Soybean FT1A gene mutation

Abstract

Soybean (Glycine max) is a globally important legume crop due to its ability to fix atmospheric nitrogen, and agricultural practices such as water and nutrient management can help farmers improve efficiency, increase crop productivity, conserve water, improve soil quality, improve soil nutrient efficiency, and produce sustainable soybean crops. This disclosure discloses novel plants, soybean plant parts including stems, roots, leaves, flowers, pods, seeds, etc., and nucleotide sequences of soybean varieties containing a mutated FT1a gene, as well as methods for producing the same by growing soybean plants or lots, and methods for using the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 479,312, filed January 10, 2023, the entire contents of which, including the specification, claims, figures, and sequence listing, are incorporated herein by reference in their entirety.

[0002] Sequence table XML This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML file, created on December 26, 2023, is named P14293WO00.xml and is 26,209 bytes in size.

[0003] Disclosed herein are novel plants, plant parts, and nucleotide sequences of soybean varieties containing a mutated FT1a gene, as well as methods for producing same by growing soybean plants or lots, and methods for using same. [Background technology]

[0004] Agriculture is a vital industry for the global economy, particularly in the United States. Soybeans (Glycine max) are a globally important legume crop due to their ability to fix atmospheric nitrogen. Soybeans are a major source of animal feed protein, and soybean oil is used in a wide variety of industries, including food and beverage, biodiesel, and other industries. Summary of the Invention [Problem to be solved by the invention]

[0005] Soybean sustainability is a priority for farmers worldwide. Agricultural practices such as water and nutrient management can help farmers improve efficiency, increase crop productivity, conserve water, improve soil quality, improve soil nutrient efficiency, and produce sustainable soybean crops. Benefits of bioengineering for soybean farmers include increased yields and resistance to extreme weather events. [Means for solving the problem]

[0006] Disclosed herein are soybean plant cells comprising a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. Also provided are soybean plant parts comprising said soybean plant cells, such as stems, roots, leaves, flowers, pods, and seeds. Also provided are soybean seed lots comprising said seeds. Also provided are soybean plants comprising said soybean plant cells.

[0007] Also provided is a biological sample comprising a nucleic acid comprising a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. Polynucleotides are provided comprising loss-of-function alleles of the soybean FT1a gene set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8. Also provided are polynucleotides encoding the polypeptides of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 10. In some embodiments, the foregoing polynucleotides are isolated.

[0008] Also disclosed is a method of producing a soybean seed lot, the method comprising: (i) growing a population of soybean plants comprising the soybean plants described above; and (ii) harvesting seeds at maturity from the soybean plant population of step (i). A method of producing a soybean crop is provided, the method comprising planting the seed lot described above.

[0009] Guide RNA molecules are provided that comprise a spacer RNA molecule that targets exon 1 of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. Also provided are guide RNA molecules that comprise a spacer RNA encoded by SEQ ID NO: 11.

[0010] Also disclosed are methods of producing the above-described soybean plant cells, soybean plant parts, and soybean plants. In some embodiments, the methods comprise introducing a loss-of-function allele into the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. In some embodiments, the methods comprise (i) screening a population of soybean plant cells, parts, or plants for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, and (ii) isolating soybean plant cells, soybean plant parts, or soybean plants comprising the loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0011] Methods are provided for determining whether a soybean plant cell, soybean plant part, or soybean plant contains a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments, the methods comprise analyzing a polynucleotide comprising a portion of SEQ ID NO: 3 or an allelic variant thereof, or analyzing RNA encoded by a portion of SEQ ID NO: 3 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 indicates the presence of a loss-of-function allele. In certain embodiments, the methods comprise analyzing a polypeptide encoded by SEQ ID NO: 3, a portion thereof, or an allelic variant thereof, from the soybean plant cell, soybean plant part, or soybean plant, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide or a change in the biological or biochemical activity of the polypeptide indicates the presence of a loss-of-function allele. [Brief explanation of the drawings]

[0012] [Figure 1A]1 shows the wild-type FT1a gene (SEQ ID NO: 3) with the exons of the mRNA splice variant (Glyma.18G298900.1) transcript encoding the FT1a protein of SEQ ID NO: 1. All exons are in bold, translated bases (codons) are in uppercase, introns are in lowercase, and both the 5' and 3' untranslated regions (UTRs) are in lowercase and underlined. [Figure 1B] Same as above. [Figure 2A] Shown is the wild-type FT1a gene (SEQ ID NO: 3) with the exons of the mRNA splice variant (Glyma.18G298900.3) transcript encoding the FT1a protein of SEQ ID NO: 2. All exons are in bold, translated bases (codons) are in uppercase, introns are in lowercase, and both the 5' and 3' untranslated regions (UTRs) are in lowercase and underlined. [Figure 2B] Same as above. [Figure 3A] Shown is a mutant ft1a gene (SEQ ID NO: 5) with exons of the transcript encoding the mutant ft1a protein of SEQ ID NO: 6. All exons are in bold, translated bases (codons) are in uppercase, introns are in lowercase, and both the 5' and 3' untranslated regions (UTRs) are in lowercase and underlined. [Figure 3B] Same as above. [Figure 4A] Shown is an ft1a gene variant (SEQ ID NO: 8) with the exons of the transcript encoding the mutant ft1a protein of SEQ ID NO: 9. All exons are in bold, translated bases (codons) are in uppercase, introns are in lowercase, and both the 5' and 3' untranslated regions (UTRs) are in lowercase and underlined. [Figure 4B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, the phrase "allelic variant" refers to polynucleotide or polypeptide sequence variants present within a particular gene at a particular locus in different strains, varieties or isolates of a given organism.

[0014] As used herein, the phrase "amorphic allele" refers to an allele of a gene that has no gene activity compared to the wild-type allele of the gene. Amorphic alleles are also known as null alleles.

[0015] The term "and / or," as used herein, should be considered a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or," when used in phrases such as "A and / or B," is intended herein to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in phrases 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).

[0016] As used herein, the phrase "biological sample" refers to either intact or non-intact soybean plant tissue (e.g., crushed soybean seeds or soybean plant tissue, shredded soybean plant tissue, frozen dried tissue). It can also be an extract containing intact or non-intact seeds or soybean plant tissue. Biological samples can include flour, powder, syrup, oil, starch, and cereals manufactured to contain all or part of a soybean plant by-product. In certain embodiments, the biological sample is "non-renewable" (i.e., cannot be regenerated into a soybean plant or soybean plant part).

[0017] As used herein, the terms "correspond," "corresponding," and the like, when used in the context of nucleotide positions, mutations, and / or substitutions in any given polynucleotide (e.g., an allelic variant of SEQ ID NO: 3) relative to a reference polynucleotide sequence (e.g., SEQ ID NO: 3), all refer to nucleotide positions in the given sequence that have identity to nucleotides in the reference nucleotide sequence when the given polynucleotide is aligned with the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL O 1.2.4 with default parameters).

[0018] As used herein, the terms "Cpf1" and "Cas12a" are used interchangeably to refer to the same RNA-dependent DNA endonuclease (RdDe).

[0019] As used herein, the phrase "endogenous gene" refers to the native form of a genetic unit in its natural location in the genome of an organism.

[0020] As used herein, the term "expression" refers to the production of a functional end-product (e.g., mRNA, guide RNA, or protein) in either a precursor or mature form.

[0021] As used herein, the phrase "hypomorphic allele" refers to an allele of a gene that has less gene activity than the wild-type allele, but more gene activity than the amorphic allele.

[0022] As used herein, the terms "include," "includes," and "including" are to be interpreted as including at least the features they refer to, while not excluding additional, unspecified features.

[0023] As used herein, the term "homozygous allele" refers to an allele of a gene that has wild-type gene activity.

[0024] As used herein, the term "isolated" means removed from its natural environment.

[0025] As used herein, the term "introduced" means providing a nucleic acid (e.g., an expression construct) or protein to 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 a 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," where the nucleic acid fragment comprises the genome of the cell (e.g., a nuclear chromosome, a plasmid, a plastid, a chloroplast, or a mitochondrial DNA), is converted into an autonomous replicon, or is transiently expressed (e.g., a transfected mRNA).

[0026] As used herein, a "loss-of-function allele" can include an amorphic allele or a hypomorphic allele of a gene.

[0027] As used herein, the term "plant" includes reference to the entire immature or mature soybean plant, including the plant from which the seeds or grains or anthers have been removed. Any seed or embryo that will produce a plant is also considered to be a soybean plant.

[0028] As used herein, the term "mutated FT1a gene" or "ft1a gene" refers to an endogenous soybean FT1a gene comprising a loss-of-function allele. The term "ft1a protein" refers to a protein encoded by an endogenous soybean FT1a gene comprising a loss-of-function allele.

[0029] As used herein, the term "plant" includes the entire soybean plant and any progeny, cell, tissue, part, or parts of the plant. Thus, as used herein, the term "plant" includes reference to the entire immature or mature soybean plant, including plants from which the seeds or grains or anthers have been removed.

[0030] The term "plant part" includes, for example, but is not limited to, any part of a plant: seeds (including mature and immature seeds); grains; stems; plant cuttings; plant cells; plant cell cultures; or plant organs (e.g., pollen, embryos, pods; flowers, fruits, buds, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells organized into a structural or functional unit. A plant cell or tissue culture can regenerate plants having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and can regenerate plants 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 stems. In contrast, some plant cells cannot regenerate to produce plants and are referred to herein as "non-regenerable" plant cells.

[0031] To the extent that any of the preceding definitions conflicts with a definition provided in any patent or non-patent document incorporated by reference herein, any patent or non-patent document cited herein, or any patent or non-patent document found elsewhere, it is understood that the preceding definition controls herein.

[0032] The present disclosure provides soybean plant cells, plant parts including seeds, plants, seed lots, and biological samples that contain a mutated FT1a gene (i.e., containing a loss-of-function allele of the endogenous FT1a gene). These soybean plants and parts may be used for human consumption, livestock feed, industrial feedstock, or as breeding material for the development of other soybean varieties.

[0033] The targeted endogenous FT1a gene includes the genomic DNA of SEQ ID NO: 3 and its allelic variants located on soybean chromosome 18. The endogenous soybean FT1a gene is located at nucleotides 57,922,912 to 57,928,648 on chromosome 18 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.18G298900) available 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). Alternative splicing of the FT1a gene transcript results in mRNA splice variant 1 (Glyma.18G298900.1), which encodes the FT1a protein of SEQ ID NO: 1, as shown in Figures 1A and 1B, and mRNA splice variant 2 (Glyma.18G298900.3), which encodes the FT1a protein of SEQ ID NO: 2, as shown in Figures 2A and 2B. Allelic variants of the endogenous soybean FT1a gene include variants encoding FT1a 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: 1 or SEQ ID NO: 2. Allelic variants of the endogenous soybean FT1a gene also include variants consisting of 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: 3. In certain embodiments, allelic variants of the endogenous soybean FT1a gene are homozygous alleles of the endogenous soybean FT1a gene. The wild-type soybean FT1a gene encodes a member of the phosphatidylethanolamine-binding protein (PEBP) family, which is described in Serre et al., Structure (1998), 6:1255-1265.In the soybean FT1a wild-type protein of SEQ ID NO: 1, amino acid residues that are conserved with other PEBP family members include residues 65-76, P80, H87, G116, and R119. In the soybean FT1a wild-type protein of SEQ ID NO: 2, amino acid residues that are conserved with other PEBP family members include residues 65-76, P80, and H87.

[0034] Soybean plant cells, soybean plant parts, and soybean plants are provided that contain a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. Examples of loss-of-function alleles can include deletions, insertions, and / or substitutions of one or more nucleotides in the endogenous FT1a gene. The insertions, deletions, and / or substitutions can occur anywhere in the FT1a gene, including, for example, the promoter region, exons, introns, and / or untranslated regions (5'UTR or 3'UTR). In certain embodiments, the loss-of-function alleles comprise deletions, insertions, and / or substitutions within the coding region of the FT1a gene. In certain embodiments, the loss-of-function alleles of the FT1a gene can include deletions of the entire coding region or any portion of the coding region required for biological activity. In certain embodiments, the loss-of-function allele comprises one or more deletions, insertions, and / or substitutions of nucleotides corresponding to nucleotides in exon 1 of mRNA splice variants 1 and 2 (i.e., nucleotides 229 to 429 of SEQ ID NO: 3, or the equivalent position in an allelic variant of SEQ ID NO: 3), exon 2 of mRNA splice variants 1 and 2 (i.e., nucleotides 596 to 657 of SEQ ID NO: 3, or the equivalent position in an allelic variant of SEQ ID NO: 3), exon 3 of mRNA splice variants 1 and 2 (i.e., nucleotides 3643 to 3683 of SEQ ID NO: 3, or the equivalent position in an allelic variant of SEQ ID NO: 3), or in exon 4 of mRNA splice variant 1 of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof (i.e., nucleotides 4999 to 5225 or nucleotides 5104 to 5225 of SEQ ID NO: 3, or the equivalent position in an allelic variant of SEQ ID NO: 3). In certain embodiments, the loss-of-function allele comprises a deletion or substitution of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides of the endogenous soybean FT1a gene of SEQ ID NO: 3 located at nucleotides 331 to 356 of SEQ ID NO: 3 or an allelic variant thereof.

[0035] In certain embodiments, the loss-of-function allele comprises a deletion, insertion, and / or substitution resulting in a frameshift and / or nonsense mutation in the coding region of the FT1a gene. In certain embodiments, the loss-of-function allele of the FT1a gene can comprise a deletion of any number of nucleotides not divisible by three in an exon of the FT1a gene. In certain embodiments, the loss-of-function allele of the FT1a gene comprises a deletion of nucleotides 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 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, The FT1a gene fragment may comprise a deletion of 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, or 200 nucleotides, resulting in a frameshift mutation. In certain embodiments, the frameshift mutation occurs at nucleotides corresponding to one or more of nucleotides 229 to 429 of SEQ ID NO: 3, or an allelic variant thereof. In certain embodiments, the frameshift mutation occurs at nucleotides corresponding to one or more of nucleotides 340 to 343 of SEQ ID NO: 3, or an allelic variant thereof. In certain embodiments, a mutated FT1a gene comprising a loss-of-function allele having a frameshift mutation may comprise the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or an allelic variant thereof.In certain embodiments, such an allelic variant of SEQ ID NO:4 or SEQ ID NO:5 may comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity over the entire length of SEQ ID NO:4 or SEQ ID NO:5. In certain embodiments, a mutated FT1a gene, including a loss-of-function allele having a frameshift mutation, may encode a polypeptide comprising the amino acid sequence of SEQ ID NO:6 or an allelic variant thereof. In certain embodiments, such an allelic variant of SEQ ID NO:6 may comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity over the entire length of SEQ ID NO:6.

[0036] In certain embodiments, a loss-of-function allele comprises an internal deletion that removes at least one, two, or three codons while retaining the reading frame of the encoded FT1a protein, resulting in a mutant ft1a protein lacking at least one, two, or three amino acid residues. In certain embodiments, a loss-of-function allele of the FT1a gene can comprise a deletion of any number of nucleotides divisible by three in an exon of the FT1a gene. In certain embodiments, a loss-of-function allele of the FT1a gene is a deletion of nucleotides 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, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 1 , 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, or 201 nucleotide deletions and maintain the reading frame. In certain embodiments, the loss-of-function allele comprises an internal deletion including at least nucleotides corresponding to at least nucleotides 343 to 351 of SEQ ID NO:3, or an allelic variant thereof that preserves the reading frame. In certain embodiments, the loss-of-function allele comprises an internal deletion comprising at least the nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO:1 and SEQ ID NO:2, or an allelic variant thereof that maintains the reading frame.In these embodiments, a loss-of-function allele may comprise an internal deletion of nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO:1 and SEQ ID NO:2, and further comprise a deletion of nucleotides encoding P2, R3, S4, T5, D6, P7, L8, V9, 110, G11, G12, V13, 114, G15, D16, V17, L18, E19, P20, F21, F22, F23, F24, F25, F26, F27, F28, F29, F30, F31, F32, F33, F34, F35, F36, F37, F38, F39, F40, F41, F42, F43, F44, F45, F46, F47, F48, F49, F50, F51, F52, F53, F54, F55, F56, F57, F58, F59, F60, F61, F62, F63, F64, F65, F66, F67, F68, F69, F70, F71, F72, F73, F74, F75, F76, F77, F78, F79, F80, F81, F82, F83, F84, F85, F86, F87, F88, F89, F90, F91, F92, F93, F94, F95, F96, F97, F98, F99, F100, F101, F102, F103, F104, F105, F106, F107, F108, F110, F111, F112, F113, F1 The FT1a gene may comprise a deletion of nucleotides encoding T22, S23, S24, V25, S26, M27, G28, 129, V30, Y31, N32, N33, C34, P35, Q36, V37, 138, E42, L43, K44, P45, S46, K47, 148, L49, n50, R51, P52, R53, 154, E55, 156, G57, G58, D59, D60, L61, R62, T63, F64, Y65, T66, and / or L67. In certain embodiments, a mutated FT1a gene comprising a loss-of-function allele with an internal deletion may comprise the nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:8, or an allelic variant thereof. In certain embodiments, such allelic variants of SEQ ID NO:7 or SEQ ID NO:8 may comprise a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity over the entire length of SEQ ID NO:7 or SEQ ID NO:8. In certain embodiments, a mutated FT1a gene comprising a loss-of-function allele having an internal deletion may encode a polypeptide comprising the amino acid sequence of SEQ ID NO:9 and / or SEQ ID NO:10, or an allelic variant thereof. In certain embodiments, such allelic variants of SEQ ID NO:9 and / or SEQ ID NO:10 may comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity over the entire length of SEQ ID NO:9 and / or SEQ ID NO:10.

[0037] In certain embodiments, the yield of soybean plants containing a loss-of-function allele of the endogenous soybean FT1a gene is increased compared to the yield of wild-type control soybean plants lacking the loss-of-function allele. Increased yield of soybean plants can be measured in many ways, including the number of pods per plant, the number of seeds per plant, the total harvested seed weight per plant, or the total harvested seed weight per unit area (e.g., seed weight per acre or seed weight per hectare). In certain embodiments, increased yield can result from improved response to stress, including abiotic stress (e.g., drought, heat, cold, and / or salt stress).

[0038] In certain embodiments, the number of pods per soybean plant comprising a loss-of-function allele in the FT1a gene is increased compared to the number of pods per plant for a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the number of pods 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% compared to the number of pods per plant for a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the number of seeds per plant comprising a loss-of-function allele in the FT1a gene is increased compared to the number of seeds per plant for a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the number of seeds per plant comprising a loss-of-function allele in the FT1a 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% compared to the number of seeds per plant from a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the total seed weight harvested per plant comprising a loss-of-function allele in the FT1a gene is increased compared to the total seed weight harvested per plant for a wild-type control soybean plant lacking the loss-of-function allele. 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% compared to the total harvested seed weight per plant from a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the total harvested seed weight per unit area of ​​soybean plants comprising a loss-of-function allele in the FT1a gene is increased compared to the total harvested seed weight per unit area of ​​a wild-type control soybean plant lacking the loss-of-function allele.In certain embodiments, the total harvested seed weight per unit area of ​​soybean plants comprising a loss-of-function allele in the FT1a 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% compared to the total harvested seed weight per unit area from a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the average weight of 1000 seeds obtained from the soybean plants is equivalent to or essentially the same as the average weight of 1000 seeds obtained from a wild-type control soybean plant lacking the loss-of-function allele.

[0039] In certain embodiments, soybean plants containing a loss-of-function allele in the FT1a gene, when grown under stress, exhibit an increased number of pods per plant, increased number of seeds per plant, increased total number of seeds per plant, increased total harvested seed weight per plant, and / or increased total harvested seed weight per unit area compared to wild-type control soybean plants lacking the loss-of-function allele grown under stress. Non-limiting examples of stress include drought, cold, heat, salt, shade, nutrient deficiency, high planting density, and the presence of pests or pathogens. In certain embodiments, stress includes abiotic stress. In certain embodiments, abiotic stress includes drought, cold, heat, salt, and stress. In these embodiments, when plants containing a loss-of-function allele of the FT1a gene are grown under drought stress, the number of pods per plant, the number of seeds per plant, the total number of seeds per plant, the total number of harvested seed weights per plant, and / or the total number of harvested seed weights per unit area can be increased compared to the number of pods per plant, the number of seeds per plant, the total number of harvested seed weights per plant, and / or the total number of harvested seed weights per unit area of ​​wild-type control soybean plants lacking the loss-of-function allele grown under drought stress.

[0040] In certain embodiments, the number of pods per plant, the number of seeds per plant, the total harvested seed weight per plant, and / or the total harvested seed weight per unit area are increased compared to a control (e.g., a check) in which soybean plants containing a loss-of-function allele in the FT1a gene are grown for the full growing season. In certain embodiments, the seed plants containing a loss-of-function allele in the FT1a gene are planted on or after (e.g., within one week of) the earliest first planting date provided by the USDA Risk Management Agency for the maturity zone in which they are planted. Non-limiting examples of Risk Management Agency (RMA) crop insurance replant dates can range from about April 1 in the southeastern United States to about May 5 in the upper Midwest United States (see internet site "soybeanresearchinfo.com / wp-content / uploads / 2022 / 01 / 2700-003-23_Planting-Date-V1.pdf"). In certain embodiments, at least 50%, 70%, 80%, or 90% of the plants in a soybean crop containing a loss-of-function allele in the FT1a gene have 95% of their pods at full maturity color at harvest. The full maturity color is cultivar dependent and can be gray, tan, or brown. In certain embodiments, the soybean crop containing a loss-of-function allele in the FT1a gene is a full-season cultivar of the soybean maturity group zone in which it is grown, and the seed is harvested during the full growing season of the full-season cultivar or after the full growing season of the full-season cultivar. Soybeans containing a loss-of-function allele in the FT1a gene are classified into one of 13 maturity group designations: 000, 00, 0, or I through X, where maturity groups can also be represented by an Arabic numeral and one decimal point (e.g., "5.8"). Soybeans are typically grown in maturity groups 00 through VIII in the United States (see the internet site "soybeanresearchinfo.com / research-highlight / delineating-optimal-soybean-maturity-groups-across-the-united-states / ").

[0041] Soybean seed lots are provided that include soybean seeds that include a loss-of-function allele in the FT1a gene. In certain embodiments, soybean plants that include a mutated FT1a gene can be used to obtain seed lots in which the average weight of 1,000 seeds in the seed lot is equal to or essentially the same as the average weight of 1,000 FT1a seeds in a control seed lot obtained from a wild-type control plant that lacks the loss-of-function allele in the FT1a gene (e.g., a wild-type soybean plant that is homozygous for the wild-type FT1a 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 control soybean plant that lacks the loss-of-function allele in the FT1a gene. In certain embodiments, the seed lots are packaged in lots containing about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms) of seeds.

[0042] Also provided are polynucleotides comprising any of the aforementioned mutated FT1a genes or fragments thereof. In certain embodiments, polynucleotides comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, or an allelic variant thereof, are provided. In certain embodiments, an allelic variant of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 comprises a sequence having at least 95%, 96, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity over the entire length of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, provided that the sequence is not identical over the entire length to SEQ ID NO:3. In certain embodiments, a polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10, or an allelic variant thereof. In certain embodiments, the encoded allelic variant comprises a polypeptide having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity over the entire length of SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10, provided that the sequence is not identical to SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, the polynucleotide is an isolated polynucleotide.

[0043] Also provided are biological samples and soybean by-products comprising any of the foregoing polynucleotides. In certain embodiments, the by-products are processed products produced from the soybean plants or seeds thereof of the present disclosure, including (a) soybean seed flour (defatted or non-defatted); (b) extracted soy proteins, oils, sugars, syrups, and starches; (c) fermented soy products; (d) soy-based animal feeds or human foods (e.g., soybean seed flour (defatted or non-defatted) and other ingredients (e.g., other grains, other seed meals, other protein meals, other oils, other starches, other sugars, binders, preservatives, humectants, vitamins, and / or minerals)); (e) pharmaceuticals; (f) raw or processed biomass (e.g., cellulosic and / or lignocellulosic materials; silage); and (g) various industrial products.

[0044] Also provided are methods for producing soybean by-products using the soybean plants, seeds, and seed lots of the present disclosure. Such methods typically include at least one processing step of cleaning, disintegrating, flaking, crushing, soaking, pressing, extracting, draining, and / or extruding the seeds.

[0045] The present disclosure also relates to a method for producing a soybean plant having a loss-of-function allele of an endogenous soybean FT1a gene by crossing a first parent soybean plant with a second parent soybean plant, wherein the first parent soybean plant or the second parent soybean plant contains the loss-of-function allele. Furthermore, both the first and second parent soybean plants can contain the loss-of-function allele. Such methods using soybean plants containing the loss-of-function allele, including selfing, backcrossing, hybrid production, and crossing into populations, are all part of the present disclosure. All plants produced using a soybean plant containing the loss-of-function allele as a parent are within the scope of the present disclosure, including plants derived from the soybean plant containing the loss-of-function allele. Also provided are F1 progeny soybean plants, F1 seeds, and various parts of F1 soybean plants produced from crosses of soybean plants containing the loss-of-function allele with other soybean plants. Below are described breeding methods that can be used with the soybean plants of the present disclosure in the development of further soybean plants. One such embodiment is a method for developing progeny soybean plants in a soybean plant breeding program, comprising the steps of: obtaining a soybean plant or part thereof containing a loss-of-function allele of an endogenous soybean FT1a gene and using the plant or plant part as a source of breeding material; and selecting progeny plants having the loss-of-function allele. Breeding processes that can be used in soybean plant breeding programs include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. These processes can be combined with techniques such as restriction fragment polymorphism-enhanced selection, genetic marker-enhanced selection (e.g., SNP and SSR markers), and double haploid generation.

[0046] Field crops are bred using techniques that utilize plant pollination methods. The soybean plants of the present disclosure can be self-pollinating, sib-pollinating, or cross-pollinating to create pedigree soybean plants. A plant is self-pollinating when pollen from one flower is transferred to the same flower or another flower on the same plant. A plant is sib-pollinating when individuals from the same line or variety are used for pollination. A plant is cross-pollinating when pollen is shed from the flower of a different plant from a different line or variety. As used herein, the terms "cross-pollination" and "outcrossing" do not include self-pollination or sib-pollination. Soybean (Glycine max) is a self-pollinating plant in nature, and although cross-breeding is possible, it rarely occurs in nature. Although some researchers have reported that insects carry pollen from one soybean plant to another, it is generally estimated that less than 1% of soybean seeds formed in open fields have the potential to hybridize, i.e., produce F1 hybrid soybean plants.

[0047] Other suitable breeding, selection, and cultivation methods may also be used. The choice of a particular breeding or selection method will vary depending on environmental factors, population size, etc.

[0048] A method for producing a soybean seed lot, the method comprising: (i) growing to maturity a population of soybean plants comprising a mutated FT1a gene; and (ii) harvesting seeds at maturity from the population of soybean plants of step (i), thereby producing a soybean seed lot, wherein the soybean plants are homozygous for the mutated FT1a gene. In certain embodiments, the seed lot is packaged in lots containing about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms).

[0049] Also provided herein are methods for treating the disclosed soybean seeds and seed lots, as well as the resulting treated seeds and seed lots. Seeds can be treated with such fertilizers, biological agents, nematicides, insecticides, and fungicides by methods including in-furrow application or by coating (e.g., using a drum coater, rotary coater, tumble drum, fluidized bed, and / or pouring bed equipment). Methods and compositions including various binders, fillers, film coating agents, and active ingredients such as fertilizers, surfactants, plant growth regulators, crop desiccants, fungicides, bactericides, bacteriostats, insecticides, and insect repellents for coating seeds that can be adapted for use with the seeds provided herein are disclosed in U.S. Patent No. 10,745,578, the entire contents of which are incorporated herein by reference.

[0050] The present disclosure also provides methods for generating soybean plants containing a mutated FT1a gene. In certain embodiments, the methods can include making a deletion, insertion, and / or substitution that results in a mutated FT1a gene. Gene editing molecules used in the methods provided herein include molecules that can introduce double-stranded breaks ("DSBs") or single-stranded breaks ("SSBs") at specific sites or sequences in double-stranded DNA, such as a target gene and an associated guide RNA located in or within genomic DNA. In certain embodiments, a loss-of-function allele is generated by introducing a DSB at a target site in the FT1a gene (e.g., SEQ ID NO: 3 or an allelic variant thereof) to induce non-homologous end joining (NHEJ) at the break site, followed by restoring the desired loss-of-function allele. In certain embodiments, the loss-of-function allele results from introducing a DSB at a target site in the FT1a gene (e.g., SEQ ID NO: 3 or an allelic variant thereof), followed by introducing a desired donor or other DNA template polynucleotide into the DSB by homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ, and then recovering the desired loss-of-function allele.Examples of such gene editing molecules include (a) RNA-guided nucleases, RNA-guided DNA endonucleases or RNA-directed DNA endonucleases (RdDes), nucleases including class 1 CRISPR-type nuclease systems, type II Cas nucleases, Cas9, nCas9 nickase, type V Cas nucleases, Cas12a nucleases, nCas12a nickases, Cas12d (Ca sY), Cas12e (CasX), Cas12b (C2c1), Cas12c (C2c3), Cas12i, Cas12j, Cas14, engineered nucleases, codon-optimized nucleases, zinc finger nucleases (ZFNs) or nickases, transcription activator-like effector nucleases (TAL-effector nucleases or TALENs) or nickases (TALE-nickases), Argonaute and a meganuclease or modified meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effecting site-specific modification of a target nucleotide sequence (including the introduction of a DSB or SSB); (c) a guide RNA (gRNA) for use with an RNA-guided nuclease, or DNA encoding a gRNA for use with an RRNA-guided nuclease; (d) optionally, a donor DNA template polynucleotide suitable for insertion into 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 a combination thereof) suitable for insertion into a break in genomic DNA (e.g., by non-homologous end joining (NHEJ)).

[0051] In certain embodiments, the mutated FT1a gene, and plant cells, parts including seeds, and plants comprising the mutated FT1a gene are generated by CRISPR technology. CRISPR technology for editing eukaryotic genes is disclosed in U.S. Patent Application Publication Nos. 2016 / 0138008A1 and 2015 / 0344912A1, as well as U.S. Patent Nos. 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. The Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1. Plant RNA promoters for expressing CRISPR guide RNAs and plant codon-optimized CRISPR Cas9 endonucleases are disclosed in International Patent Application No. PCT / US2015 / 018104 (published as WO 2015 / 131101, which claims priority to U.S. Provisional Patent Application No. 61 / 945,700). The method of using CRISPR technology for genome editing in plants is disclosed in US Patent Application Publication No. 2015 / 0082478A1 and US Patent Application Publication No. 2015 / 0059010A1 and International Patent Application No. PCT / US2015 / 038767A1 (published as International Publication No. WO2016 / 007347 and claims priority to US Provisional Patent Application No. 62 / 023,246).All patent publications mentioned in this paragraph are incorporated herein by reference in their entirety.In certain embodiments, RNA-guided endonuclease is used, which leaves blunt ends after cutting target site.Blunt-end cutting RNA-guided endonucleases include Cas9, Cas12c, Cas12i, and Cas12h (Yan et al., 2019). In certain embodiments, RNA-guided endonucleases that leave sticky single-stranded DNA overhang ends after cleaving the target site are used. Sticky-end cutting RNA-guided endonucleases include Cas12a, Cas12b, and Cas12e.

[0052] Guide RNA molecules are provided that include spacer RNA molecules that target the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments, the spacer RNA molecule targets a portion of nucleotides corresponding to exon 1 (i.e., nucleotides 229 to 429 of SEQ ID NO: 3, or an equivalent position in an allelic variant of SEQ ID NO: 3), exon 2 (i.e., nucleotides 596 to 657 of SEQ ID NO: 3, or an equivalent position in an allelic variant of SEQ ID NO: 3), exon 3 (i.e., nucleotides 3643 to 3683 of SEQ ID NO: 3, or an equivalent position in an allelic variant of SEQ ID NO: 3), or exon 4 (i.e., nucleotides 4999 to 5225 or nucleotides 5104 to 5225 of SEQ ID NO: 3, or an equivalent position in an allelic variant of SEQ ID NO: 3) of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments, the spacer RNA molecule comprises an RNA encoded by SEQ ID NO: 11. A guide RNA comprising a spacer RNA molecule encoded by SEQ ID NO: 11, in combination with a Cas12a nuclease, comprises: (i) a deletion of the endogenous FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; (ii) a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides of the endogenous soybean FT1a gene of SEQ ID NO: 3 located at nucleotides 331 to 356 of SEQ ID NO: 3. It can be used to generate a mutated FT1a gene that contains a deletion or a deletion of nucleotides located at the equivalent positions of an allelic variant of SEQ ID NO:3; (iii) the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, or an allelic variant thereof; or the sequence of SEQ ID NO:4 or SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8; or (iv) encodes a polypeptide of SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10, or an allelic variant thereof.

[0053] CRISPR-based 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 a CRISPR endonuclease, and CRISPR guide RNAs comprising a single guide RNA or guide RNA in combination with a tracrRNA or scoutRNA, or polynucleotides encoding same, are useful for performing genome editing without remnants of CRISPR elements or selectable genetic markers occurring in progeny. In certain embodiments, CRISPR elements are provided directly to eukaryotic cells (e.g., soybean plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of cell-free synthetic processes (e.g., in vitro translation), or as isolated or semi-purified products of cell-based synthetic processes (e.g., in bacterial or other cell lysates). In certain embodiments, soybean plants or soybean plant cells used in the systems, methods, and compositions provided herein may contain a transgene expressing a CRISPR endonuclease (e.g., Cas9, Cpf1-type, or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNA or crRNA and tracrRNA) are used to form an RNA-guided endonuclease / guide RNA complex that can specifically bind to a sequence in the gDNA target site adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically determines the location of the appropriate PAM site and the design of the crRNA or sgRNA. G-rich PAM sites, e.g., 5'-NGG, are typically targeted in the design of crRNAs and sgRNAs used with the Cas9 protein.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 typical targets for designing crRNAs or sgRNAs to be used with Cas12a proteins. In some instances, Cas12a can also recognize a 5'-CTA PAM motif. Other examples of potential Cas12a 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 (where N is defined as any nucleotide). The Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1, which is incorporated herein by reference for its disclosure of DNA encoding the Cpfl endonuclease and guide RNAs and PAM sites.

[0054] In certain embodiments, mutated FT1a genes, as well as plant cells, plant parts including seeds, and plants containing mutated FT1a genes, are produced by using zinc finger nucleases or zinc finger nickases. Zinc finger nucleases are site-specific endonucleases that have two protein domains: a DNA-binding domain consisting of multiple zinc finger repeats, each recognizing 9 to 18 base pairs, and a DNA-cleavage domain containing a nuclease domain (typically Fokl). The cleavage domains dimerize to cleave DNA. Therefore, a pair of ZFNs is required to target a non-palindromic target polynucleotide. In certain embodiments, previously described methods for designing zinc finger nucleases and zinc finger nickases (Urnov 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 described herein. The zinc finger binding domain of a zinc finger nuclease or nickase provides specificity and can be designed to specifically recognize any desired target DNA sequence. Zinc finger DNA binding domains are derived from the DNA binding domains of a broad class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of a ZFP typically contains a tandem array of at least three zinc "fingers," each of which recognizes a specific triplet of DNA. Several strategies can be used to engineer the binding specificity of zinc finger binding domains. One approach, called "modular assembly," relies on the functional autonomy of individual zinc fingers with DNA. This approach involves identifying the zinc fingers of each component triplet in the sequence and linking them into a multi-finger peptide to target a given sequence.Several alternative strategies for designing zinc finger DNA-binding domains have also been developed. These methods are designed to adapt the ability of a zinc finger to contact adjacent fingers and nucleotide bases outside of its target triplet. Typically, the engineered zinc finger DNA-binding domain has novel binding specificities compared to naturally occurring zinc finger proteins. 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 the nucleotide sequence of each triplet or quadruplet is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, both of which are incorporated by reference in their entireties. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) can be adapted for use with the methods described herein. Additionally, enhanced binding specificity for zinc finger binding domains is described in U.S. Pat. No. 6,794,136, which is incorporated herein by reference in its entirety. Additionally, individual zinc finger domains can be linked together using any suitable linker sequence. Examples of linker sequences are known; see, for example, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, which are incorporated herein by reference in their entirety. The nucleic acid cleavage domain is nonspecific and typically a restriction endonuclease such as Fokl. This endonuclease must dimerize to cleave DNA. Therefore, cleavage by Fokl as part of a ZFN requires two adjacent, independent binding events, which must occur in both the correct orientation and with appropriate spacing to allow dimer formation. The requirement of two DNA binding events allows for more specific targeting of long, potentially unique recognition sites.Fokl variants with enhanced activity have been described and can be used in the methods described herein. See, e.g., Guo et al. (2010) J. Mol. Biol., 400:96-107.

[0055] In certain embodiments, mutated FT1a genes, as well as plant cells, parts including seeds, and plants containing mutated FT1a genes, are generated by using TAL effector nucleases or TALENs. Transcription activator-like effectors (TALEs) are proteins secreted by certain Xanthomonas bacteria that regulate gene expression in host plants and promote bacterial colonization and survival. TALEs act as transcription factors and regulate the expression of plant resistance genes. Recent studies of TALEs have revealed a code linking the repeat region of TALEs to their target DNA binding sites. TALEs contain highly conserved repeat regions, mostly composed of tandem repeats of 33 or 34 amino acid segments. The repeat monomers differ from each other primarily at amino acid positions 12 and 13. A strong correlation was found between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotides in the TALE binding site. The simple relationship between the amino acid sequence of a TALE binding domain and DNA recognition allows for the design of DNA binding domains with any desired specificity. TALEs can be linked to non-specific DNA cleavage domains to prepare genome editing proteins called TAL-effector nucleases or TALENs. As with ZFNs, restriction endonucleases such as Fokl can be conveniently used. Methods for using TALENs in plants have already been described and can be adapted to 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 the methods described herein (Wu et al.; Biochem Biophys Res Commun. (2014); 446(1):261-6; Luo et al; Scientific Reports 6, Article number: 20657 (2016)).

[0056] A variety of therapeutic methods can be used to deliver gene editing molecules and / or other molecules to plant cells. In certain embodiments, one or more processes are employed to deliver gene editing or other molecules (e.g., including polynucleotides, polypeptides, or combinations thereof) to plant cells through barriers, such as cell walls, cell membranes, nuclear membranes, and / or other lipid bilayers. In certain embodiments, polynucleotide-, polypeptide-, or RNP (ribonucleoprotein)-containing compositions containing molecules are delivered directly, for example, by direct contact of the composition with plant cells. The compositions can be provided in the form of a liquid, solution, suspension, emulsion, inverse emulsion, colloid, dispersion, gel, liposome, micelle, injectable material, aerosol, solid, powder, microparticles, nanoparticles, or combinations thereof, and can be applied directly to plants, plant parts, plant cells, or plant explants (e.g., by microinjection, by scraping, puncturing, or otherwise disrupting the cell wall or cell membrane, by spraying, dipping, soaking, or other direct contact). For example, plant cells or plant protoplasts are immersed in a liquid genome editing molecule-containing composition. In certain embodiments, the composition is delivered using negative or positive pressure, for example, by vacuum infiltration or by applying hydrodynamic or fluid pressure. In certain embodiments, the composition is introduced into plant cells or plant protoplasts by, for example, microinjection, or by disruption or deformation of the cell wall or membrane, for example, by physical treatment, such as application of negative or positive pressure, shear force, or treatment with a chemical or physical delivery agent, such as a surfactant, liposome, or nanoparticle.See, e.g., delivery of substances to cells using microfluidic flow through a cell deformation constriction, as described in U.S. Patent Application Publication No. 2014 / 0287509, which is incorporated by reference in its entirety. Other techniques useful for delivering compositions to eukaryotic cells, plant cells, or plant protoplasts include ultrasound or sonication; vibration, friction, shear stress, vortex flow, cavitation; centrifugation or application of mechanical force; mechanical deformation or disruption of cell walls or cell membranes; enzymatic cell wall or cell membrane disruption or permeabilization; abrasion or mechanical scarring (e.g., abrasion with carborundum or other particulate abrasives, or scarring with files or sandpaper) or chemical scarring (e.g., treatment with acids or caustic agents); and electroporation. In certain embodiments, the compositions are provided by transfecting plant cells or plant protoplasts with a polynucleotide encoding a genome-editing molecule (e.g., an RNA-dependent DNA endonuclease, an RNA-dependent DNA-binding protein, an RNA-dependent nickase, an ABE, or a CBE, and / or a guide RNA) via bacteria (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azotobacter sp., Phyllobacterium sp.). See, e.g., Broothaerts et al. (2005) Nature, 433:629-633. Any of these techniques, or a combination thereof, may alternatively be used on plant explants, plant parts or tissues, or intact plants (or seeds) from which plant cells are optionally subsequently obtained or isolated; in certain embodiments, the composition is delivered in a separate step after the plant cells have been isolated.

[0057] In certain embodiments, a method for producing a soybean plant cell, soybean plant part, or soybean plant comprises (i) screening a population of soybean plant cells, parts, or plants for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising the loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0058] In certain embodiments, a population of soybean plant cells, parts, or plants screened for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 is first pre-screened by screening for phenotypically characteristic plants having a loss-of-function mutation in the FT1a gene of SEQ ID NO: 3, or allelic variants thereof. In certain embodiments, such phenotypic traits include an increase in the number of pods per plant; the number of seeds per plant; and / or the total seed weight harvested per plant compared to a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, such phenotypic traits include an increase in the number of pods per plant, the number of seeds per plant, and / or the total harvested seed weight per plant compared to wild-type control soybean plants lacking the loss-of-function allele, where the screened plants and control plants are grown under stress conditions (e.g., abiotic stress such as drought, cold, heat, or salt stress). In certain embodiments, plants exhibiting one or more of the foregoing phenotypic traits are then subjected to screening for the presence of the loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO:3 to identify and / or select soybean plants comprising the loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO:3.

[0059] In a specific embodiment, a population of soybean plant cells, parts, or plants screened for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 is subjected to one or more mutagenesis treatments. Loss-of-function alleles of the endogenous soybean FT1a gene can be created 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 rays.

[0060] Soybean plant cells, parts, or plants containing loss-of-function alleles of the endogenous FT1a gene can be generated using mutagenesis and identified by TILLING (targeted local lesions induced in the genome) or by EcoTILLING. TILLING is a common reverse genetics technique that uses mutagenesis to generate a library of mutant individuals, which are then subjected to high-throughput screening to discover mutations. In addition to being able to efficiently detect induced mutations, high-throughput TILLING technology is also ideal for detecting natural mutations. EcoTILLING is a method that uses TILLING technology to explore natural mutations in individuals (Barkley and Wang. Current genomics vol. 9, 4(2008):212-26. doi:10.2174 / 138920208784533656). Identified mutations can be introduced into a desired genetic background by crossing the mutant with a plant of the desired genetic background and performing an appropriate number of backcrosses to remove the original undesirable parental background. A more detailed description of methods and compositions for TILLING is disclosed in U.S. Patent Application Publication No. 2004 / 0053236A1, which is incorporated herein by reference in its entirety, and may be adapted for use in the methods provided herein for identifying soybean plant cells, parts, or plants that contain a loss-of-function allele of an endogenous FT1a gene.

[0061] In certain embodiments, screening involves analyzing the number of pods per plant, the number of seeds per plant, the total harvested seed weight per plant, and / or the total harvested seed weight per unit area in one or more candidate plants or one or more populations of candidate plants. In these embodiments, an increase in the number of pods per plant, the number of seeds per plant, the total harvested seed weight per plant, and / or the total harvested seed weight per unit area compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant containing the loss-of-function allele. In certain embodiments, screening is performed on a population of plants grown under stress. Suitable examples of stress conditions include drought, salinity, cold, heat, salt, shade, nutrient deficiency, high planting density, and the presence of pests or pathogens.

[0062] Methods are provided for determining whether a soybean plant cell, soybean plant part, or soybean plant contains a loss-of-function allele of an endogenous soybean FT1a gene. Methods for determining the presence or absence of a loss-of-function allele can be used in breeding programs, for example, for identification, selection, introgression, etc.

[0063] In certain embodiments, the method comprises analyzing a polynucleotide comprising a portion of SEQ ID NO:3 or an allelic variant thereof, or analyzing RNA encoded by a portion of SEQ ID NO:3 or an allelic variant thereof, from a plant cell, plant part, or plant. In certain embodiments, an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA indicates the presence of a loss-of-function allele. Detection of a loss-of-function allele 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 primers (e.g., selective amplification primers) and / or probes (e.g., capable of selective hybridization or generation of specific primer extension products) that specifically recognize the FT1a gene (e.g., a portion of SEQ ID NO:3 or an allelic variant thereof). Such primers and / or probes may comprise or consist of about 15, 20, 25, 30, 40, 45, or 50 or more contiguous nucleotides of SEQ ID NO:3 or its allelic variants. In certain embodiments, primers or probes may 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:3 or its allelic variants. In certain embodiments, hybridization probes (e.g., polynucleotides consisting of at least about 15-30 base pairs of SEQ ID NO:3 or its allelic variants) may comprise a detectable label (e.g., fluorescent label, radioactive label, epitope label, and chemiluminescent label). In certain embodiments, the FT1a gene can be directly sequenced using nucleic acid sequencing techniques, including whole genome sequencing.

[0064] In certain embodiments, the method comprises analyzing a polypeptide encoded by SEQ ID NO:3, a portion thereof, or an allelic variant thereof from a soybean plant cell, a soybean plant part, or a soybean 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 biological or biochemical activity of the polypeptide indicates the presence of a loss-of-function allele. Detection of a polypeptide-based loss-of-function allele can be determined by methods well known in the art, such as activity assays, Western blots using antibodies capable of specifically binding to the polypeptide, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunohistochemistry, immunocytochemistry, immunofluorescence, and the like.

[0065] 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 propagation material (e.g., seeds, seedlings, embryos, pollen, roots, stems, leaves, shoots, explants, or calluses), and soybean plants provided herein are not produced exclusively by biological processes. In certain optional embodiments provided herein, the soybean plant cells, soybean plant propagation material (e.g., seeds, seedlings, embryos, pollen, roots, stems, leaves, shoots, explants, or calluses), and soybean plants provided herein are not produced exclusively by biological processes.

[0066] The following numbered embodiments also form part of this disclosure:

[0067] 1. A soybean plant cell comprising a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, optionally not produced by an exclusive biological process.

[0068] 2. The soybean plant cell of embodiment 1, wherein the plant cell is homozygous for a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0069] 3. The soybean plant cell of embodiment 1 or embodiment 2, wherein the loss-of-function allele comprises an amorphic allele of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0070] 4. The soybean plant cell of any one of embodiments 1 to 3, wherein the loss-of-function allele comprises a hypomorphic allele of the FT1a gene.

[0071] 5. A soybean plant cell according to any one of embodiments 1 to 4, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the coding region of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0072] 6. The soybean plant cell of any one of embodiments 1 to 5, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the first exon of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; optionally, the frameshift or nonsense mutation occurs at a nucleotide corresponding to nucleotides 340 to 343 of SEQ ID NO: 3 or an allelic variant thereof.

[0073] 7. A soybean plant cell according to any one of embodiments 1 to 6, wherein the loss-of-function allele comprises a frameshift mutation of SEQ ID NO: 4 or SEQ ID NO: 5, or wherein the ft1a gene comprising the frameshift mutation encodes the protein of SEQ ID NO: 6.

[0074] 8. A soybean plant cell described in any one of embodiments 1 to 7, wherein the loss-of-function allele comprises (i) at least nucleotides corresponding to nucleotides 343 to 351 of SEQ ID NO: 3 or an allelic variant thereof; or (ii) an internal deletion including at least nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO: 1 or SEQ ID NO: 2; and optionally, in either (i) or (ii), the internal deletion maintains the reading frame of the encoded ft1a mutant protein, including the loss-of-function allele with respect to amino acid residues of the ft1a mutant protein that have not been deleted.

[0075] 9. The soybean plant cell of embodiment 8, wherein the internal deletion comprises: (i) an internal deletion of SEQ ID NO: 7 or SEQ ID NO: 8; or (ii) an internal deletion encoding a protein corresponding to a protein of SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0076] 10. The soybean plant cell of any one of embodiments 1 to 9, with the proviso that the soybean plant cell is not produced exclusively by essentially biological methods.

[0077] 11. The soybean plant cell of any one of embodiments 1 to 10, wherein the soybean plant cell comprises a superior soybean embryo.

[0078] 12. The soybean plant cell of any one of embodiments 1 to 11, wherein the soybean plant cell further comprises: (i) one or more mutations in separate soybean genes; and / or (ii) one or more transgenes, optionally wherein the transgenes encode proteins or RNAs that confer herbicide resistance or pest resistance.

[0079] 13. A soybean plant part comprising a soybean plant cell according to any one of embodiments 1 to 12, optionally wherein the soybean plant part is not produced exclusively by a biological process.

[0080] 14. The soybean plant part of embodiment 13, wherein the plant part is a stem, root, leaf, flower, pod, or seed.

[0081] 15. The soybean plant part of embodiment 13 or embodiment 14, wherein the part is a seed.

[0082] 16. The soybean plant part of any one of embodiments 13-15, wherein the part is a seed and wherein a population of soybean plants grown from the seeds has an increased total seed weight harvested per acre or hectare compared to the total seed weight harvested per acre or hectare of a population of wild-type control soybean plants lacking the loss-of-function allele.

[0083] 17. The soybean plant part of any one of embodiments 13-16, wherein the part is a seed, and the average weight of the seed is equivalent to the average weight of a wild-type control seed lacking the loss-of-function allele.

[0084] 18. A soybean plant part according to any one of embodiments 13 to 17, wherein the part is a pod; and optionally is homozygous for a loss-of-function mutation, such that the number of pods in the soybean plant from which the pods are obtained is increased compared to the number of pods in a wild-type control plant lacking the loss-of-function allele.

[0085] 19. A soybean seed lot comprising the seeds of any one of embodiments 14-17, optionally wherein the soybean seed lot is not produced exclusively by a biological process.

[0086] 20. The soybean seed lot of embodiment 19, wherein the average weight of 1000 seeds in the seed lot is equivalent to the average weight of 1000 seeds in a control seed lot obtained from a wild-type control plant lacking the loss-of-function allele.

[0087] 21. The soybean seed lot of embodiment 19 or embodiment 20, wherein the average number of seeds per kg of seeds in the seed lot is equivalent to the average number of seeds per kg of seeds in a control seed lot obtained from a wild-type control soybean plant lacking the loss-of-function allele.

[0088] 22. A soybean plant comprising a soybean plant cell according to any one of embodiments 1 to 12, optionally wherein the soybean plant is not produced exclusively by a biological process.

[0089] 23. The soybean plant of embodiment 22, wherein the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant is increased compared to the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant of a wild-type control soybean plant lacking the loss-of-function allele.

[0090] 24. The soybean plant of embodiment 22 or embodiment 23, wherein the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant is increased when the plant is grown under stress compared to the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant of a wild-type control soybean plant lacking the loss-of-function allele grown under stress, and optionally the stress comprises an abiotic stress.

[0091] 25. A soybean plant according to any one of embodiments 22 to 24, wherein the average weight of 1000 seeds obtained from the soybean plant is equivalent to the average weight of 1000 seeds obtained from a wild-type control soybean plant lacking the loss-of-function allele.

[0092] 26. A biological sample comprising a nucleic acid comprising a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0093] 27. The biological sample of embodiment 26, wherein the sample comprises a homogenate of a seed powder or tissue sample, and optionally the tissue sample comprises a sample of leaf, flower, pod, seed, stem, or root tissue.

[0094] 28. The biological sample of embodiment 26 or embodiment 27, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the coding region of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0095] 29. A biological sample according to any one of embodiments 26 to 28, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the first exon of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; optionally, the frameshift or nonsense mutation occurs at a nucleotide corresponding to nucleotides 340 to 343 of SEQ ID NO: 3 or an allelic variant thereof, and optionally, the frameshift mutation comprises a frameshift mutation of SEQ ID NO: 4 or SEQ ID NO: 5, or the ft1a gene comprising the frameshift mutation encodes the protein of SEQ ID NO: 6.

[0096] 30. A biological sample described in any one of embodiments 26 to 29, wherein the loss-of-function allele comprises (i) at least nucleotides corresponding to nucleotides 343 to 351 of SEQ ID NO: 3 or an allelic variant thereof; or (ii) an internal deletion including at least nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO: 1 or SEQ ID NO: 2; and optionally, in either (i) or (ii), the internal deletion maintains the reading frame of the encoded ft1a mutant protein, including the loss-of-function allele with respect to amino acid residues of the ft1a mutant protein that are not deleted.

[0097] 31. The biological sample of embodiment 30, wherein the internal deletion comprises (i) an internal deletion of SEQ ID NO: 7 or SEQ ID NO: 8; or (ii) an internal deletion encoding a protein corresponding to a protein of SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0098] 32. The biological sample of any one of embodiments 26 to 31, wherein said sample lacks nucleic acids comprising the wild-type allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0099] 33. A polynucleotide comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8, optionally wherein said polynucleotide is isolated.

[0100] 34. A polynucleotide encoding the polypeptide of SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10, optionally wherein said polynucleotide is isolated.

[0101] 35. A method for producing a soybean seed lot, comprising: (i) growing a population of soybean plants comprising the soybean plant of any one of embodiments 22-25; and (ii) harvesting seeds at maturity from the population of soybean plants of step (i), thereby producing a soybean seed lot.

[0102] 36. The method of embodiment 35, wherein the total harvested seed weight per acre or hectare of the population of soybean plants is increased compared to the total harvested seed weight per acre or hectare of a population of wild-type control soybean plants lacking the loss-of-function allele.

[0103] 37. The method of embodiment 35 or embodiment 36, wherein the total harvested seed weight per acre or hectare of a population of soybean plants grown under stress is increased compared to the total harvested seed weight per acre or hectare of a population of wild-type control soybean plants lacking the loss-of-function allele grown under stress, and optionally, the stress comprises an abiotic stress.

[0104] 38. The method of any one of embodiments 35-37, wherein the average weight of 1000 seeds of the soybean seed lot is equivalent to the average weight of 1000 seeds of a wild-type control soybean seed lot lacking the loss-of-function allele.

[0105] 39. The method of any one of embodiments 35-38, wherein at least 50%, 70%, 80%, or 90% of the plants in the population of soybean plants have 95% of their pods at full maturity color when harvested in step (i).

[0106] 40. The method of any one of embodiments 35-39, wherein the population of soybean plants is a full-season variety of the Soybean Maturity Group Zone in which they are grown, and the seed is harvested during the full growing season of the full-season variety or after the full growing season of the full-season variety.

[0107] 41. A method of producing a soybean crop, comprising planting a seed lot according to any one of embodiments 19-21.

[0108] 42. The method of embodiment 41, further comprising harvesting seeds from the soybean crop grown from the planted seeds.

[0109] 43. The method of embodiment 41 or embodiment 42, wherein the total seeds harvested per acre or hectare of the soybean crop is increased compared to the total seeds harvested per acre or hectare of a wild-type control soybean crop lacking the loss-of-function allele.

[0110] 44. The method of any one of embodiments 41-43, wherein the total seed harvest per acre or hectare of a soybean crop grown under stress is increased compared to the total seed harvest per acre or hectare of a wild-type control soybean crop lacking the loss-of-function allele grown under stress, and optionally, the stress comprises an abiotic stress.

[0111] 45. The method of any one of embodiments 41-44, wherein the average weight of 1000 harvested seeds is equivalent to the average weight of 1000 harvested wild-type control soybean seeds lacking the loss-of-function allele.

[0112] 46. ​​The method of any one of embodiments 41 to 45, wherein the seed is planted on or within one week of the earliest first planting date provided by the USDA Risk Management Service for the maturity zone in which the seed is planted.

[0113] 47. The method of any one of embodiments 41 to 46, wherein at least 50%, 70%, 80%, or 90% of the plants in the soybean crop have 95% of their pods at full maturity color at harvest.

[0114] 48. The method of any one of embodiments 41 to 47, wherein the soybean crop is a full-season variety for the soybean maturity group zone in which it is grown, and the seed is harvested during the full growing season for the full-season variety or after the full growing season for the full-season variety.

[0115] 49. A guide RNA molecule comprising a spacer RNA molecule targeting exon 1 of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, optionally wherein the spacer RNA molecule comprises RNA encoded by SEQ ID NO: 11.

[0116] 50. A method for producing a soybean plant cell of any one of embodiments 1-12, a soybean plant part of any one of embodiments 13-18, or a soybean plant of any one of embodiments 22-25, comprising introducing a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0117] 51. The method of embodiment 50, wherein the loss-of-function allele is introduced by (i) directing both: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule targeting the endogenous FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, exon 1 of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, or a spacer RNA molecule comprising RNA encoded by SEQ ID NO: 11; and (b) an RNA-dependent endonuclease (RDE) that recognizes the gRNA molecule to the genome of the target soybean plant cell; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising the loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0118] 52. The method of embodiment 51, wherein directing the gRNA and RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, the gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.

[0119] 53. The method of embodiment 51 or embodiment 52, wherein a soybean plant cell, soybean plant part, or soybean plant containing the loss-of-function allele is identified by (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 3 or an allelic variant thereof encoded by a portion of SEQ ID NO: 3 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: 3 in one or more candidate plant cells, plant parts, or plants; and / or (iii) analyzing the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant in one or more candidate plants, wherein an increase in the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant containing the loss-of-function allele.

[0120] 54. The method of embodiment 45, wherein the loss-of-function allele is introduced by crossing the soybean plant with a second soybean plant and harvesting F1 seeds containing the loss-of-function allele, thereby producing progeny soybean seeds containing the loss-of-function allele.

[0121] 55. A method for producing the soybean plant cell of any one of embodiments 1-12, the soybean plant part of any one of embodiments 13-18, or the soybean plant of any one of embodiments 22-25, comprising: (i) screening a population of soybean plant cells, parts, or plants for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising the loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0122] 56. The method of embodiment 55, wherein the population of soybean plant cells, parts, or plants is subjected to one or more mutagenesis treatments, and optionally, the mutagenesis procedure comprises chemical mutagenesis.

[0123] 57. The method of embodiment 55 or embodiment 56, wherein the screening comprises (i) analyzing a polynucleotide comprising a portion of SEQ ID NO: 3 or an allelic variant thereof, or analyzing RNA encoded by a portion of SEQ ID NO: 3 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 indicates the presence of a loss-of-function allele; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO: 3, 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 in the polypeptide, or a change in the biological or biochemical activity of the polypeptide, indicates the presence of a loss-of-function allele.

[0124] 58. The method of any one of embodiments 55-57, wherein said screening further comprises analyzing the number of pods per plant, the number of seeds per plant, and / or the total seed weight harvested per plant in one or more candidate plants, wherein an increase in the number of pods per plant, the number of seeds per plant, and / or the total seed weight harvested per plant compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the loss-of-function allele.

[0125] 59. The method of any one of embodiments 55-58, wherein said screening is carried out on a population of plants grown under stress.

[0126] 60. The method of embodiment 59, wherein the stress comprises an abiotic stress.

[0127] 61. A method for determining whether a soybean plant cell, soybean plant part, or soybean plant contains a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO:3 or an allelic variant thereof, the method comprising: (i) analyzing from the plant cell, plant part, or plant a polynucleotide comprising a portion of SEQ ID NO:3 or an allelic variant thereof, or analyzing RNA encoded by a portion of SEQ ID NO:3 or an allelic variant thereof, wherein an insertion, deletion, and / or substitution of one or more nucleotides in the polynucleotide or RNA indicates the presence of a loss-of-function allele; and / or (ii) analyzing from the soybean plant cell, soybean plant part, or soybean plant a polypeptide encoded by SEQ ID NO:3, a portion thereof, or an allelic variant thereof, wherein an insertion, deletion, and / or substitution of one or more amino acid residues in the polypeptide, or a change in the biological or biochemical activity of the polypeptide, indicates the presence of a loss-of-function allele.

[0128] 62. The method of embodiment 61, wherein the method further comprises analyzing the number of pods per plant, the number of seeds per plant, and / or the total harvested seed weight per plant in one or more of the soybean plants, wherein an increase in the number of pods per plant, the number of seeds per plant, and / or the total harvested seed weight per plant compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising the loss-of-function allele.

[0129] 63. The method of embodiment 61 or 62, wherein the analysis is performed on a population of plants grown under stress.

[0130] 64. The method of embodiment 63, wherein the stress comprises an abiotic stress.

[0131] 65. The method of embodiment 64, wherein the abiotic stress comprises drought stress. [Example]

[0132] Example 1. Creation of soybean with a mutated FT1a gene We transformed soybean plants and created a vector to disrupt the open reading frame of the GmFTA1a gene (Glyma10g38970; SEQ ID NO: 3) by CRISPR-mediated gene editing. A CRISPR guide RNA containing a crRNA fused to a spacer RNA (SEQ ID NO: 11) was designed to target exon 1 of the Glycine max FT1a gene.

[0133] The recovered genotypes were -10:13D and -4:9D. The -10:13D genotype has a 13-bp deletion (SEQ ID NO:5) that results in a frameshift mutation and introduces a premature stop codon. The -10:13D genotype is predicted to encode a truncated 84-amino acid polypeptide (SEQ ID NO:6). The -4:9D genotype has a 9-bp deletion (SEQ ID NO:8) that is predicted to encode a polypeptide with a 3-amino acid internal deletion (SEQ ID NOs:9 and 10).

[0134] Example 2. Performance of soybeans with a mutated FT1a gene Seeds of the homozygous 4:9D mutant line SENF2228 and the homozygous 10:13D mutant line SENF2229 were propagated and planted in field furrows along with lines with check and unrelated edits. Total yield data from the field trials showed that SENF2228 and SENF2229 tended to have higher yields per plot compared to the wild type, null isolates, and several unrelated mutants grown adjacent to them.

[0135] Some field-grown plants were also subjected to more detailed phenotyping. SENF2228 and SENF2229 consistently showed a trend toward higher yields compared to controls grown at similar densities. This trend was evident in yield components such as pod number, seed number, and total seed weight, although seed weight was comparable to that of the null isolate and wild-type check.

[0136] Example 3. Performance of soybeans with a mutated FT1a gene in field trials Seeds of the homozygous 4:9D mutant line SENF2228 and the homozygous 10:13D mutant line SENF2229 were propagated and planted in furrows in the field together with the NINF1170 check. The results of the field trials are shown in Table 1. The phenotypic characteristics of SENF2228, SENF2229 and the comparison variety NINF1170 are shown in Table 1.

[0137] [Table 1]

[0138] All cited patents and patent publications mentioned 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 indicated by the above disclosure and illustrated by the examples. While the materials and methods of the present disclosure have been described in terms of embodiments and illustrative examples, it will be apparent to those skilled in the art that substitutions and variations can be made to the materials and methods described herein without departing from the concept, spirit, and scope of the present 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 present disclosure as encompassed by the disclosed embodiments recited herein and the specification and appended claims.

Claims

1. A soybean plant cell comprising a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO:3 or an allelic variant thereof.

2. 2. The soybean plant cell of claim 1, wherein the plant cell is homozygous for a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

3. 2. The soybean plant cell of claim 1, wherein the loss-of-function allele comprises an amorphic allele of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

4. 2. The soybean plant cell of claim 1, wherein the loss-of-function allele comprises a hypomorphic allele of the FT1a gene.

5. 2. The soybean plant cell of claim 1, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the coding region of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

6. 6. The soybean plant cell of claim 5, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the first exon of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; and optionally, the frameshift or nonsense mutation occurs at a nucleotide corresponding to nucleotides 340-343 of SEQ ID NO: 3 or an allelic variant thereof.

7. 7. The soybean plant cell of claim 6, wherein the loss-of-function allele comprises the frameshift mutation of SEQ ID NO: 4 or SEQ ID NO: 5, or the ft1a gene comprising the frameshift mutation encodes the protein of SEQ ID NO:

6.

8. 2. The soybean plant cell of claim 1, wherein the loss-of-function allele comprises an internal deletion including (i) at least nucleotides corresponding to nucleotides 343 to 351 of SEQ ID NO: 3 or an allelic variant thereof; or (ii) at least nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO: 1 or SEQ ID NO: 2; and optionally, in either (i) or (ii), the internal deletion maintains the reading frame of the encoded ft1a mutant protein, including the loss-of-function allele, with respect to amino acid residues of the non-deleted ft1a mutant protein.

9. 9. The soybean plant cell of claim 8, wherein the internal deletion comprises: (i) the internal deletion of SEQ ID NO: 7 or SEQ ID NO: 8; or (ii) the internal deletion encoding a protein corresponding to a protein of SEQ ID NO: 9 and / or SEQ ID NO:

10.

10. 2. The soybean plant cell of claim 1, provided that said soybean plant cell is not produced exclusively by essentially biological methods.

11. 2. The soybean plant cell of claim 1, wherein the soybean plant cell comprises a superior soybean embryo.

12. 2. The soybean plant cell of claim 1, wherein the soybean plant cell further comprises: (i) one or more mutations in separate soybean genes; and / or (ii) one or more transgenes, optionally wherein the transgenes encode proteins or RNAs that confer herbicide resistance or pest resistance.

13. A soybean plant part comprising the soybean plant cell of any one of claims 1 to 12.

14. 14. The soybean plant part of claim 13, wherein the plant part is a stem, root, leaf, flower, pod, or seed.

15. 15. The soybean plant part of claim 14, wherein the part is a seed.

16. 15. The soybean plant part of claim 14, wherein the part is a seed and the total seed weight harvested per acre or hectare of a population of soybean plants grown from the seeds is increased compared to the total seed weight harvested per acre or hectare of a population of wild-type control soybean plants lacking the loss-of-function allele.

17. 15. The soybean plant part of claim 14, wherein said part is a seed, and wherein the average weight of said seed is equivalent to the average weight of a wild-type control seed lacking said loss-of-function allele.

18. 15. The soybean plant part of claim 14, wherein the part is a pod; and optionally is homozygous for the loss-of-function mutation, such that the number of pods in the soybean plant from which the pods are obtained is increased compared to the number of pods in a wild-type control plant lacking the loss-of-function allele.

19. 16. A soybean seed lot comprising the seeds of claim 15.

20. 20. The soybean seed lot of claim 19, wherein the average weight of 1000 seeds in the seed lot is equivalent to the average weight of 1000 seeds in a control seed lot obtained from a wild-type control plant lacking the loss-of-function allele.

21. 20. The soybean seed lot of claim 19, wherein the average number of seeds per kg of seeds in said seed lot is equivalent to the average number of seeds per kg of seeds in a control seed lot obtained from a wild-type control soybean plant lacking said loss-of-function allele.

22. A soybean plant comprising the soybean plant cell of any one of claims 1 to 12.

23. 23. The soybean plant of claim 22, wherein the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant is increased compared to the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant of a wild-type control soybean plant lacking the loss-of-function allele.

24. 23. The soybean plant of claim 22, wherein the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant is increased when the plant is grown under stress compared to the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant of a wild-type control soybean plant lacking the loss-of-function allele grown under stress, and optionally the stress comprises drought stress.

25. 23. The soybean plant of claim 22, wherein the average weight of 1000 seeds obtained from said soybean plant is equivalent to the average weight of 1000 seeds obtained from a wild-type control soybean plant lacking said loss-of-function allele.

26. A biological sample comprising a nucleic acid comprising a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

27. 27. The biological sample of claim 26, wherein the sample comprises a seed powder or a homogenate of a tissue sample, and optionally the tissue sample comprises a sample of leaf, flower, pod, seed, stem, or root tissue.

28. 27. The biological sample of claim 26, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the coding region of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

29. 29. The biological sample of claim 28, wherein the loss-of-function allele comprises a frameshift or nonsense mutation in the first exon of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; optionally, the frameshift or nonsense mutation occurs at a nucleotide corresponding to nucleotides 340 to 343 of SEQ ID NO: 3 or an allelic variant thereof, and optionally, the frameshift mutation comprises the frameshift mutation of SEQ ID NO: 4 or SEQ ID NO: 5, or the ft1a gene comprising the frameshift mutation encodes the protein of SEQ ID NO:

6.

30. The biological sample of claim 26, wherein the loss-of-function allele comprises (i) at least nucleotides corresponding to nucleotides 343 to 351 of SEQ ID NO: 3 or an allelic variant thereof; or (ii) an internal deletion including at least nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO: 1 or SEQ ID NO: 2; and optionally, in either (i) or (ii), the internal deletion maintains the reading frame of the encoded ft1a mutant protein, including the loss-of-function allele, with respect to amino acid residues of the ft1a mutant protein that are not deleted.

31. 31. The biological sample of claim 30, wherein the internal deletion comprises: (i) the internal deletion of SEQ ID NO: 7 or SEQ ID NO: 8; or (ii) the internal deletion encoding a protein corresponding to a protein of SEQ ID NO: 9 and / or SEQ ID NO:

10.

32. 27. The biological sample of claim 26, wherein the sample lacks nucleic acid comprising the wild-type allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

33. A polynucleotide comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8, optionally wherein said polynucleotide is isolated.

34. A polynucleotide encoding the polypeptide of SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10, optionally wherein said polynucleotide is isolated.

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

36. 36. The method of claim 35, wherein the total harvested seed weight per acre or hectare of the population of soybean plants is increased compared to the total harvested seed weight per acre or hectare of a population of wild-type control soybean plants lacking the loss-of-function allele.

37. 36. The method of claim 35, wherein the total harvested seed weight per acre or hectare of the population of soybean plants grown under stress is increased compared to the total harvested seed weight per acre or hectare of a population of wild-type control soybean plants lacking the loss-of-function allele grown under stress, and optionally the stress comprises drought stress.

38. 36. The method of claim 35, wherein the average weight of 1000 seeds of the soybean seed lot is equivalent to the average weight of 1000 seeds of a wild-type control soybean seed lot lacking the loss-of-function allele.

39. 36. The method of claim 35, wherein at least 50%, 70%, 80%, or 90% of the plants in the population of soybean plants have 95% of their pods that are full-maturity color when harvested in step (i).

40. 36. The method of claim 35, wherein the population of soybean plants is a full season variety of the Soybean Maturity Group Zone in which they are grown and the seed is harvested during or after the full growing season of the full season variety.

41. 20. A method of producing a soybean crop comprising planting the seed lot of claim 19.

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

43. 42. The method of claim 41, wherein the total seeds harvested per acre or hectare of the soybean crop is increased compared to the total seeds harvested per acre or hectare of a wild-type control soybean crop lacking the loss-of-function allele.

44. 42. The method of claim 41, wherein the total seeds harvested per acre or hectare of the soybean crop grown under stress is increased compared to the total seeds harvested per acre or hectare of a wild-type control soybean crop lacking the loss-of-function allele grown under stress, and optionally the stress comprises drought stress.

45. 42. The method of claim 41, wherein the average weight of 1000 of the harvested seeds is equivalent to the average weight of 1000 of harvested wild-type control soybean seeds lacking the loss-of-function allele.

46. 42. The method of claim 41, wherein the seed is planted on or within one week of the earliest first planting date provided by the USDA Risk Management Service for the maturity zone in which the seed is planted.

47. 42. The method of claim 41, wherein at least 50%, 70%, 80%, or 90% of the plants in the soybean crop have 95% of their pods at full maturity color at harvest.

48. 42. The method of claim 41, wherein the soybean crop is a full season variety for the Soybean Maturity Group Zone in which it is grown and the seed is harvested during the full growing season for a full season variety or after the full growing season for a full season variety.

49. A guide RNA molecule comprising a spacer RNA molecule targeting exon 1 of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, optionally wherein the spacer RNA molecule comprises the RNA encoded by SEQ ID NO:

11.

50. A method for producing the soybean plant cell of claim 1, the soybean plant part of claim 13, or the soybean plant of claim 22, comprising introducing a loss-of-function allele into the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

51. the loss-of-function allele is (i) by directing both (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule targeting the endogenous FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, exon 1 of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, or a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 11; and (b) an RNA-dependent endonuclease (RDE) that recognizes the gRNA molecule to the genome of a target soybean plant cell; and (ii) the method of claim 50 is introduced by isolating a soybean plant cell, soybean plant part, or soybean plant comprising a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

52. 51. The method of claim 50, wherein directing the gRNA and the RDE to the genome of the target maize 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 maize plant cell.

53. 51. The method of claim 50, wherein the soybean plant cell, soybean plant part, or soybean plant containing a loss-of-function allele is identified by (i) analyzing in one or more candidate plant cells, plant parts, or plants a polynucleotide comprising a portion of SEQ ID NO: 3 or an allelic variant thereof encoded by a portion of SEQ ID NO: 3 or an allelic variant thereof, (ii) analyzing in one or more candidate plant cells, plant parts, or plants a polypeptide encoded by a portion of SEQ ID NO: 3; and / or (iii) analyzing in one or more candidate plants the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant, wherein an increase in the number of pods per plant; the number of seeds per plant; and / or the total harvested seed weight per plant compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant containing the loss-of-function allele.

54. 51. The method of claim 50, wherein the loss-of-function allele is introduced by crossing a soybean plant containing the loss-of-function allele with a second soybean plant and harvesting F1 seed containing the loss-of-function allele, thereby producing progeny soybean seed containing the loss-of-function allele.

55. 23. A method for producing the soybean plant cell of claim 1, the soybean plant part of claim 13, or the soybean plant of claim 22, comprising: (i) screening a population of soybean plant cells, parts, or plants for the presence of a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

56. 56. The method of claim 55, wherein a population of soybean plant cells, parts, or plants is subjected to one or more mutagenesis treatments, optionally wherein the mutagenesis procedure comprises chemical mutagenesis.

57. 56. The method of claim 55, wherein the screening comprises: (i) analyzing a polynucleotide comprising a portion of SEQ ID NO:3 or an allelic variant thereof, or analyzing RNA encoded by a portion of SEQ ID NO:3 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 indicates the presence of the loss-of-function allele; and / or (ii) analyzing a polypeptide encoded by SEQ ID NO:3, 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 in the polypeptide, or a change in the biological or biochemical activity of the polypeptide, indicates the presence of the loss-of-function allele.

58. 56. The method of claim 55, wherein said screening further comprises analyzing the number of pods per plant, the number of seeds per plant, and / or the total seed weight harvested per plant in one or more candidate plants, wherein an increase in the number of pods per plant, the number of seeds per plant, and / or the total seed weight harvested per plant compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant that contains the loss-of-function allele.

59. 59. The method of claim 58, wherein the screening is performed on a population of plants grown under stress.

60. 60. The method of claim 59, wherein the stress comprises drought stress.

61. 1. A method for determining whether a soybean plant cell, soybean plant part, or soybean plant contains a loss-of-function allele of the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, said method comprising: (i) analyzing from said plant cell, plant part, or plant a polynucleotide comprising a portion of SEQ ID NO:3 or an allelic variant thereof, or analyzing RNA encoded by a portion of SEQ ID NO:3 or an allelic variant thereof, wherein an insertion, deletion, and / or substitution of one or more nucleotides in said polynucleotide or RNA indicates the presence of said loss-of-function allele; and / or (ii) analyzing from said soybean plant cell, soybean plant part, or soybean plant a polypeptide encoded by SEQ ID NO:3, a portion thereof, or an allelic variant thereof, wherein an insertion, deletion, and / or substitution of one or more amino acid residues in said polypeptide, or a change in the biological or biochemical activity of said polypeptide, indicates the presence of said loss-of-function allele.

62. 62. The method of claim 61, wherein the method further comprises analyzing one or more of the soybean plants for the number of pods per plant, the number of seeds per plant, and / or the total seed weight harvested per plant, and wherein an increased number of pods per plant, the number of seeds per plant, and / or the total seed weight harvested per plant compared to a wild-type control soybean plant lacking the loss-of-function allele is indicative of a soybean plant cell, soybean plant part, or soybean plant that comprises the loss-of-function allele.

63. 63. The method of claim 62, wherein the analysis is performed on a population of plants grown under stress.

64. 64. The method of claim 63, wherein the stress comprises an abiotic stress.

65. 65. The method of claim 64, wherein the abiotic stress comprises drought stress.

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