of individual zinc finger repeats that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain that comprises a nuclease domain (typically Fokl). The cleavage domain dimerizes in order to cleave DNA; therefore, a pair of ZFNs are required to target non-palindromic target polynucleotides. In certain embodiments, zinc finger nuclease and zinc finger nickase design methods which have been described (Umov et al. (2010) Nature Rev. Genet., 11:636 - 646; Mohanta et al. (2017) Genes vol. 8,12: 399; Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560-5568; Liu et al. (2013) Nature Communications, 4: 2565) can be adapted for use in the methods set forth herein.The zinc finger binding domains of the zinc finger nuclease or nickase provide specificity and can be engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc “fingers” each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity of the zinc finger binding domain. One approach, termed “modular assembly”, relies on the functional autonomy of individual zinc fingers with DNA. In this approach, a given sequence is targeted by identifying zinc fingers for each component triplet in the sequence and linking them into a multifinger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, e.g., US Patents 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) can be adapted for use in the methods described herein. In addition, enhancement of binding specificity for zinc finger binding domains has been described in US Patent 6,794,136, incorporated herein by reference in its entirety. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. Examples of linker sequences are publicly known, e.g., see US Patents 6,479,626; 6,903,185; and 7,153,949, incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fokl as part of a ZFN requires two adjacent and independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fokl variants with enhanced activities have been described and can be adapted for use in the methods described herein; see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96 - 107.

[0070] In certain embodiments, the mutated RPF1 gene and plant cells, parts including seeds, and plants comprising the mutated RPF1 gene are generated by use of TAL-effector nucleases or TALENs. Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a nonspecific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl, can be conveniently used. Methods for use of TALENs in plants have been described and can be adapted for use in the methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623 - 2628; Mahfouz (2011) GM Crops, 2:99 - 103; and Mohanta et al. (2017) Genes vol. 8,12: 399). TALE nickases have also been described and can be adapted for use in methods described herein (Wu et al.; Biochem Biophys Res Commun. (2014);446(l):261-6; Luo et al; Scientific Reports 6, Article number: 20657 (2016)).

[0071] Various treatments can be used for delivery of gene editing molecules and / or other molecules to a plant cell. In certain embodiments, one or more treatments is employed to deliver the gene editing or other molecules (e.g., comprising a polynucleotide, polypeptide or combination thereof) into a plant cell, e.g., through barriers such as a cell wall, a plasma membrane, a nuclear envelope, and / or other lipid bilayer. In certain embodiments, a polynucleotide-, polypeptide-, or RNP (ribonucleoprotein) -containing composition comprising the molecules are delivered directly, for example by direct contact of the composition with a plant cell. Aforementioned compositions can be provided in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant, plant part, plant cell, or plant explant (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). Forexample, a plant cell or plant protoplast is soaked in a liquid genome editing moleculecontaining composition. In certain embodiments, the composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In certain embodiments, the composition is introduced into a plant cell or plant protoplast, e.g, by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in US Published Patent Application 2014 / 0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the composition to a eukaryotic cell, plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In certain embodiments, the composition is provided by bacterially mediated (e.g. , Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the genome editing molecules (e.g, RNA dependent DNA endonuclease, RNA dependent DNA binding protein, RNA dependent nickase, ABE, or CBE, and / or guide RNA); see, e.g., Broothaerts et al. (2005) Nature, 433 :629 - 633). Any of these techniques or a combination thereof are alternatively employed on a plant explant, plant part or tissue or intact plant (or seed) from which a plant cell is optionally subsequently obtained or isolated; in certain embodiments, the composition is delivered in a separate step after the plant cell has been isolated.

[0072] In certain embodiments, the methods for generating the soybean plant cell, soybean plant parts, or soybean plants comprise: (i) screening a population of soybean plant cells, parts, or plants for the presence of at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising at least one mutation in the soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0073] In certain embodiments, the population of soybean plant cells, parts, or plants which are screened for the presence of at least one mutation in the endogenous soybean RPF1 gene arefirst pre-screened by screening for phenotypic characteristics plants having at least one mutation in a RPF1 gene. In certain embodiments, the population of soybean plant cells, parts, or plants which are screened for the presence of at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1, an allelic variant thereof, and / or at least one mutation in a RPF1 gene are first pre-screened by screening of phenotypic characteristics plants having at least one mutation in a RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments, such phenotypic characteristics include increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield (e.g., pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area) in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield for a wild-type or control soybean plant lacking the at least one mutation(s). In certain embodiments, such phenotypic characteristics include increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield (e.g., pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area) in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield per plant for a wild-type or control soybean plant lacking the at least one mutation where the screened and wild-type or control plants are grown under nitrogen deficient conditions. In certain embodiments, plants exhibiting one or more of the aforementioned phenotypic characteristics are then subjected to screening for the presence of at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or 14 and soybean plants comprising at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or 14 are identified and / or selected. In certain embodiments, plants exhibiting one or more of the aforementioned phenotypic characteristics are then subjected to screening for the presence of at least one mutation in the RPF1 gene and soybean plants comprising at least one mutation in the endogenous soybean RPF1 are identified and / or selected.

[0074] In certain embodiments, the population of soybean plant cells, parts, or plants which are screened for the presence of at least one mutation in the endogenous soybean RPF1 gene (e.g., of SEQ ID NO: 1 or an allelic variant thereof) have been subjected to one or more mutagenesis treatments. Mutations of the endogenous soybean RPF1 gene can be generated by mutagenesis methods known in the art, such as chemical mutagenesis or radiation mutagenesis. Suitable chemical mutagens include ethyl methanesulfonate (EMS), sodium azide, methylnitrosourea (MNU), and diepoxybutane (DEB). Suitable radiation includes x-rays, fast neutron radiation, and gamma radiation.

[0075] Soybean plant cells, parts, or plants comprising at least one mutation in the endogenous RPF1 gene can be generated using mutagenesis and identified by TILLING (Targeting Induced Local Lesions IN Genomes) or identified using EcoTILLING. TILLING is a general reverse genetics technique that uses mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations. In addition to allowing efficient detection of induced mutations, high-throughput TILLING technology is ideal for the detection of natural mutations. EcoTILLING is a method that uses TILLING techniques to look for natural mutations in individuals (Barkley and Wang. Current genomics vol. 9,4 (2008): 212-26. doi: 10.2174 / 138920208784533656). Identified mutations can then be introduced into desirable genetic backgrounds by crossing the mutant with a plant of the desired genetic background and performing a suitable number of backcrosses to cross out the originally undesired parent background. A more detailed description of methods and compositions for TILLING are disclosed in US Patent Application Publication 2004 / 0053236 Al, which is incorporated herein by reference in its entirety and can be adapted for use in the methods provided herein for identifying soybean plant cells, parts, or plants comprising at least one mutation in the endogenous RPF1 gene.

[0076] In certain embodiments, the screening comprises analyzing pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area in one or more candidate plants or one or more candidate plant populations. In these embodiments, an increase in pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area in comparison to a wild-type or control soybean plant lacking the at least one mutation is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation. In certain embodiments, the screening is conducted on a population of plants grown under stress. Suitable examples of stress conditions include drought, salt, cold, heat, salt, shade, nutrient deficiency, high planting density, and the presence of pests or pathogens.

[0077] Methods for determining whether a soybean plant cell, plant part, or plant comprises at least one mutation in the endogenous soybean RPF1 gene are provided. Methods for determining the presence or absence of the at least one mutation can be used in, for example, breeding programs for identification, selection, introgression, and the like.

[0078] In certain embodiments, the methods comprise analyzing a polynucleotide comprising a portion of a RPF1 gene (e.g., of SEQ ID NO: 1, 2, 9, 10, 14 or an allelic variant thereof) or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from the plant cell, plant part, or plant. In certain embodiments, an insertion, deletion, and / or substitutionof one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation. Detection of the at least one mutation in a nucleic acid sample (e.g., DNA, RNA, or cDNA) can be achieved by any combination of nucleic acid amplification (e.g, PCR amplification), hybridization, sequencing, and / or mass-spectrometry based techniques. In certain embodiments, such detection is achieved by amplification and / or hybridization-based detection methods using a primer (e.g., selective amplification primers) and / or probe (e.g., capable of selective hybridization or generation of a specific primer extension product) which specifically recognizes the RPF1 gene (e.g., a portion of SEQ ID NO: 1 or an allelic variant thereof). Such primers and / or probes can comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous nucleotides of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments, the primers or probes can comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides or about 15 to 30 contiguous nucleotides of SEQ ID NO: 1 or an allelic variant thereof. In certain embodiments, the hybridization probes (e.g., polynucleotides comprising at least about 15 to 30 base pairs of SEQ ID NO: 1 or an allelic variant thereof) can comprise detectable labels (e.g., fluorescent, radioactive, epitope, and chemiluminescent labels). In certain embodiments, the RPF1 gene can be directly sequenced using nucleic acid sequencing technologies, including whole genome sequencing.

[0079] In certain embodiments, the methods comprise analyzing a RPF1 polypeptide encoded by SEQ ID NO: 1 or 14, a portion thereof, or an allelic variant thereof from the soybean plant cell, plant part, or plant. In certain embodiments, an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation. Detection of the at least one mutation based on the polypeptide can be determined by methods well known in the art such as activity assays, western blots using antibodies capable of specifically binding the polypeptide, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence, and the like.

[0080] In certain optional embodiments, the soybean plant cells disclosed herein are non-regenerable soybean plant cells. In certain optional embodiments provided herein, the soybean plant cells, soybean plant propagules (e.g., a seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, explant, or callus), and soybean plants provided herein are not produced by an exclusively biological process. In certain optional embodiments provided herein, the methods for producing soybean plant cells, soybean plant propagules (e.g, a seed, seedling, ovule,embryo, pollen, root, stem, leaf, shoot, explant, or callus), and soybean plants provided herein are not exclusively biological processes.

[0081] The following numbered embodiments also form part of the present disclosure:

[0082] 1. A soybean plant cell comprising at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0083] 2. The soybean plant cell of embodiment 1, wherein the plant cell is homozygous for the at least one mutation in the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof.

[0084] 3. The soybean plant cell of embodiment 1 or embodiment 2, wherein the at least one mutation comprises a loss-of-function allele of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof, wherein the loss-of-function allele reduces expression of the RPF1 gene relative to a wild-type or control soybean plant cell lacking the at least one mutation.

[0085] 4. The soybean plant cell of any one of embodiments 1-3, wherein the at least one mutation is a non-natural mutation.

[0086] 5. The soybean plant cell of any one of embodiments 1-4, wherein the at least one mutation comprises an amorphic allele of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof.

[0087] 6. The soybean plant cell of any one of embodiments 1-5, wherein the at least one mutation comprises a hypomorphic allele of the RPF1 gene.

[0088] 7. The soybean plant cell of any one of embodiments 1-6, wherein the at least one mutation comprises a frameshift mutation or a nonsense mutation in the coding region of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof.

[0089] 8. The soybean plant cell of any one of embodiments 1-7, wherein the at least one mutation comprises an internal deletion in the coding region of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof, optionally wherein the internal deletion preserves the reading frame of the encoded mutant RPF1 protein comprising the at least one mutation with respect to amino acid residues of the mutant RPF1 protein which have not been deleted.

[0090] 9. The soybean plant cell of any one of embodiments 1-8, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, or 3939 nucleotides of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0091] 10. The soybean plant cell of any one of embodiments 1-9, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, or 1836 nucleotides corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0092] 11. The soybean plant cell of any one of embodiments 1-10, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0093] 12. The soybean plant cell of any one of embodiments 1-11, with the proviso that the soybean plant cell is not exclusively produced by an essentially biological method, optionally wherein the soybean plant cell is produced by introducing one or more gene editing molecules into a soybean plant cell and selecting the soybean plant cell comprising at least one non-natural mutation.

[0094] 13. The soybean plant cell of any one of embodiments 1-12, wherein the soybean plant cell comprises elite soybean germplasm, optionally wherein the elite soybean germplasm comprises germplasm of a soybean cultivar or variety.

[0095] 14. The soybean plant cell of any one of embodiments 1-13, wherein the soybean plant cell further comprises at least one mutation in a distinct soybean gene.

[0096] 15. The soybean plant cell of any one of embodiments 1-14, wherein the soybean plant cell further comprises at least one mutation in the soybean AlPlOa gene, AlPlOb gene, AML4 gene, RIC1 gene, RIC2 gene, FTla gene, JAG1 gene, JAG2 gene, TCP5-L gene, BS1 gene, BS2 gene, CRN gene, TFLlb gene, CYP76C gene, and / or NF -YC4 gene.

[0097] 16. The soybean plant cell of any one of embodiments 1-15, wherein the soybean plant cell further comprises one or more transgenes, optionally wherein the transgenes encode proteins or RNAs conferring herbicide tolerance or pest tolerance.

[0098] 17. The soybean plant cell of any one of embodiments 1-16, wherein the soybean plant cell further comprises an A2704-12, A5547-127, BPS-CV127-9, DAS44406-6, DAS68416-4, DAS81419-2, DP305423, GTS 40- 3-2, HOS, A5547-127, MON87701, MON87705, MON87708, MON87769, MON89788, MON98788, MST-FG072-3, or SYHT0H210 transgenic event or modification thereof.

[0099] 18. A soybean plant part comprising the soybean plant cell of any one of embodiments 1-17.

[0100] 19. The soybean plant part of embodiment 18, wherein the part is a stem, root, leaf, flower, pod, or seed.

[0101] 20. The soybean plant part of embodiment 18 or embodiment 19, wherein the part is a seed.

[0102] 21. A soybean seed lot comprising the seed of embodiment 20, optionally wherein the seed lot comprises elite soybean germplasm, a soybean cultivar, a soybean variety, and / or is homozygous for the mutation.

[0103] 22. A soybean plant comprising the soybean plant cell of any one of embodiments 1-17.

[0104] 23. The soybean plant of embodiment 22, wherein the soybean plant comprises increased seeds per pod, percentage of 3 seeded pods, and / or percentage of 4 seeded pods in comparison to seeds per pod, percentage of 3 seeded pods, and / or percentage of 4 seeded pods for a wild-type or control soybean plant lacking the at least one mutation, optionally wherein the increased seeds per pod, percentage of 3 seeded pods, and / or percentage of 4 seeded pods is on the main stem and / or branches.

[0105] 24. The soybean plant of embodiment 22 or embodiment 23, wherein yield is increased in comparison to yield for a wild-type or control soybean plant lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0106] 25. A plant or plant part of an elite soybean plant, cultivar, or variety with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield comprising at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, wherein the increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is in comparison to a wild-type or control soybean plant lacking the at least one mutation, and optionally wherein the plant part is a seed.

[0107] 26. The plant or plant part of embodiment 25, wherein the elite soybean plant, cultivar, or variety is homozygous for the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0108] 27. The plant or plant part of embodiment 25 or embodiment 26, wherein the at least one mutation comprises a loss-of-function allele of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof, wherein the loss-of-function allele reduces expression of the RPF1 gene relative to a wild-type or control plant or plant part lacking the at least one mutation.

[0109] 28. The plant or plant part of any one of embodiments 25-27, wherein the at least one mutation is a non-natural mutation.

[0110] 29. A biological sample comprising a nucleic acid containing at least one mutation in the soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.[OHl] 30. The biological sample of embodiment 29, wherein the sample comprises seed meal or a tissue sample homogenate, optionally wherein the tissue sample comprises a sample of leaf, flower, pod, seed, stem, or root tissue.

[0112] 31. The biological sample of embodiment 29 or embodiment 30, wherein the at least one mutation is a non-natural mutation.

[0113] 32. The biological sample of any one of embodiment 29-31, wherein the at least one mutation comprises a frameshift mutation or a nonsense mutation in the coding region of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof.

[0114] 33. The biological sample of any one of embodiments 29-32, wherein the at least one mutation comprises an internal deletion in the coding region of the RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof, optionally wherein the internal deletion preserves the reading frame of the encoded mutant RPF1 protein comprising the at least one mutation with respect to amino acid residues of the mutant RPF1 protein which have not been deleted.

[0115] 34. The biological sample of any one of embodiments 29-33, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, or 1836 nucleotides corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0116] 35. The biological sample of any one of embodiments 29-34, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consistsessentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0117] 36. The biological sample of any one of embodiments 29-35, wherein the sample lacks a nucleic acid comprising the wild-type allele of the soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0118] 37. A polynucleotide comprising at least one mutation relative to the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the polynucleotide is isolated.

[0119] 38. The polynucleotide of embodiment 37, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0120] 39. A method of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising the soybean plant of any one of embodiments 22-28; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot.

[0121] 40. The method of embodiment 39, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of wild-type or control soybean plants lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0122] 41. A method of producing a soybean crop comprising planting the seed lot of embodiment 21.

[0123] 42. The method of embodiment 41, further comprising harvesting seed from soybean crop grown from the planted seed.

[0124] 43. The method of embodiment 41 or embodiment 42, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean crop lacking the at least one mutation, optionallywherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0125] 44. A method for producing a soybean by-product comprising at least one processing step of cleaning, cracking, flaking, crushing, macerating, pressing, extracting, expelling, and / or extruding the seed lot of embodiment 21.

[0126] 45. The method of embodiment 44, wherein the by-product is soybean protein and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; (ii) extracting the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) with an organic solvent to produce defatted soymeal; and (iii) extracting the defatted soymeal from step (ii) with an aqueous solvent to produce an aqueous fraction comprising soybean protein.

[0127] 46. The method of embodiment 44 or embodiment 45, wherein the by-product is soybean oil and wherein the soybean seed lot is pressed to produce the oil.

[0128] 47. The method of any one of embodiments 44-46, wherein the by-product is soybean oil and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; and (ii) solvent extracting, expelling, and / or extruding step the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) to produce the oil.

[0129] 48. A guide RNA molecule comprising a spacer RNA molecule which targets the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the spacer RNA molecule comprises the RNA encoded by SEQ ID NO: 4, 5, or 6.

[0130] 49. A guide RNA molecule comprising a Casl2 direct repeat element which is operably linked to the spacer RNA of embodiment 48.

[0131] 50. A gene editing system comprising: a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to the endogenous soybean RPF1 gene of SEQ ID NO: 1, 14, or an allelic variant thereof.

[0132] 51. An expression cassette comprising: a polynucleotide encoding CRISPR-Cas effector protein comprising a cleavage domain and the guide RNA molecule of embodiment 48 or embodiment 49.

[0133] 52. A method for generating a soybean plant cell of any one of embodiments 1-17, soybean plant part of any one of embodiments 18-20, or soybean plant of any one of embodiments 22-24 comprising introducing at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof of a soybean plant cell.

[0134] 53. The method of embodiment 52, further comprising regenerating a soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof from the soybean plant cell, and optionally selecting a soybean plant with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield, optionally wherein increased yield comprises increased pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0135] 54. The method of embodiment 52 or embodiment 53, wherein the at least one mutation is introduced by: (i) directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, or a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6; and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0136] 55. The method of any one of embodiments 52-54, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.

[0137] 56. The method of any one of embodiments 52-55, wherein the soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation is identified by: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof in one or more candidate plant cells, plant parts, or plants; (ii) analyzing a polypeptide encoded by a portion of SEQ ID NO: 1 in one or more candidate plant cells, plant parts, or plants; and / or (iii) analyzing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in comparison to a wild-type or control soybean plant lacking the at least one mutation is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0138] 57. The method of any one of embodiments 52-56, wherein the at least one mutation is introduced by crossing a soybean plant comprising the at least one mutation with a second soybean plant and harvesting Fl seed comprising the at least one mutation, thereby producing progeny soybean seed comprising the at least one mutation.

[0139] 58. A method for generating a soybean plant cell of any one of embodiments 1-17, soybean plant part of any one of embodiments 18-20, or soybean plant of any one of embodiments 22-24 comprising: (i) screening a population of soybean plant cells, parts, or plants for the presence of the at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0140] 59. The method of embodiment 58, wherein the population of soybean plant cells, parts, or plants have been subjected to one or more mutagenesis treatments, optionally wherein the mutagenesis procedure comprises chemical mutagenesis.

[0141] 60. The method of embodiment 58 or embodiment 59, wherein the screening comprises: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from one or more candidate plant cells, plant parts, or plants, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from one or more candidate plant cells, plant parts, or plants, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation.

[0142] 61. The method of any one of embodiments 58-60, wherein the screening further comprising analyzing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in comparison to a wild-type or control soybean plant lacking the at least one mutation, wherein increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0143] 62. A method for increasing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in a soybean plant, comprising: introducing at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof of a soybean plant cell.

[0144] 63. The method of embodiment 62, further comprising regenerating a soybean plant comprising the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 oran allelic variant thereof from the soybean plant cell, and optionally selecting a soybean plant with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield.

[0145] 64. The method of embodiment 62 or embodiment 63, wherein the at least one mutation is introduced by: (i) directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, or a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6; and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and (ii) isolating a soybean plant cell or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0146] 65. The method of any one of embodiments 62-64, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.

[0147] 66. The method of any one of embodiments 62-65, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean crop lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0148] 67. A soybean plant or plant cell produced by the method of any one of embodiments 62-66.

[0149] 68. A method for determining whether a soybean plant cell, plant part, or plant comprises at least one mutation of the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, the method comprising: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from the plant cell, plant part, or plant, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from the soybean plant cell, plant part, or plant, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation.

[0150] 69. The method of embodiment 68, wherein the method further comprises analyzing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in comparison to a wild-type or control soybean plant lacking the at least one mutation, wherein increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0151] 70. A method for determining whether a soybean seed lot comprises soybean seed comprising at least one mutation of the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, the method comprising: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from a sample of the soybean seed lot, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from a sample of the soybean seed lot, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation.

[0152] 71. Use of the soybean plant cell of any one of embodiments 1-17, the soybean plant part of any one of embodiments 18-20, the soybean seed lot of embodiment 21, or the soybean plant of any one of embodiments 22-28 to produce soybean seed.

[0153] 72. Use of the soybean plant cell of any one of embodiments 1-17, the soybean plant part of any one of embodiments 18-20, the soybean seed lot of embodiment 21, or the soybean plant of any one of embodiments 22-28 to grow a soybean crop.

[0154] 73. Use of the soybean plant cell of any one of embodiments 1-17, the soybean plant part of any one of embodiments 18-20, the soybean seed lot of embodiment 21, or the soybean plant of any one of embodiments 22-28 to obtain a soybean by-product.

[0155] 74. The use of embodiment 73, wherein the soybean by-product comprises soybean flour, meal, protein, oil, syrup, or starch.

[0156] 75. Use of the guide RNA molecule of embodiment 48 or embodiment 49, the gene editing system of embodiment 50, or the expression cassette of embodiment 51 to introduce at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

[0157] 76. A soybean plant cell, soybean plant part, or soybean plant obtainable by the process comprising: (i) directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising at least one mutation in the RPF1 gene of SEQ ID NO. 1 or an allelic variant thereof.

[0158] 77. The soybean plant cell, soybean plant part, or soybean plant of embodiment 76, wherein the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6.

[0159] 78. The soybean plant cell, soybean plant part, or soybean plant of embodiment 76 or embodiment 77, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.

[0160] 79. The soybean plant cell, soybean plant part, or soybean plant of any one of embodiments 76-78, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean crop lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

[0161] 80. A soybean genome characterized by the fact that it comprises the nucleotide sequence of SEQ ID NO: 1 or 14 which contains a non-natural mutation.

[0162] 81. The soybean genome of embodiment 80, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide of SEQ ID NO: 1, optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500 or 3939 nucleotides of SEQ ID NO: 1.

[0163] 82. The soybean genome of embodiment 80 or embodiment 81, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 120-1955 of SEQ ID NO: 1, optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, or 1836 nucleotides corresponding to nucleotides 120-1955 of SEQ ID NO: 1.

[0164] 83. The soybean genome of any one of embodiments 80-82, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of SEQ ID NO: 1.EXAMPLESExample 1. Generation of soybean with an amorphic allele of a RPF1 gene

[0165] A vector was created to transform soybean plants and disrupt the open reading frame of the RPF1 gene (SEQ ID NO: 1) through CRISPR-mediated gene editing. At least one CRISPR guide RNA comprising a crRNA fused to a spacer RNA (encoded by SEQ ID NO: 4, 5, and / or 6) was designed to target the of the Glycine max RPF1 gene (Figure 1).

[0166] The plasmids are transformed w Agrobacterium tumefaciens EHA105 (Hood el al., 1993, Transgenic Research. 2: 208-218. doi:10.1007 / BF01977351) by electroporation following standard techniques. Frozen glycerol stocks are prepared for use in plant transformation.

[0167] Transgenic TO soybean events are made by d z zcVcvv / zzzz-mediated transformation with plasmids each encoding a single guide RNAs to create deletions in the coding region. Sterilized soybean seeds are imbibed in water overnight, and explants are prepared as mature cotyledon halves with trimmed hypocotyls. The explants went through the typical transformation and regeneration steps of infection and co-cultivation, shoot induction and elongation and selection, rooting, and transplanting to soil to produce T1 seeds (see, for example, Li et al, Optimization of d zYz / zcVcvvzzzzz-Mediated Transformation in Soybean (2017) Frontiers in Plant Science v8 Article 246; Pareddy et al. Transgenic Res. 2020 Jun;29(3):267-281. doi: 10.1007 / sl 1248-020-00198-8).

[0168] TO plants are grown and genotyped by amplicon sequencing (AmpSeq).Example 2. Performance of soybean with an amorphic allele of a RPF1 gene

[0169] Seeds of the soybean plants homozygous for the amorphic alleles of the RPF1 gene are increased and planted in rows in the field along with checks and lines having unrelatedmutations in soybean genes other than RPF1 gene. In instances where the plants are homozygous for the amorphic alleles of the RPF1 gene and further comprise a hypomorphic or amorphic allele of at least one of an AlPlOa gene, AlPlOb gene, AML4 gene, CRN gene, JAG1 gene, JAG2 gene, TCP5-L gene, RIC1 gene, RIC2 gene, FTla gene, BS1 gene, BS2 gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene, certain checks will lack the amorphic alleles of the RPF1 gene and lack the hypomorphic or amorphic allele of the AlPlOa gene, AlPlOb gene, AML4 gene, CRN gene, JAG1 gene, JAG2 gene, TCP5-L gene RIC1 gene, RIC2 gene, FTla gene, BS1 gene, BS2 gene, TFLlb gene, and / or NF-YC4 gene. Data on seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, pod count per plant, seed count per plant, total harvested seed weight per plant, and total harvested seed weight per unit area from field trials are collected and compared to adjacently grown checks. Plants homozygous for the amorphic alleles of the RPF1 gene will have increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or seed yield in comparison to the checks lacking the amorphic alleles of the RPF1 gene.Example 3. Summary of Biological Sequences.Table 2. Summary of Biological SequencesSequence Type Description SEQ ID NODNA / PRT RPF1 Wild type genomic sequence (sense, 5'-3')(Glyma.08G108400.1) 1 DNARPF1 Coding sequence 2 DNARPF1 Encoded wild type polypeptide 3 PRTRPF1 Spacer (adjacent to PAM) 4 DNARPF1 Spacer (adjacent to PAM) 5 DNARPF1 Spacer (adjacent to PAM) 6 DNARPF1 Promoter + Gene (Glyma.08G108400.1) 7 DNARPF1 Promoter 8 DNARPF1 5' UTR (5'-3') (Glyma.08G108400.1) 9 DNARPF1 3' UTR (5'-3') (cDNA) 10 DNARPF1 3' UTR (5'-3') (Glyma.08G108400.1) 11 DNARPF1 3' UTR (5'-3') (Glyma.08G108400.1) 12 DNARPF1 3' UTR (5'-3') (Glyma.08G108400.1) 13 DNARPF1 Wild type genomic sequence (sense, 5'-3')(Glyma.08G108400.2) 14 DNARPF1 5' UTR (5'-3') (Glyma.08G108400.2) 15 DNARPF1 3' UTR (5'-3') (Glyma.08G108400.2) 16 DNARPF1 3' UTR (5'-3') (Glyma.08G108400.2) 17 DNA

[0170] All cited patents and patent publications referred to in this application are incorporated herein by reference in their entirety. All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure and illustrated by the examples. Although the materials and methods of this disclosure have been described in terms of embodiments and illustrative examples, it will be apparent to those of skill in the art that substitutions and variations can be applied to the materials and methods described herein without departing from the concept, spirit, and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as encompassed by the embodiments of the disclosures recited herein and the specification and appended claims.

Claims

What is claimed is:

1. A soybean plant cell comprising at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

2. The soybean plant cell of claim 1, wherein the plant cell is homozygous for the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.3 The soybean plant cell of claim 1, wherein the at least one mutation comprises a loss-of-function allele of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, wherein the loss-of-function allele reduces expression of the RPF1 gene relative to a wild-type or control soybean plant cell lacking the at least one mutation.4 The soybean plant cell of claim 1, wherein the at least one mutation is a non-natural mutation.5 The soybean plant cell of claim 1, wherein the at least one mutation comprises an amorphic allele of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.6 The soybean plant cell of claim 1, wherein the at least one mutation comprises a hypom orphic allele of the RPF1 gene.7 The soybean plant cell of claim 1, wherein the at least one mutation comprises a frameshift mutation or a nonsense mutation in the coding region of the RPF1 gene of SEQ ID O: 1 or an allelic variant thereof.8 The soybean plant cell of claim 1, wherein the at least one mutation comprises an internal deletion in the coding region of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the internal deletion preserves the reading frame of the encoded mutant RPF1 protein comprising the at least one mutation with respect to amino acid residues of the mutant RPF1 protein which have not been deleted.9 The soybean plant cell of claim 1, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide of the RPF1 gene of SEQ ID O: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises,consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, or 3939 nucleotides of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

10. The soybean plant cell of claim 1, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600 1700, 1800, or 1836 nucleotides corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.11 The soybean plant cell of claim 1, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.12 The soybean plant cell of claim 1, with the proviso that the soybean plant cell is not exclusively produced by an essentially biological method, optionally wherein the soybean plant cell is produced by introducing one or more gene editing molecules into a soybean plant cell and selecting the soybean plant cell comprising at least one non-natural mutation.13 The soybean plant cell of claim 1, wherein the soybean plant cell comprises elite soybean germplasm, optionally wherein the elite soybean germplasm comprises germplasm of a soybean cultivar or variety.

14. The soybean plant cell of claim 1, wherein the soybean plant cell further comprises at least one mutation in a distinct soybean gene.

15. The soybean plant cell of claim 14, wherein the soybean plant cell further comprises at least one mutation in the soybean AlPlOa gene, AlPlOb gene, AML4 gene, RIC1 gene, RIC2 gene, FT la gene, JAG1 gene, JAG2 gene, TCP5-L gene, BS1 gene, BS2 gene, CRN gene, TFLlb gene, CYP76C gene, and / or NF-YC4 gene.

16. The soybean plant cell of claim 1, wherein the soybean plant cell further comprises one or more transgenes, optionally wherein the transgenes encode proteins or RNAs conferring herbicide tolerance or pest tolerance.

17. The soybean plant cell of claim 16, wherein the soybean plant cell further comprises an A2704-12, A5547-127, BPS-CV127-9, DAS44406-6, DAS68416-4, DAS81419-2, DP305423, GTS 40- 3-2, HOS, A5547-127, MON87701, MON87705, MON87708, MON87769, MON89788, MON98788, MST-FG072-3, or SYHT0H210 transgenic event or modification thereof.

18. A soybean plant part comprising the soybean plant cell of any one of claims 1-17.

19. The soybean plant part of claim 18, wherein the part is a stem, root, leaf, flower, pod, or seed.

20. The soybean plant part of claim 19, wherein the part is a seed.

21. A soybean seed lot comprising the seed of claim 20, optionally wherein the seed lot comprises elite soybean germplasm, a soybean cultivar, a soybean variety, and / or is homozygous for the mutation.

22. A soybean plant comprising the soybean plant cell of any one of claims 1-17.

23. The soybean plant of claim 22, wherein the soybean plant comprises increased seeds per pod, percentage of 3 seeded pods, and / or percentage of 4 seeded pods in comparison to seeds per pod, percentage of 3 seeded pods, and / or percentage of 4 seeded pods for a wild-type or controlsoybean plant lacking the at least one mutation, optionally wherein the increased seeds per pod, percentage of 3 seeded pods, and / or percentage of 4 seeded pods is on the main stem and / or branches.

24. The soybean plant of claim 22, wherein yield is increased in comparison to yield for a wild-type or control soybean plant lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

25. A plant or plant part of an elite soybean plant, cultivar, or variety with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield comprising at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, wherein the increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is in comparison to a wild-type or control soybean plant lacking the at least one mutation, and optionally wherein the plant part is a seed.

26. The plant or plant part of claim 25, wherein the elite soybean plant, cultivar, or variety is homozygous for the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

27. The plant or plant part of claim 25, wherein the at least one mutation comprises a loss-of-function allele of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, wherein the loss-of-function allele reduces expression of the RPF1 gene relative to a wild-type or control plant or plant part lacking the at least one mutation.

28. The plant or plant part of claim 25, wherein the at least one mutation is a non-natural mutation.

29. A biological sample comprising a nucleic acid containing at least one mutation in the soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

30. The biological sample of claim 29, wherein the sample comprises seed meal or a tissue sample homogenate, optionally wherein the tissue sample comprises a sample of leaf, flower, pod, seed, stem, or root tissue.

31. The biological sample of claim 29, wherein the at least one mutation is a non-natural mutation.

32. The biological sample of claim 29, wherein the at least one mutation comprises a frameshift mutation or a nonsense mutation in the coding region of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

33. The biological sample of claim 29, wherein the at least one mutation comprises an internal deletion in the coding region of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the internal deletion preserves the reading frame of the encoded mutant RPF1 protein comprising the at least one mutation with respect to amino acid residues of the mutant RPF1 protein which have not been deleted.

34. The biological sample of claim 29, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600 1700, 1800, or 1836 nucleotides corresponding to nucleotides 120-1955 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.35 The biological sample of claim 29, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.36 The biological sample of claim 29, wherein the sample lacks a nucleic acid comprising the wild-type allele of the soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

37. A polynucleotide comprising at least one mutation relative to the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the polynucleotide is isolated.

38. The polynucleotide of claim 37, wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the at least one mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

39. A method of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising the soybean plant of claim 22; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot.

40. The method of claim 39, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of wild-type or control soybean plants lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

41. A method of producing a soybean crop comprising planting the seed lot of claim 21.

42. The method of claim 41, further comprising harvesting seed from soybean crop grown from the planted seed.

43. The method of claim 41, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean crop lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

44. A method for producing a soybean by-product comprising at least one processing step of cleaning, cracking, flaking, crushing, macerating, pressing, extracting, expelling, and / or extruding the seed lot of claim 21.

45. The method of claim 44, wherein the by-product is soybean protein and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; (ii) extracting the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) with an organic solvent to produce defatted soymeal; and (iii) extracting the defatted soymeal from step (ii) with an aqueous solvent to produce an aqueous fraction comprising soybean protein.

46. The method of claim 44, wherein the by-product is soybean oil and wherein the soybean seed lot is pressed to produce the oil.

47. The method of claim 44, wherein the by-product is soybean oil and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; and (ii) solvent extracting, expelling, and / or extruding step the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) to produce the oil.

48. A guide RNA molecule comprising a spacer RNA molecule which targets the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the spacer RNA molecule comprises the RNA encoded by SEQ ID NO: 4, 5, or 6.

49. A guide RNA molecule comprising a Casl2 direct repeat element which is operably linked to the spacer RNA of claim 48.

50. A gene editing system comprising:a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

51. An expression cassette comprising:a polynucleotide encoding CRISPR-Cas effector protein comprising a cleavage domain and the guide RNA molecule of claim 48.

52. A method for generating a soybean plant cell of claim 1, soybean plant part of claim 18, or soybean plant of claim 22 comprising introducing at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof of a soybean plant cell.

53. The method of claim 52, further comprising regenerating a soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof from the soybean plant cell, and optionally selecting a soybean plant with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield, optionally wherein increased yield comprises increased pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

54. The method of claim 52, wherein the at least one mutation is introduced by:(i directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, or a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6; and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and(ii isolating a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.55 The method of claim 54, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.56 The method of claim 54, wherein the soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation is identified by: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof in one or more candidate plant cells, plant parts, or plants; (ii analyzing a polypeptide encoded by a portion of SEQ ID NO: 1 in one or more candidateplant cells, plant parts, or plants; and / or (iii) analyzing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in comparison to a wild-type or control soybean plant lacking the at least one mutation is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

57. The method of claim 52, wherein the at least one mutation is introduced by crossing a soybean plant comprising the at least one mutation with a second soybean plant and harvesting Fl seed comprising the at least one mutation, thereby producing progeny soybean seed comprising the at least one mutation.

58. A method for generating a soybean plant cell of claim 1, soybean plant part of claim 18, or soybean plant of claim 22 comprising:(i screening a population of soybean plant cells, parts, or plants for the presence of the at least one mutation in the endogenous RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof; and (ii isolating a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.59 The method of claim 58, wherein the population of soybean plant cells, parts, or plants have been subjected to one or more mutagenesis treatments, optionally wherein the mutagenesis procedure comprises chemical mutagenesis.60 The method of claim 58, wherein the screening comprises: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from one or more candidate plant cells, plant parts, or plants, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from one or more candidate plant cells, plant parts, or plants, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation.

61. The method of claim 58, wherein the screening further comprising analyzing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in comparison to a wild-type or control soybean plant lacking the at least one mutation, wherein increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

62. A method for increasing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in a soybean plant, comprising:introducing at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof of a soybean plant cell.

63. The method of claim 62, further comprising regenerating a soybean plant comprising the at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof from the soybean plant cell, and optionally selecting a soybean plant with increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield.

64. The method of claim 62, wherein the at least one mutation is introduced by:(i directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, or a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6; and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and(ii isolating a soybean plant cell or soybean plant comprising the at least one mutation in the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.65 The method of claim 64, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.66 The method of claim 62, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod,percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean crop lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

67. A soybean plant or plant cell produced by the method of claim 62.

68. A method for determining whether a soybean plant cell, plant part, or plant comprises at least one mutation of the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, the method comprising:(i analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from the plant cell, plant part, or plant, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from the soybean plant cell, plant part, or plant, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation.69 The method of claim 68, wherein the method further comprises analyzing seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield in comparison to a wildtype or control soybean plant lacking the at least one mutation, wherein increased seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.70 A method for determining whether a soybean seed lot comprises soybean seed comprising at least one mutation of the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof, the method comprising:(i analyzing a polynucleotide comprising a portion of SEQ ID NO: 1 or an allelic variant thereof or analyzing an RNA encoded by a portion of SEQ ID NO: 1 or an allelic variant thereof from a sample of the soybean seed lot, wherein an insertion, deletion, and / or substitution of oneor more nucleotides in the polynucleotide or RNA is indicative of the presence of the at least one mutation; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 1, a portion thereof, or an allelic variant thereof from a sample of the soybean seed lot, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biologic or biochemical activity of the polypeptide is indicative of the presence of the at least one mutation.

71. Use of the soybean plant cell of claim 1, the soybean plant part of claim 18, the soybean seed lot of claim 21, or the soybean plant of claim 22 to produce soybean seed.

72. Use of the soybean plant cell of claim 1, the soybean plant part of claim 18, the soybean seed lot of claim 21, or the soybean plant of claim 22 to grow a soybean crop.

73. Use of the soybean plant cell of claim 1, the soybean plant part of claim 18, the soybean seed lot of claim 21, or the soybean plant of claim 22 to obtain a soybean by-product.

74. The use of claim 73, wherein the soybean by-product comprises soybean flour, meal, protein, oil, syrup, or starch.

75. Use of the guide RNA molecule of claim 48, the gene editing system of claim 50, or the expression cassette of claim 51 to introduce at least one mutation in the endogenous soybean RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof.

76. A soybean plant cell, soybean plant part, or soybean plant obtainable by the process comprising:(i directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule which targets the RPF1 gene of SEQ ID NO: 1 or an allelic variant thereof and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule to the genome of a target soybean plant cell; and(ii isolating a soybean plant cell, soybean plant part, or soybean plant comprising at least one mutation in the RPF1 gene of SEQ ID NO. 1 or an allelic variant thereof.

77. The soybean plant cell, soybean plant part, or soybean plant of claim 76, wherein the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 4, 5, or 6.

78. The soybean plant cell, soybean plant part, or soybean plant of claim 76, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.

79. The soybean plant cell, soybean plant part, or soybean plant of claim 76, wherein the seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield is increased in comparison to seeds per pod, percentage of 3 seeded pods, percentage of 4 seeded pods, and / or yield of a wild-type or control soybean crop lacking the at least one mutation, optionally wherein yield comprises pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area.

80. A soybean genome characterized by the fact that it comprises the nucleotide sequence of SEQ ID NO: 1 which contains a non-natural mutation.

81. The soybean genome of claim 80, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide of SEQ ID NO: 1, optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, or 3939 nucleotides of SEQ ID NO: 1.

82. The soybean genome of claim 80, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 120-1955 of SEQ ID NO: 1, optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, or 1836 nucleotides corresponding to nucleotides 120-1955 of SEQ ID NO: 1.

83. The soybean genome of claim 80, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide corresponding to nucleotides 415-439, 675-699, or 770-794 of SEQ ID NO: 1 or an allelic variant thereof, optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides corresponding to nucleotides 415-439, 675-699, or 770-794 of SEQ ID NO: 1.

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