Soybean FT1a gene mutation

Abstract

The present disclosure relates to novel soybean plants, plant parts, and nucleotide sequences in soybean plants comprising a mutated FT1a gene, as well as methods of use and preparation thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international patent application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 479,312, filed on January 10, 2023, the entire contents of which (including specification, claims, drawings, and sequence listing) are incorporated herein by reference in their entirety. Sequence Listing XML

[0002] This application contains a sequence listing, which has been electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML file was created on December 26, 2023, is named P14293WO00.xml, and is 26,209 bytes in size. Technical Field

[0003] Disclosed herein are nucleotide sequences in novel plants, plant parts, and soybean varieties comprising a mutated FT1a gene, as well as methods of making them by growing soybean plants or batches, and methods of using them. Background Art

[0004] Agriculture is a vital sector in the global economy, particularly in the United States. Soybeans (Glycine max) are a crucial legume crop worldwide due to their ability to fix atmospheric nitrogen. Soybeans are a major source of protein in animal feed, and soybean oil is widely used in various industries, including food and beverages, biodiesel, and other sectors.

[0005] Soy 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, enrich soil quality, improve soil nutrient efficiency, and produce sustainable soybean crops. Bioengineering benefits for soybean farmers include increased yields and resilience to extreme weather conditions. Summary of the Invention

[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 the aforementioned soybean plant cell, including stems, roots, leaves, flowers, pods, and seeds. Also provided are soybean seed lots comprising the seeds. Also provided are soybean plants comprising the aforementioned soybean plant cells.

[0007] Also provided are biological samples 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. Also provided are polynucleotides comprising the 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 for producing a soybean seed lot, the method comprising: (i) growing a soybean plant population comprising the aforementioned soybean plants; and (ii) harvesting seeds from the soybean plant population of step (i) at maturity. A method for producing a soybean crop is provided, the method comprising planting the aforementioned seed lot.

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

[0010] Also disclosed are methods for generating the aforementioned 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 soybean plant cells, parts, or a population of plants for the presence of the 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 that contain 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, plant part, or plant comprises a loss-of-function allele of an 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, from a plant cell, plant part, or plant, 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, 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 a soybean plant cell, plant part, or plant, wherein an insertion, deletion, and / or substitution of one or more amino acid residues of the polypeptide, or a change in the biological or biochemical activity of the polypeptide, indicates the presence of a loss-of-function allele. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A 、 Figure 1B Shown is the wild-type FT1a gene (SEQ ID NO: 3) with exons encoding the mRNA splice variant 1 (Glyma.18G298900.1) transcript of the FT1a protein of SEQ ID NO: 1. All exons are shown in bold, translated bases (codons) are shown in uppercase, introns are shown in lowercase, and the 5' and 3' untranslated regions (UTRs) are shown in lowercase and underlined.

[0013] Figure 2A 、 Figure 2B The wild-type FT1a gene (SEQ ID NO: 3) is shown with exons of the mRNA splice variant 2 (Glyma.18G298900.3) transcript encoding the FT1a protein of SEQ ID NO: 2. All exons are shown in bold, translated bases (codons) are shown in uppercase, introns are shown in lowercase, and the 5' and 3' untranslated regions (UTRs) are shown in lowercase and underlined.

[0014] Figure 3A 、 Figure 3B Shown is an ft1a gene mutant (SEQ ID NO: 5) having exons encoding a transcript of the mutant ft1a protein of SEQ ID NO: 6. All exons are shown in bold, translated bases (codons) are shown in uppercase, introns are shown in lowercase, and the 5' and 3' untranslated regions (UTRs) are shown in lowercase and underlined.

[0015] Figure 4A 、 Figure 4B Shown is an ft1a gene mutant (SEQ ID NO: 8) having exons encoding a transcript of the mutant ft1a protein of SEQ ID NO: 9. All exons are shown in bold, translated bases (codons) are shown in uppercase, introns are shown in lowercase, and the 5' and 3' untranslated regions (UTRs) are shown in lowercase and underlined. DETAILED DESCRIPTION

[0016] As used herein, the phrase "allelic variant" refers to polynucleotide or polypeptide sequence variations that occur in a particular gene at a particular locus in different strains, varieties, or isolates of a given organism.

[0017] As used herein, the phrase "amorphous allele" refers to an allele of a gene that has no gene activity compared to the wild-type allele of the gene. Amorphous alleles are also called null alleles.

[0018] Furthermore, as used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without each other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover 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).

[0019] As used herein, the phrase "biological sample" refers to complete or incomplete (e.g., ground soybean seeds or soybean plant tissue, chopped soybean plant tissue, freeze-dried tissue) soybean plant tissue. It can also be an extract comprising complete or incomplete seeds or soybean plant tissue. Biological samples can include flour, meal, syrup, oil, starch, and cereals, all or part of which are manufactured to contain soybean plant by-products. In certain embodiments, the biological sample is "non-renewable" (i.e., cannot be regenerated into a soybean plant or soybean plant part).

[0020] For example, the terms "corresponding," "corresponding," and the like, when used in the context of nucleotide positions, mutations, and / or substitutions of 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 the positions of nucleotides in the given sequence that are identical to nucleotides in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL 0 1.2.4 with default parameters).

[0021] As used herein, the terms "Cpf1" and "Cas12a" are used interchangeably to refer to the same RNA-dependent DNA endonuclease (RdDe).

[0022] As used herein, the phrase "endogenous gene" refers to a gene unit in its native form in its natural location in the genome of an organism.

[0023] As used herein, the term "expression" refers to the production of a functional end-product (e.g., mRNA, guide RNA, or protein) in either precursor or mature form.

[0024] As used herein, the phrase "hypomorphic allele" refers to an allele of a gene that has lower gene activity than the wild-type allele but higher than the amorphous allele.

[0025] As used herein, the terms "include," "includes," and "including" should be interpreted as implying at least the features they specify without excluding any additional unspecified features.

[0026] As used herein, the term "isomorphic allele" refers to an allele of a gene that has wild-type gene activity.

[0027] As used herein, the term "isolated" means having been removed from its natural environment.

[0028] As used herein, the term "introducing" means that nucleic acid (e.g., expression construct) or protein is provided into a cell. Introducing includes mentioning that nucleic acid is incorporated into a eukaryotic cell or prokaryotic cell, wherein the nucleic acid can be incorporated into the genome of the cell, and includes mentioning that the nucleic acid or protein is transiently provided into the cell. Introducing includes mentioning stable or transient transformation methods. Therefore, in the context of inserting nucleic acid fragments (e.g., recombinant DNA construct / expression construct) into a cell, "introducing" means "transfection" or "conversion" or "transduction", and includes mentioning that nucleic acid fragments are incorporated into a eukaryotic cell or prokaryotic cell, wherein the nucleic acid fragments can be incorporated into the genome (e.g., nuclear chromosome, plasmid, plastid, chloroplast or mitochondrial DNA) of the cell, converted into autonomous replicons or transient expression (e.g., transfected mRNA).

[0029] As used herein, a "loss-of-function allele" can include an amorphous allele or a hypomorphic allele of a gene.

[0030] As used herein, the term "plant" includes reference to immature or mature whole soybean plants, including plants from which seeds or kernels or anthers have been removed. Any seed or embryo that will produce a plant is also considered a soybean plant.

[0031] 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.

[0032] As used herein, the term "plant" includes the entire soybean plant and any progeny, cell, tissue, or part or parts of a plant. Thus, the term "plant" includes reference to an immature or mature whole soybean plant, including plants from which seeds, kernels, or anthers have been removed.

[0033] The term "plant part" includes any one or more parts of a plant, including, for example, but not limited to, seeds (including mature seeds and immature seeds), grains, stalks, plant cuttings, plant cells, plant cell cultures, or plant organs (e.g., pollen, embryos, pods, flowers, fruits, buds, leaves, roots, stems, and explants). Plant tissues or plant organs can be seeds, protoplasts, callus, or any other plant cell mass organized into structural or functional units. Plant cells or tissue cultures can be capable of regenerating plants having the physiological and morphological characteristics of the plant from which the cells or tissues are obtained, and can regenerate plants having the substantially identical genotype to the plant. Regenerable cells in plant cells or tissue cultures can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stalks. In contrast, some plant cells cannot be regenerated to produce plants, and are referred to herein as "non-regenerable" plant cells.

[0034] To the extent that any of the foregoing definitions are inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, in any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the foregoing definitions will control herein.

[0035] The present disclosure provides soybean plant cells, plant parts (including seeds, plants, seed batches, and biological samples) comprising a mutant FT1a gene (i.e., comprising a loss-of-function allele of the endogenous FT1a gene). These soybean plants and parts can be used for human food, livestock feed, as industrial raw materials, or as breeding material for developing additional soybean varieties.

[0036] The target endogenous FT1a gene comprises 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 of chromosome 18 of the soybean Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.18G298900 on the world wide web internet website "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 produces Figure 1A 、 Figure 1B The mRNA splice variant 1 (Glyma.18G298900.1) encoding the FT1a protein of SEQ ID NO: 1 is shown, and Figure 2A 、 Figure 2B mRNA splice variant 2 (Glyma.18G298900.3) encoding the FT1a protein of SEQ ID NO: 2 is shown in . Allelic variants of the endogenous soybean FT1a gene include variants encoding an FT1a protein 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 comprising 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, the allelic variant of the endogenous soybean FT1a gene is an isomorphic allele of the endogenous soybean FT1a gene. The wild-type soybean FT1a gene encodes a member of the phosphatidylethanolamine binding protein (PEBP) family, described in Serre et al. Structure (1998), 6:1255–1265. Residues conserved in the soybean FT1a wild-type protein of SEQ ID NO: 1 with other PEBP family members include amino acid residues (including residues 65-76, P80, H87, G116, and R119). Residues conserved in the soybean FT1a wild-type protein of SEQ ID NO: 2 with other PEBP family members include amino acid residues (including residues 65-76, P80, and H87).

[0037] Soybean plant cells, plant parts, and plants comprising a loss-of-function allele of the soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof are provided. Examples of loss-of-function alleles can include deletions, insertions, and / or substitutions of one or more nucleotides of the endogenous FT1a gene. The insertions, deletions, and / or substitutions can be made anywhere in the FT1a gene, including, for example, in the promoter region, exons, introns, and / or untranslated regions (5'UTR or 3'UTR). In certain embodiments, the loss-of-function allele comprises a deletion, insertion, and / or substitution 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 the entire coding region or any portion of the coding region required for biological activity. In certain embodiments, the loss-of-function allele comprises a deletion, insertion and / or substitution of one or more nucleotides of: exon 1 of mRNA splice variants 1 and 2 of the FT1a gene of SEQ ID NO: 3 or its allelic variants (i.e., nucleotides 229 to 429 of SEQ ID NO: 3, or at an equivalent position of 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 at an equivalent position of 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 at an equivalent position of an allelic variant of SEQ ID NO: 3), or nucleotides corresponding to those of exon 4 of mRNA splice variant 1 (i.e., nucleotides 4999 to 5225 or nucleotides 5104 to 5225 of SEQ ID NO: 3, or at an equivalent position of 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, or an allelic variant thereof, located at nucleotides 331 to 356 of SEQ ID NO: 3.

[0038] In certain embodiments, the loss-of-function allele comprises a deletion, insertion, and / or substitution that results in a frameshift mutation and / or nonsense mutation in the coding region of the FT1a gene. In certain embodiments, the loss-of-function allele of the FT1a gene may include a deletion of any number of nucleotides in an exon of the FT1a gene that is not divisible by 3. In certain embodiments, the loss-of-function allele of the FT1a gene may include a deletion of SEQ ID NO: 3 at nucleotides 229 to 429 (i.e., the first exon). 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98, 100, 101, 103, 104, 106, 107, 109, 110, 112, 113 , 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, 151, 152, 154, 155, 157, 158, 160, 161, 163, 164, 166, 167, 169, 170, 172, 173, 175, 176, 178, 179, 181, 182, 184, 185, 187, 188, 190, 191, 193, 194, 196, 197, 199, or 200 nucleotide deletions resulting in frameshift mutations. In certain embodiments, the frameshift mutation occurs at a nucleotide 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 a nucleotide corresponding to one or more of nucleotides 340 to 343 of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments, the FT1a gene comprising a mutation of 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 allelic variants 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 to the full length of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, a mutated FT1a gene comprising 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 allelic variants 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 to the full length of SEQ ID NO: 6.

[0039] In certain embodiments, the loss-of-function allele comprises an internal deletion that retains the reading frame of the encoded FT1a protein while removing at least one, two, or three codons, thereby resulting in a mutant FT1a protein lacking at least one, two, or three amino acid residues. In certain embodiments, the loss-of-function allele of the FT1a gene may include a deletion of any number of nucleotides in an exon of the FT1a gene that is divisible by 3. In certain embodiments, the loss-of-function allele of the FT1a gene may include SEQ ID NO:3 located at nucleotides 229 to 429 (i.e., within the first exon of mRNA splice forms 1 and 2). 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114 of the endogenous soybean FT1a gene of NO:3 In some embodiments, the loss-of-function allele comprises an internal deletion comprising nucleotides corresponding to at least nucleotides 343 to 351 of SEQ ID NO: 3 or an allelic variant thereof, and the reading frame is preserved. In some embodiments, the loss-of-function allele comprises an internal deletion comprising nucleotides encoding at least amino acids corresponding to N39 to C41 of SEQ ID NO: 1 and SEQ ID NO: 2 or an allelic variant thereof, and the reading frame is preserved.In these embodiments, the loss-of-function allele can comprise an internal deletion of nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NOs: 1 and 2, and further comprises P2, R3, S4, T5, D6, P7, L8, V9, I10, G11, G12, V13, I14, G15, D16, V17, L18, E19, P20, F21, T22, S23, S24, V25, S26, M27, G28, I29, V30, Y31, N32, N33, C34, In some embodiments, the FT1a gene comprising a mutation of a loss-of-function allele having 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 to the full length of SEQ ID NO: 7 or SEQ ID NO: 8. In certain embodiments, a FT1a gene comprising a mutation of 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 to the full length of SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0040] In certain embodiments, the yield of a soybean plant comprising a loss-of-function allele of the endogenous soybean FT1a gene is increased compared to the yield of a wild-type control soybean plant lacking the loss-of-function allele. The increased yield of the soybean plant can be measured in a variety of ways, including pod count per plant, seed count per plant, total weight of seeds harvested per plant, or total weight of seeds harvested per unit area (e.g., seed weight per acre or seed weight per hectare). In certain embodiments, the increased yield can be caused by an improved response to stress, including abiotic stress (e.g., drought, heat, cold, and / or salt stress).

[0041] In certain embodiments, the pod count per soybean plant comprising the loss-of-function allele in the FT1a gene is increased compared to the pod count per plant of a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the pod count per plant is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to the pod count per plant from a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the seed count per plant comprising the loss-of-function allele in the FT1a gene is increased compared to the seed count per plant of a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the seed count per plant comprising the 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 seed count per plant from a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the total weight of seeds harvested per plant comprising the loss-of-function allele in the FT1a gene is increased compared to the total weight of seeds harvested per plant from a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the total weight of seeds harvested 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 weight of seeds harvested per plant from a corresponding wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the total weight of seeds harvested per unit area of ​​a soybean plant comprising a loss-of-function allele in the FT1a gene is increased compared to the total weight of seeds harvested per unit area of ​​a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, the total weight of seeds harvested per unit area of ​​a soybean 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 total weight of seeds harvested 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 plant is comparable to or substantially equivalent to the average weight of 1000 seeds obtained from a wild-type control soybean plant lacking the loss-of-function allele.

[0042] In certain embodiments, the pod count per plant, seed count per plant, total seed weight per plant, and / or total seed weight per unit area harvested of soybean plants comprising a loss-of-function allele in the FT1a gene are increased when grown under stress, as compared to the pod count per plant, seed count per plant, total seed weight per plant harvested, and / or total seed weight per unit area harvested of 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 comprises abiotic stress. In certain embodiments, abiotic stress comprises drought, cold, heat, and salt stress. In these embodiments, the pod count per plant, seed count per plant, total seed weight per plant, and / or total seed weight per unit area harvested of plants comprising a loss-of-function allele in the FT1a gene can be increased when grown under drought stress, as compared to the pod count per plant, seed count per plant, total seed weight harvested per plant, and / or total seed weight harvested per unit area of ​​wild-type control soybean plants lacking the loss-of-function allele grown under drought stress.

[0043] In certain embodiments, when soybean plants comprising a loss-of-function allele in the FT1a gene are grown for a full growing season, the pod count per plant, the seed count per plant, the total weight of seeds harvested per plant, and / or the total weight of seeds harvested per unit area are increased compared to a control (e.g., a check). In certain embodiments, seed plants comprising a loss-of-function allele in the FT1a gene are planted on or after (e.g., on the same day or within a week) the earliest initial planting date specified by the USDA Risk Management Agency for the mature zone in which they are planted. Non-limiting examples of the RMA replanting crop insurance date can range from about April 1 in the southeastern United States to about May 5 in the northern Midwest of the United States (see https internet website "soybeanresearchinfo.com / wp-content / uploads / 2022 / 01 / 2700-003-23_Planting-Date-V1.pdf"). In certain embodiments, at harvest, 95% of the pods of at least 50%, 70%, 80% or 90% of the plants in a soybean crop comprising a loss-of-function allele in the FT1a gene have a fully mature color. The fully mature color depends on the variety and can be gray, tan or brown. In certain embodiments, the soybean crop comprising the loss-of-function allele in the FT1a gene is an all-season variety of the soybean maturity group region in which it is grown, and wherein the seed is harvested during or after the full growing season of the all-season variety. Soybeans comprising the loss-of-function allele in the FT1a gene can be assigned to any of 13 maturity group designations ranging from 000, 00, 0 or I to X. Maturity groups can also be represented by Arabic numerals followed by decimals (e.g., "5.8"). Soybean maturity groups 00 to VIII are typically grown in the United States (see https: / / www.soybeanresearchinfo.com / research-highlight / delineating-optimal-soybean-maturity-groups-across-the-united-states / ).

[0044] A soybean seed batch comprising soybean seeds containing a loss-of-function allele in the FT1a gene is provided. In certain embodiments, the soybean plants comprising a mutated FT1a gene can produce a seed batch wherein the average weight of 1000 seeds in the seed batch is equivalent to or substantially equal to the average weight of 1000 seeds in a control seed batch obtained from a wild-type control plant lacking 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 batch is equivalent to or substantially equal to the average number of seeds per kilogram of seeds in a control seed batch obtained from a wild-type control soybean plant lacking the loss-of-function allele in the FT1a gene. In certain embodiments, the seed batch is packaged in batches comprising approximately 50 to 60 pounds (i.e., approximately 22.7 to 27.2 kilograms) of seeds.

[0045] Also provided are polynucleotides comprising any of the aforementioned mutations of the FT1a gene or fragments thereof. In certain embodiments, polynucleotides comprising the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, or allelic variants thereof, are provided. In certain embodiments, allelic variants of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 will comprise a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the full 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 to SEQ ID NO:3 over its full length. In certain embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:10, or allelic variants thereof. In certain embodiments, the encoded allelic variant will comprise a polypeptide having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the full 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.

[0046] Also provided are biological samples and soybean by-products comprising any of the aforementioned polynucleotides. In certain embodiments, by-products are processed products made from soybean plants or seeds of the present disclosure, including: (a) soybean seed meal (defatted or not defatted); (b) extracted soy protein, oil, sugar, syrup, and starch; (c) soybean fermentation products; (d) soybean-based animal feed or human food (e.g., feed and food comprising soybean seed meal (defatted or not defatted) and other ingredients (e.g., other grains, other seed meals, other protein meals, other oils, other starches, other sugars, adhesives, preservatives, wetting agents, vitamins, and / or minerals); (e) pharmaceuticals; (f) raw or processed biomass (e.g., cellulose and / or lignocellulosic materials; silage); and (g) various industrial products.

[0047] Also provided are methods of producing soybean by-products using the soybean plants, seeds, and seed lots of the present disclosure. Such methods will typically include at least one processing step of: cleaning, cracking, flaking, crushing, macerating, pressing, extracting, draining, and / or extruding the seeds.

[0048] 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 comprises the loss-of-function allele. In addition, both the first parent soybean plant and the second parent soybean plant can comprise the loss-of-function allele. Any such method of using a soybean plant comprising a loss-of-function allele is part of the present disclosure: selfing, backcrossing, hybrid production, population hybridization, etc. All plants produced using a soybean plant comprising a loss-of-function allele as a parent are within the scope of the present disclosure, including plants derived from a soybean plant having a loss-of-function allele. Also provided are F1 progeny soybean plants, F1 seeds, and different parts of an F1 soybean plant produced by crossing a soybean plant comprising a loss-of-function allele with any other soybean plant. Breeding methods that can be used with the soybean plants of the present disclosure in the development of additional soybean plants are described below. One such embodiment is a method for developing progeny soybean plants in a soybean plant breeding program, the method comprising: obtaining a soybean plant or part thereof comprising a loss-of-function allele of an endogenous soybean FT1a gene, and utilizing the plant or plant part as a source of breeding material; and selecting progeny plants having the loss-of-function allele. Breeding steps that can be used in a soybean plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as restriction fragment polymorphism enhanced selection, genetic marker enhanced selection (e.g., SNP or SSR markers), and preparation of doubled haploids can be utilized.

[0049] Field crops are cultivated by utilizing the technology of plant pollination method. The soybean plants of the present disclosure can be self-pollinated, sibling-pollinated or cross-pollinated to produce pedigree soybean plants. If the pollen from one flower is transferred to the same or another flower of the same plant, the plant is self-pollinated. When individuals within the same family or variety are used for pollination, the plant is sibling-pollinated. If the pollen comes from flowers on different plants of different families or varieties, the plant is cross-pollinated. As used herein, the terms "cross-pollination" and "outcrossing" do not include self-pollination or sibling-pollination. Soybean plants (soybeans) are considered to be naturally self-pollinating plants. Although they are capable of cross-pollination, they rarely do so in nature. Some researchers report that insects carry pollen from one soybean plant to another soybean plant, and it is generally estimated that less than 1% of soybean seeds formed in open-air plantings can be traced back to cross-pollination, that is, less than 1% of soybean seeds formed in open-air plantings can produce F1 hybrid soybean plants.

[0050] Any other suitable breeding, selection or growing method may be used. The choice of a particular breeding or selection method will vary depending on environmental factors, population size, etc.

[0051] A method for producing a soybean seed lot, the method comprising: (i) growing a population of soybean plants comprising a mutant FT1a gene to maturity; and (ii) harvesting seeds from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot, wherein the soybean plants are homozygous for the mutant FT1a gene. In certain embodiments, the seed lot is packaged in batches comprising about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms).

[0052] Also provided herein are methods for treating soybean seeds and seed batches of the present disclosure and the resulting treated seeds and seed batches. Such fertilizers, biopharmaceuticals, nematicides, insecticides, and fungicides can be used by methods including in-ditch application or by coating (e.g., with a drum coater, rotary coater, tumbling drum, fluidized bed, and / or spouted bed apparatus) to treat seeds. Methods and compositions for coating seeds that can be applied to seeds provided herein are disclosed in U.S. Patent No. 10,745,578, which is incorporated herein by reference in its entirety, including various adhesives, fillers, film coatings, and active ingredients such as fertilizers, surfactants, plant growth regulators, crop desiccants, fungicides, bactericides, bacterial inhibitors, insecticides, and insect repellents.

[0053] The present disclosure also provides methods for preparing soybean plants comprising a mutated FT1a gene. In certain embodiments, the methods can include performing deletions, insertions, and / or substitutions, resulting in a mutated FT1a gene. The gene editing molecules used in the methods provided herein include molecules capable of introducing double-strand breaks ("DSBs") or single-strand breaks ("SSBs") at specific sites or sequences in double-stranded DNA, such as in genomic DNA or a target gene located within genomic DNA and an accompanying guide RNA. In certain embodiments, the loss-of-function allele is derived from 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 recovery of the desired loss-of-function allele. In certain embodiments, the loss-of-function allele is derived from the introduction of a DSB at a target site in the FT1a gene (e.g., SEQ ID NO: 3 or an allelic variant thereof), followed by homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ to introduce the desired donor or other DNA template polynucleotide at the DSB, followed by recovery of the desired loss-of-function allele. Examples of such gene editing molecules include: (a) nucleases, including RNA-guided nucleases, RNA-guided DNA endonucleases or RNA-guided DNA endonucleases (RdDe), class 1 CRISPR-type nuclease systems, type II Cas nucleases, Cas9, nCas9 nickases, type V Cas nucleases, Cas12a nucleases, nCas12a nickases, Cas12d (CasY), 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 (T-effector nucleases); The invention provides a method for preparing a nucleic acid sequence of at least one nucleotide sequence of the present invention, wherein the nucleic acid sequence of the present invention is selected from the group consisting of: (a) an ALE-nickase), an Argonaute, and a mega-nuclease or an engineered mega-nuclease; (b) a polynucleotide encoding one or more nucleases capable of effecting a site-specific alteration of a target nucleotide sequence (including the introduction of a DSB or SSB); (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for an RNA-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 nonhomologous end joining (NHEJ)).

[0054] In certain embodiments, mutant FT1a genes and plant cells, parts (including seeds), and plants comprising mutant FT1a genes are generated using CRISPR technology. CRISPR technology for editing genes in eukaryotic organisms is disclosed in U.S. Patent Application Publication Nos. 2016 / 0138008A1 and 2015 / 0344912A1, and U.S. Patents 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 RNA and PAM site are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1. Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonucleases are disclosed in International Patent Application No. PCT / US2015 / 018104 (published as WO 2015 / 131101 and claiming priority to U.S. Provisional Patent Application No. 61 / 945,700). Methods for genome editing using CRISPR technology in plants are disclosed in U.S. Patent Application Publications No. US2015 / 0082478A1 and US2015 / 0059010A1 and International Patent Application No. PCT / US2015 / 038767A1 (published as WO 2016 / 007347 and claiming priority to U.S. Provisional Patent Application No. 62 / 023,246). All patent publications cited in this paragraph are incorporated herein by reference in their entirety. In certain embodiments, RNA-guided endonucleases are used that leave blunt ends after cleaving the target site. Blunt-end-cutting RNA-guided endonucleases include Cas9, Cas12c, Cas12i, and Cas 12h (Yan et al., 2019). In certain embodiments, RNA-guided endonucleases are used that leave staggered single-stranded DNA overhangs after cleaving the target site. Wrong-end-cutting RNA-guided endonucleases include Cas12a, Cas12b, and Cas12e.

[0055] Provided are guide RNA molecules comprising a spacer RNA molecule that targets the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof. In certain embodiments, the spacer RNA molecule targets a portion of exon 1 (i.e., nucleotides 229 to 429 of SEQ ID NO: 3, or an equivalent position of 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 of 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 of an allelic variant of SEQ ID NO: 3), or nucleotides corresponding to exon 4 (i.e., nucleotides 4999 to 5225 or nucleotides 5104 to 5225 of SEQ ID NO: 3, or an equivalent position of 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 RNA encoded by SEQ ID NO: 11. A guide RNA comprising a spacer RNA molecule encoded by SEQ ID NO: 11 can be used in combination with a Cas12a nuclease to generate a mutant FT1a gene that: (i) comprises a deletion in the endogenous FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; (ii) comprises 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 or at an equivalent position of an allelic variant of SEQ ID NO: 3; (iii) comprises 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 SEQ ID NO: 4 or SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 8 or an allelic variant thereof. NO:7 or SEQ ID NO:8; or (iv) a polypeptide encoding SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO:10 or an allelic variant thereof.

[0056] CRISPR type genome editing can be applicable to plant cells and methods provided herein in several ways.CRISPR elements, such as gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs (including single guide RNAs or guide RNAs combined with tracrRNA or scoutRNA, or polynucleotides encoding thereof), can be used to achieve genome editing without the residues of CRISPR elements or selective 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 separation or semi-purified products of cell-free synthesis processes (e.g., in in vitro translation), or as separation or semi-purified products of cell-based synthesis processes (e.g., as in bacteria or other cell lysates). In certain embodiments, the soybean plants or soybean plant cells used in the systems, methods, and compositions provided herein can include transgenics expressing CRISPR endonucleases (e.g., Cas9, Cpf1 types, or other CRISPR endonucleases). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. The guide RNA (sgRNA or crRNA and tracrRNA) forms an RNA-guided endonuclease / guide RNA complex that can specifically bind to a sequence adjacent to the protospacer adjacent motif (PAM) sequence in the gDNA target site. The type of RNA-guided endonuclease typically informs the location of the appropriate PAM site and the design of the crRNA or sgRNA. G-rich PAM sites, such as 5'-NGG, are typically targeted for designing crRNA or sgRNA for use 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, wherein "V" is A, C or G) are typically targeted for designing crRNA or sgRNA for use with Cas12a proteins. In some cases, Cas12a can also recognize 5'-CTA PAM motifs.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 (wherein N is defined as any nucleotide). Cpf1 endonuclease and corresponding guide RNA and PAM site are disclosed in U.S. Patent Application Publication No. 2016 / 0208243A1, the DNA encoding Cpf1 endonuclease and guide RNA and PAM site disclosed therein are incorporated herein by reference.

[0057] In certain embodiments, mutant FT1a genes and plant cells, parts (including seeds), and plants comprising mutant FT1a genes are generated by using zinc finger nucleases or zinc finger nickases. Zinc finger nucleases are site-specific endonucleases that contain two protein domains: a DNA binding domain that contains multiple individual zinc finger repeats, each of which recognizes 9 to 18 base pairs, and a DNA cleavage domain that contains a nuclease domain (typically Fok1). The cleavage domains dimerize to cleave DNA; therefore, a pair of ZFNs is required to target a non-palindromic target polynucleotide. In certain embodiments, the methods for designing zinc finger nucleases and zinc finger nickases that have been described (Urnov et al. (2010) Nature Rev. Genet., 11:636–646; Mohanta et al. (2017) Genes 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 engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domain is derived from the DNA binding domain of a large class of eukaryotic transcription factors known as 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 DNA triplet. Many strategies can be used to design the binding specificity of zinc finger binding domains. One method, called "modular assembly," relies on the functional autonomy of a single zinc finger with DNA. In this method, a given sequence is targeted by identifying the zinc fingers of each triplet component in the sequence and connecting them into a multi-finger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to adapt to the ability of zinc fingers to contact adjacent fingers and nucleotide bases outside their target triplet. Typically, engineered zinc finger DNA binding domains have new 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, using a database of triplet (or quadruple) nucleotide sequences and single zinc finger amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with one or more amino acid sequences of a zinc finger that binds to a specific triplet or quadruple sequence. See, for example, U.S. Patents 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (eg, phage display and yeast two-hybrid systems) can be adapted for use with the methods described herein.In addition, enhancing the binding specificity of zinc finger binding domains has been described in U.S. Patent 6,794,136, which is incorporated herein by reference in its entirety. In addition, any suitable linker sequence can be used to link individual zinc finger domains together. Examples of linker sequences are well known, for example, see U.S. Patents 6,479,626; 6,903,185; and 7,153,949, which are incorporated herein by reference in their entirety. Nucleic acid cleavage domains are non-specific and are typically restriction endonucleases, such as Fok1. Such endonucleases must dimerize in order to cleave DNA. Therefore, cleavage by Fok1 as part of a ZFN requires two adjacent and independent binding events, which must occur in the correct orientation and at appropriate intervals to allow dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fok1 variants with enhanced activity have been described and may be suitable for use in the methods described herein; see, for example, Guo et al. (2010) J. Mol. Biol., 400:96-107.

[0058] In certain embodiments, mutant FT1a genes and plant cells, parts (including seeds), and plants comprising mutant FT1a genes are generated by using TAL-effector nucleases or TALENs. Transcription activator-like effectors (TALEs) are proteins secreted by certain Xanthomonas species that regulate gene expression in host plants and promote bacterial colonization and survival. TALEs act as transcription factors and regulate the expression of resistance genes in plants. Recent studies on TALEs have revealed a code that connects the repeat region of TALEs to their target DNA binding sites. TALEs contain highly conserved and repetitive regions consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other primarily at amino acid positions 12 and 13. A strong correlation has been found between the unique amino acid pairs at positions 12 and 13 and the corresponding nucleotides in the TALE binding site. The simple relationship between the amino acid sequence and the DNA recognition of the TALE binding domain allows the design of a DNA binding domain of any desired specificity. TALEs can be linked to non-specific DNA cleavage domains to create genome editing proteins, known as TAL effector nucleases or TALENs. As in the case of ZFNs, restriction endonucleases such as Fok1 can be conveniently used. Methods for using TALENs in plants have been described and can be adapted for use in the methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 108: 2623–2628; Mahfouz (2011) GM Crops [Transgenic Crops], 2: 99-103; and Mohanta et al. (2017) Genes [Gene] Vol. 8, 12: 399. TALE nickases have also been described and may 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 No. 20657 (2016)).

[0059] Various treatments can be used to deliver gene editing molecules and / or other molecules to plant cells. In certain embodiments, one or more treatments are used to deliver gene editing or other molecules (e.g., comprising polynucleotides, polypeptides, or combinations thereof) to plant cells, for example, through barriers such as cell walls, plasma membranes, nuclear envelopes, and / or other lipid bilayers. In certain embodiments, compositions comprising polynucleotides, polypeptides, or RNPs (ribonucleoproteins) of the molecules are delivered directly, for example, by direct contact of the compositions with plant cells. The aforementioned compositions can be provided in the form of liquids, solutions, suspensions, emulsions, reverse emulsions, colloids, dispersions, gels, liposomes, micelles, injectable materials, aerosols, solids, powders, microparticles, nanoparticles, or combinations thereof, and can be applied directly to plants, plant parts, plant cells, or plant explants (e.g., by abrasion or puncture or other means to destroy cell walls or cell membranes, by spraying or dipping or soaking or other means to directly contact, by microinjection). For example, plant cells or plant protoplasts are immersed in compositions containing liquid genome editing molecules. In certain embodiments, the composition is delivered using negative or positive pressure, for example, using vacuum infiltration or applying fluid mechanics or fluid pressure. In certain embodiments, the composition is introduced into plant cells or plant protoplasts, for example, by microinjection or by rupture or deformation of the cell wall or cell membrane, for example, by physical treatment, such as by applying negative or positive pressure, shear force, or with a chemical or physical delivery agent such as a surfactant, liposome, or nanoparticle; see, for example, U.S. Published Patent Application 2014 / 0287509, which is incorporated herein by reference in its entirety, for delivering materials to cells using microfluidic flow through cell deformation constrictions. Other techniques that can be used to deliver the composition to eukaryotic cells, plant cells, or plant protoplasts include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or disruption; enzymatic cell wall or cell membrane disruption or permeabilization; abrasion or mechanical scratching (e.g., abrasion with diamond or other abrasive particles or scratching with a file or sandpaper) or chemical scratching (e.g., treatment with acid or caustic); and electroporation.In certain embodiments, the composition is provided by bacterial-mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of 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 CBE, and / or a guide RNA; see, e.g., Broothaerts et al. (2005) Nature, 433:629–633). Any one or a combination of these techniques may alternatively be applied to a plant explant, plant part or tissue, or whole plant (or seed), and then optionally, plant cells are obtained or isolated therefrom; in certain embodiments, the composition is delivered in a separate step after the plant cells are isolated.

[0060] In certain embodiments, the method for generating a soybean plant cell, soybean plant part, or soybean plant comprises: (i) screening a soybean plant cell, part, or plant population for the presence of a loss-of-function allele in an 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 a loss-of-function allele in a soybean FT1a gene of SEQ ID NO:3, or an allelic variant thereof.

[0061] In certain embodiments, soybean plant cells, parts, or plant populations screened for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 are first pre-screened by screening for phenotypic characteristics of a loss-of-function mutation in the FT1a gene of SEQ ID NO: 3, or an allelic variant thereof. In certain embodiments, such phenotypic characteristics include increased pod count per plant, seed count per plant, and / or total harvested seed weight per plant compared to the pod count per plant, seed count per plant, and / or total harvested seed weight per plant of a wild-type control soybean plant lacking the loss-of-function allele. In certain embodiments, such phenotypic characteristics include increased pod count per plant, seed count per plant, and / or total seed weight harvested per plant compared to pod count per plant, seed count per plant, and / or total seed weight harvested per plant of a wild-type control soybean plant lacking the loss-of-function allele, wherein the screening plants and the control plants are grown under stress conditions (e.g., abiotic stress, including drought, cold, heat, or salt stress). In certain embodiments, plants exhibiting one or more of the foregoing phenotypic characteristics are then screened for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3, and soybean plants comprising the loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO: 3 are identified and / or selected.

[0062] In certain embodiments, the soybean plant cells, parts, or plant populations screened for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO:3 have been subjected to one or more mutagenesis treatments. The loss-of-function allele of the endogenous soybean FT1a gene can be generated by mutagenesis methods known in the art, such as chemical mutagenesis or radiation mutagenesis. Suitable chemical mutagens include ethyl methanesulfonate (EMS), sodium azide, methylnitrosourea (MNU), and diepoxybutane (DEB). Suitable radiation includes X-rays, fast neutron radiation, and gamma radiation.

[0063] 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 induced localized mutagenesis), or identified using EcoTILLING. TILLING is a common reverse genetics technique that uses a mutagenesis method to create a library of induced individuals, which are then subjected to high-throughput screening to find mutations. In addition to allowing for the efficient detection of induced mutations, high-throughput TILLING technology is ideal for detecting natural mutations. EcoTILLING is a method for finding individual natural mutations using TILLING technology (Barkley and Wang. Current genomics [contemporary genomics] Vol. 9, 4 (2008): 212-26. doi: 10.2174 / 138920208784533656). The identified mutation can then be introduced into the desired genetic background by hybridizing the mutant with a plant of the desired genetic background and performing an appropriate number of backcrosses to eliminate the initially undesirable parental background. A more detailed description of methods and compositions for TILLING is disclosed in U.S. Patent Application Publication 2004 / 0053236A1, which is incorporated herein by reference in its entirety, and can be adapted for use in the methods provided herein for identifying soybean plant cells, parts, or plants comprising a loss-of-function allele of an endogenous FT1a gene.

[0064] In certain embodiments, screening comprises the pod count of every strain plant in analyzing one or more candidate plants or in one or more candidate plant populations, the seed count of every strain plant, the total seed weight of every strain plant in the crops and / or the total seed weight of every unit area in the crops.In these embodiments, compare with the wild-type control soybean plant that lacks the loss of function allelic, the increase indication soybean plant cell, soybean plant part or soybean plant of the pod count of every strain plant, the seed count of every strain plant, the total seed weight of every strain plant in the crops and / or the total seed weight of every unit area in the crops comprise the loss of function allelic.In certain embodiments, screening is to carry out on the plant colony of growing under stress.The suitable example of stress condition comprises arid, salt, cold, hot, salt, shady and cool, nutrient deficiency, high planting density and has harmful organism or pathogen.

[0065] Methods for determining whether a soybean plant cell, plant part, or plant comprises a loss-of-function allele of an endogenous soybean FT1a gene are provided. Methods for determining the presence or absence of a loss-of-function allele can be used, for example, in breeding programs for identification, selection, introgression, and the like.

[0066] In certain embodiments, the method comprises analyzing a polynucleotide comprising a portion of SEQ ID NO: 3 or an allelic variant thereof from a plant cell, plant part, or plant, or analyzing an 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, insertions, deletions, and / or substitutions of one or more nucleotides in the polynucleotide or RNA indicate the presence of a loss-of-function allele. Detection of loss-of-function alleles 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 selectively hybridizing or producing 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 more consecutive nucleotides of SEQ ID NO: 3 or its allelic variants. In certain embodiments, the primer or probe may comprise or consist of about 10 to 50 consecutive nucleotides, about 10 to 40 consecutive nucleotides, about 10 to 30 consecutive nucleotides or about 15 to 30 consecutive nucleotides of SEQ ID NO: 3 or its allelic variants. In certain embodiments, the hybridization probe (e.g., a polynucleotide comprising at least about 15 to 30 base pairs of SEQ ID NO: 3 or its allelic variants) may comprise a detectable label (e.g., fluorescent, radioactive, epitope and chemiluminescent labels). In certain embodiments, the FT1a gene can be directly sequenced using nucleic acid sequencing technology (including whole genome sequencing).

[0067] In certain embodiments, the method comprises analyzing a polypeptide encoded by SEQ ID NO:3, a part thereof, or its allelic variant from a soybean plant cell, plant part, or plant. In certain embodiments, insertion, deletion, and / or substitution of one or more amino acid residues in the polypeptide or a change in the biological activity or biochemical activity of the polypeptide indicate the presence of a loss-of-function allele. Detection of a loss-of-function allele based on polypeptide can be determined by methods well known in the art (e.g., activity assays, Western blotting using antibodies that can specifically bind to the polypeptide, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence, etc.).

[0068] In some optional embodiments, the soybean plant cell disclosed herein is a non-renewable soybean plant cell. In some optional embodiments provided herein, the soybean plant cell, soybean plant propagule (for example, seed, seedling, ovule, embryo, pollen, root, stem, leaf, bud, explant or callus) and soybean plant provided herein are not just produced by biological process. In some optional embodiments provided herein, the method for producing the soybean plant cell, soybean plant propagule (for example, seed, seedling, ovule, embryo, pollen, root, stem, leaf, bud, explant or callus) and soybean plant provided herein is not just biological process.

[0069] The following numbered embodiments also form part of this disclosure:

[0070] 1. A soybean plant cell comprising a loss-of-function allele of an endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, optionally wherein the soybean plant cell is not solely produced by a biological process.

[0071] 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.

[0072] 3. The soybean plant cell of embodiment 1 or embodiment 2, wherein the loss-of-function allele comprises an amorphous allele of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0073] 4. The soybean plant cell of any one of embodiments 1-3, wherein said loss-of-function allele comprises a hypomorphic allele of said FT1a gene.

[0074] 5. The soybean plant cell of any one of embodiments 1-4, wherein the loss-of-function allele comprises a frameshift mutation or a nonsense mutation in the coding region of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0075] 6. The soybean plant cell of any one of embodiments 1-5, wherein the loss-of-function allele comprises a frameshift mutation or a nonsense mutation in the first exon of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof; optionally wherein the frameshift mutation or nonsense mutation occurs at nucleotides corresponding to nucleotides 340 to 343 of SEQ ID NO: 3 or an allelic variant thereof.

[0076] 7. The soybean plant cell of any one of embodiments 1-6, 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.

[0077] 8. The soybean plant cell of any one of embodiments 1-7, wherein the loss-of-function allele comprises an internal deletion comprising: (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; optionally in (i) or (ii), wherein the internal deletion preserves the reading frame of the encoded ft1a mutant protein comprising the loss-of-function allele relative to the amino acid residues of the ft1a mutant protein without the deletion.

[0078] 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 the protein of SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0079] 10. The soybean plant cell of any one of embodiments 1-9, wherein the soybean plant cell is not produced solely by essentially biological means.

[0080] 11. The soybean plant cell of any one of embodiments 1-10, wherein the soybean plant cell comprises elite soybean germplasm.

[0081] 12. The soybean plant cell of any one of embodiments 1-11, wherein the soybean plant cell further comprises: (i) one or more mutations in different soybean genes; and / or (ii) one or more transgenes, optionally wherein the transgene encodes a protein or RNA that confers herbicide tolerance or pest tolerance.

[0082] 13. A soybean plant part comprising the soybean plant cell of any of embodiments 1-12, optionally wherein the soybean plant part is not produced solely by a biological process.

[0083] 14. The soybean plant part of embodiment 13, wherein the part is a stem, root, leaf, flower, pod, or seed.

[0084] 15. The soybean plant part of embodiment 13 or embodiment 14, wherein the part is a seed.

[0085] 16. The soybean plant part of any one of embodiments 13-15, wherein the part is a seed, and wherein the total weight of seeds harvested per acre or hectare of a soybean plant population grown from the seeds is increased as compared to the total weight of seeds harvested per acre or hectare of a wild-type control soybean plant population lacking the loss-of-function allele.

[0086] 17. The soybean plant part of any one of embodiments 13-16, wherein the part is a seed, and wherein the average weight of the seeds is equivalent to the average weight of wild-type control seeds lacking the loss-of-function allele.

[0087] 18. The soybean plant part of any one of embodiments 13-17, wherein the part is a pod; optionally wherein the pod count of the soybean plant homozygous for the loss-of-function mutation and from which the pods are obtained is increased compared to the pod count of a wild-type control plant lacking the loss-of-function allele.

[0088] 19. A soybean seed lot comprising the seeds of any of embodiments 14-17, optionally wherein the soybean seed lot is produced solely by a biological process.

[0089] 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 wild-type control plants lacking the loss-of-function allele.

[0090] 21. The soybean seed lot of embodiment 19 or embodiment 20, wherein the average number of seeds per kilogram of seeds in the seed lot is equivalent to the average number of seeds per kilogram of seeds in a control seed lot obtained from a wild-type control soybean plant lacking the loss-of-function allele.

[0091] 22. A soybean plant comprising the soybean plant cell of any of embodiments 1-12, optionally wherein the soybean plant is produced solely by a biological process.

[0092] 23. The soybean plant of embodiment 22, wherein the pod count per plant, seed count per plant, and / or total seed weight harvested per plant is increased as compared to the pod count per plant, seed count per plant, and / or total seed weight harvested per plant of a wild-type control soybean plant lacking the loss-of-function allele.

[0093] 24. The soybean plant of embodiment 22 or embodiment 23, wherein the pod count per plant, seed count per plant, and / or total seed weight harvested per plant is increased when the plant is grown under stress, as compared to the pod count per plant, seed count per plant, and / or total seed weight harvested per plant of a wild-type control soybean plant lacking the loss-of-function allele grown under stress, optionally wherein the stress comprises an abiotic stress.

[0094] 25. The soybean plant of any one of embodiments 22-24, 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.

[0095] 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.

[0096] 27. The biological sample of embodiment 26, wherein the sample comprises seed meal or a tissue sample homogenate, optionally wherein the tissue sample comprises a sample of leaf, flower, pod, seed, stem, or root tissue.

[0097] 28. The biological sample of embodiment 26 or embodiment 27, wherein the loss-of-function allele comprises a frameshift mutation or a nonsense mutation in the coding region of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0098] 29. A biological sample as described in any of embodiments 26-28, wherein the loss-of-function allele comprises a frameshift mutation or a nonsense mutation in the first exon of the FT1a gene or its allelic variant of SEQ ID NO:3; optionally wherein the frameshift mutation or nonsense mutation occurs at nucleotides corresponding to nucleotides 340 to 343 of SEQ ID NO:3 or its allelic variant, optionally wherein the frameshift mutation comprises a frameshift mutation of SEQ ID NO:4 or SEQ ID NO:5, or wherein the ft1a gene comprising the frameshift mutation encodes a protein of SEQ ID NO:6.

[0099] 30. A biological sample as described in any of embodiments 26-29, wherein the loss-of-function allele comprises an internal deletion, the internal deletion comprising: (i) at least nucleotides corresponding to nucleotides 343 to 351 of SEQ ID NO:3 or its allelic variant; or (ii) at least nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO:1 or SEQ ID NO:2; optionally in (i) or (ii), wherein the internal deletion retains the reading frame of the ft1a mutant protein encoded by the loss-of-function allele relative to the amino acid residues of the ft1a mutant protein without the deletion.

[0100] 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 the protein of SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0101] 32. The biological sample of any one of embodiments 26-31, wherein the sample lacks nucleic acid comprising the wild-type allele of the soybean FT1a gene or an allelic variant thereof of SEQ ID NO: 3.

[0102] 33. A polynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 8, optionally wherein the polynucleotide is isolated.

[0103] 34. A polynucleotide encoding the polypeptide of SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 10, optionally wherein the polynucleotide is isolated.

[0104] 35. A method of producing a soybean seed lot, the method comprising: (i) growing a soybean plant population comprising the soybean plant of any of embodiments 22-25; and (ii) harvesting seeds from the soybean plant population of step (i) at maturity, thereby producing the soybean seed lot.

[0105] 36. The method of embodiment 35, wherein the total weight of seeds harvested per acre or hectare of the soybean plant population is increased compared to the total weight of seeds harvested per acre or hectare of a wild-type control soybean plant population lacking the loss-of-function allele.

[0106] 37. The method of embodiment 35 or embodiment 36, wherein the total weight of seeds harvested per acre or hectare of the soybean plant population grown under stress is increased compared to the total weight of seeds harvested per acre or hectare of a wild-type control soybean plant population lacking the loss-of-function allele grown under stress, optionally wherein the stress comprises an abiotic stress.

[0107] 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.

[0108] 39. The method of any one of embodiments 35-38, wherein at the time of harvest in step (i), 95% of the pods of at least 50%, 70%, 80%, or 90% of the plants in the soybean plant population have fully mature color.

[0109] 40. The method of any one of embodiments 35-39, wherein the soybean plant population is an all-season variety of the soybean maturity group region in which it is grown, and wherein the seeds are harvested during or after a full growing season of the all-season variety.

[0110] 41. A method of producing a soybean crop, the method comprising planting the seed lot of any of embodiments 19-21.

[0111] 42. The method of embodiment 41, further comprising harvesting seeds from soybean plants grown from the planted seeds.

[0112] 43. The method of embodiment 41 or embodiment 42, wherein the total number of seeds harvested per acre or hectare of the soybean crop is increased compared to the total number of seeds harvested per acre or hectare of a wild-type control soybean crop lacking the loss-of-function allele.

[0113] 44. The method of any one of embodiments 41-43, wherein the total number of seeds harvested per acre or hectare of the soybean crop grown under stress is increased compared to the total number of seeds harvested per acre or hectare of a wild-type control soybean crop lacking the loss-of-function allele grown under stress, optionally wherein the stress comprises an abiotic stress.

[0114] 45. The method of any one of embodiments 41-44, wherein the average weight of 1000 of said harvested seeds is equivalent to the average weight of 1000 of said harvested wild-type control soybean seeds lacking said loss-of-function allele.

[0115] 46. ​​The method of any one of embodiments 41 to 45, wherein the seed is planted on or within one week of the earliest initial planting date specified by the USDA Risk Management Agency for the maturity zone in which it is planted.

[0116] 47. The method of any one of embodiments 41 to 46, wherein at harvest, 95% of the pods of at least 50%, 70%, 80%, or 90% of the plants in the soybean crop have fully mature color.

[0117] 48. The method of any one of embodiments 41 to 47, wherein the soybean crop is an all-season variety of the soybean maturity group region in which it is grown, and wherein the seeds are harvested during or after a full growing season of the all-season variety.

[0118] 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.

[0119] 50. A method for generating a soybean plant cell as described in any of embodiments 1-12, a soybean plant part as described in any of embodiments 13-18, or a soybean plant as described in any of embodiments 22-25, comprising introducing a loss-of-function allele into the endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

[0120] 51. A method as described in embodiment 50, wherein the loss-of-function allele is introduced by: (i) directing both of the following: (a) a guide RNA (gRNA) molecule comprising a spacer RNA molecule that targets the endogenous FT1a gene or its allelic variant of SEQ ID NO:3, exon 1 of the FT1a gene or its allelic variant of SEQ ID NO:3, 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 a loss-of-function allele of the endogenous soybean FT1a gene or its allelic variant of SEQ ID NO:3.

[0121] 52. The method of embodiment 51, wherein directing the gRNA and the 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.

[0122] 53. The method of embodiment 51 or embodiment 52, wherein the soybean plant cell, soybean plant part, or soybean plant comprising a loss-of-function allele is identified by: (i) analyzing one or more candidate plant cells, plant parts, or plants for a polynucleotide comprising a portion of SEQ ID NO: 3 or an allelic variant thereof; (ii) analyzing one or more candidate plant cells, plant parts, or plants for a polypeptide encoded by a portion of SEQ ID NO: 3; and / or (iii) analyzing one or more candidate plants for pod count per plant, seed count per plant, and / or total seed weight harvested per plant, wherein an increase in pod count per plant, seed count per plant, and / or total seed weight harvested per plant compared to a wild-type control soybean plant lacking the loss-of-function allele indicates that the soybean plant cell, soybean plant part, or soybean plant comprises the loss-of-function allele.

[0123] 54. The method of embodiment 45, wherein the loss-of-function allele is introduced by crossing a soybean plant comprising the loss-of-function allele with a second soybean plant and harvesting F1 seeds comprising the loss-of-function allele, thereby producing progeny soybean seeds comprising the loss-of-function allele.

[0124] 55. A method for generating a soybean plant cell as described in any of Examples 1-12, a soybean plant part as described in any of Examples 13-18, or a soybean plant as described in any of Examples 22-25, the method comprising: (i) screening soybean plant cells, parts, or plant populations for the presence of a loss-of-function allele in the endogenous soybean FT1a gene of SEQ ID NO:3 or its allelic variant; and (ii) isolating soybean plant cells, soybean plant parts, or soybean plants that contain a loss-of-function allele of the soybean FT1a gene of SEQ ID NO:3 or its allelic variant.

[0125] 56. The method of embodiment 55, wherein said soybean plant cells, parts or plant populations have been subjected to one or more mutagenesis treatments, optionally wherein said mutagenesis procedure comprises chemical mutagenesis.

[0126] 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 insertions, deletions, and / or substitutions of one or more nucleotides in the polynucleotide or RNA indicate 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 insertions, deletions, and / or substitutions of one or more amino acid residues of the polypeptide, or a change in the biological or biochemical activity of the polypeptide, indicate the presence of the loss-of-function allele.

[0127] 58. The method of any one of embodiments 55-57, wherein the screening further comprises analyzing one or more candidate plants for pod count per plant, seed count per plant, and / or total weight of harvested seeds per plant, wherein an increase in pod count per plant, seed count per plant, and / or total weight of harvested seeds per plant compared to a wild-type control soybean plant lacking the loss-of-function allele indicates that the soybean plant cell, soybean plant part, or soybean plant comprises the loss-of-function allele.

[0128] 59. The method of any one of embodiments 55-58, wherein the screening is performed on a population of plants grown under stress.

[0129] 60. The method of embodiment 59, wherein the stress comprises an abiotic stress.

[0130] 61. A method for determining whether a soybean plant cell, plant part or 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 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 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 the soybean plant cell, plant part or plant, wherein an insertion, deletion and / or substitution of one or more amino acid residues of the polypeptide or a change in the biological or biochemical activity of the polypeptide indicates the presence of the loss-of-function allele.

[0131] 62. The method of embodiment 61, wherein the method further comprises analyzing one or more of the soybean plants for pod count per plant, seed count per plant, and / or total weight of harvested seeds per plant, wherein an increase in pod count per plant, seed count per plant, and / or total weight of harvested seeds per plant compared to a wild-type control soybean plant lacking the loss-of-function allele indicates that the soybean plant cell, soybean plant part, or soybean plant comprises the loss-of-function allele.

[0132] 63. The method of embodiment 61 or 62, wherein the analysis is performed on a population of plants grown under stress.

[0133] 64. The method of embodiment 63, wherein the stress comprises an abiotic stress.

[0134] 65. The method of embodiment 64, wherein the abiotic stress comprises drought stress. Examples Example 1. Generation of soybeans with a mutated FT1a gene

[0135] Vectors were created to transform soybean plants and 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 soybean FT1a gene.

[0136] The recovered genotypes included -10:13D and -4:9D. The -10:13D genotype had a 13 bp deletion (SEQ ID NO:5), which resulted in a frameshift mutation and introduced a premature stop codon. The -10:13D genotype was predicted to encode a truncated 84 amino acid polypeptide (SEQ ID NO:6). The -4:9D genotype had a 9 bp deletion (SEQ ID NO:8), which was predicted to encode a polypeptide with an internal deletion of 3 amino acids (SEQ ID NOs:9 and 10). Example 2. Performance of soybeans with a mutated FT1a gene

[0137] Seeds of the homozygous -4:9D mutant line SENF2228 and the homozygous -10:13D mutant line SENF2229 were added and planted in rows in the field alongside checkpoint and unrelated edited lines. Total yield data from the field trials showed a trend toward higher yield per plot for SENF2228 and SENF2229 compared to adjacent wild-type, null segregants, and some unrelated mutants.

[0138] Several field-grown plants were also phenotyped in more detail. When compared to controls grown at comparable densities, SENF2228 and SENF2229 showed a consistent trend toward higher yield. While individual seed weights were comparable to those of null segregants and wild-type controls, this trend was evident in yield components such as pod counts, seed counts, and total seed weight. Example 3. Performance of soybeans with a mutated FT1a gene in field trials

[0139] Seeds of the homozygous -4:9D mutant line SENF2228 and the homozygous -10:13D mutant line SENF2229 were added and planted in rows in the field along with the NINF1170 checker. The results of the field test are shown in Table 1. The phenotypic characteristics of SENF2228, SENF2229, and the comparison variety NINF1170 are shown in Table 1. Table 1 Phenotype SENF2228(-4:9D) SENF2229(-10:13D) NINF1170 Seed yield 1 53.0 52.9 49.6 Plant 2 per acre 123554 123568 105746 Plant height 3 30.9 30.4 31.0 Flowering Time 4 44 43 41 Pod formation time 61 60 59 Seed formation time 6 71 69 69 Fully mature time 7 117 115 117 1 "Seed yield" is the yield of grain (in bushels per acre) adjusted to 12% moisture content at harvest. 2 "Plants per acre" refers to the estimated number of soybean plants per acre. 3 “Plant height” is the height of the plant measured from soil level to the top node of the main stem at plant maturity and is measured in inches. 4 "Flowering time" is the number of days from planting until 50% of the plants have at least one flower open at any node on the main stem. 5 "Time to pod formation" is the number of days from planting until 50% of the plants have 3 / 16 inch long pods at one of the four uppermost nodes of the main stem with fully developed leaves. 6 "Time to seed formation" is the number of days from planting until 50% of the plants produce 1 / 8 inch long seeds in pods located at one of the four uppermost nodes of the main stem with fully developed leaves. 7 "Full maturity time" is the number of days from planting until 95% of the pods on 50% of the plants reach fully mature color.

[0140] All cited patents and patent disclosures mentioned in this application are incorporated herein by reference in their entirety. All materials and methods disclosed and claimed herein can be prepared and used as indicated by the above-mentioned disclosure and by example, without the need for excessive experimentation. Although the materials and methods disclosed herein have been described in terms of embodiments and illustrative examples, it will be apparent to those skilled in the art that, without departing from the concept, spirit and scope of the disclosure, substitutions and variations can be made to materials and methods as described herein. It will be apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope and concept of the disclosure as enumerated herein in the embodiments and description 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. 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. The soybean plant cell of claim 1, wherein the loss-of-function allele comprises an amorphous allele of the FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

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

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

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

7. The soybean plant cell of claim 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 a protein of SEQ ID NO:

6.

8. The soybean plant cell of claim 1 , wherein the loss-of-function allele comprises an internal deletion comprising: (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; optionally in (i) or (ii), wherein the internal deletion preserves the reading frame of the encoded ft1a mutant protein comprising the loss-of-function allele relative to the amino acid residues of the ft1a mutant protein without the deletion.

9. The soybean plant cell of claim 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 the protein of SEQ ID NO: 9 and / or SEQ ID NO:

10.

10. The soybean plant cell of claim 1, wherein the soybean plant cell is not produced solely by essentially biological means.

11. The soybean plant cell of claim 1 , wherein the soybean plant cell comprises elite soybean germplasm.

12. The soybean plant cell of claim 1, wherein the soybean plant cell further comprises: (i) one or more mutations in different soybean genes; and / or (ii) one or more transgenes, optionally wherein the transgene encodes a protein or RNA that confers herbicide tolerance or pest tolerance.

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

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

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

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

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

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

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

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 wild-type control plants lacking the loss-of-function allele.

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

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

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

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

25. The soybean plant of claim 22, 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.

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

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

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

6.

30. A biological sample as described in claim 26, wherein the loss-of-function allele comprises an internal deletion, the internal deletion comprising: (i) at least nucleotides corresponding to nucleotides 343 to 351 of SEQ ID NO:3 or its allelic variant; or (ii) at least nucleotides encoding amino acids corresponding to N39 to C41 of SEQ ID NO:1 or SEQ ID NO:2; optionally in (i) or (ii), wherein the internal deletion retains the reading frame of the encoded ft1a mutant protein comprising the loss-of-function allele relative to the amino acid residues of the ft1a mutant protein without the deletion.

31. The biological sample of claim 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 the protein of SEQ ID NO: 9 and / or SEQ ID NO:

10.

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

3.

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

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

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

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

38. 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. The method of claim 35, wherein at the time of harvest in step (i), 95% of the pods of at least 50%, 70%, 80%, or 90% of the plants in the soybean plant population have fully mature color.

40. The method of claim 35, wherein the soybean plant population is an all-season variety of the soybean maturity group region in which it is grown, and wherein the seeds are harvested during or after a full growing season of the all-season variety.

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

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

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

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

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

46. ​​The method of claim 41, wherein the seed is planted on or within one week of the earliest initial planting date specified by the USDA Risk Management Agency for the maturity zone in which the seed is to be planted.

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

48. The method of claim 41, wherein the soybean crop is an all-season variety of the soybean maturity group region in which it is grown, and wherein the seeds are harvested during or after a full growing season of the all-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 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 method of claim 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, 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 a target soybean plant cell; and (ii) isolating a soybean plant cell, soybean plant part, or soybean plant comprising a loss-of-function allele of said endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof.

52. The method of claim 50, wherein directing the gRNA and the 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.

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

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

55. 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, the method comprising: (i) screening soybean plant cells, parts or plant populations 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; and (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. The method of claim 55, wherein the soybean plant cells, parts or plant populations have been subjected to one or more mutagenesis treatments, optionally wherein the mutagenesis procedure comprises chemical mutagenesis.

57. 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 insertions, deletions and / or substitutions of one or more nucleotides in the polynucleotide or RNA are indicative of 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 insertions, deletions and / or substitutions of one or more amino acid residues in the polypeptide or a change in the biological or biochemical activity of the polypeptide are indicative of the presence of the loss-of-function allele.

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

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

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

61. A method for determining whether a soybean plant cell, plant part, or plant comprises a loss-of-function allele of an endogenous soybean FT1a gene of SEQ ID NO: 3 or an allelic variant thereof, the method comprising: (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 said plant cell, plant part or plant, wherein 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 a polypeptide encoded by SEQ ID NO: 3, a portion thereof or an allelic variant thereof from said soybean plant cell, plant part or plant, wherein 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. The method of claim 61, wherein the method further comprises analyzing one or more of the soybean plants for pod count per plant, seed count per plant, and / or total weight of harvested seeds per plant, wherein an increase in pod count per plant, seed count per plant, and / or total weight of harvested seeds per plant compared to a wild-type control soybean plant lacking the loss-of-function allele indicates that the soybean plant cell, soybean plant part, or soybean plant comprises the loss-of-function allele.

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

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

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

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