Compositions and methods for altering plant finitude

Abstract

Compositions and methods are provided for altering TFL1 levels in soybean plants. Methods and compositions are also provided for altering expression of genes associated with timing of terminal differentiation of the stem tip through suppression, mutagenesis and / or editing of the TFL1 gene. Further provided are modified plant cells and plants having suppressing elements or mutations that reduce expression or activity of the TFL1 gene, including reduced TFL1 levels and improved characteristics such as reduced plant height and increased lodging resistance.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 278,903, filed November 12, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Inclusion of sequence listing The sequence listing contained in the file name "MONS522US_ST26.xml", which measures 265 kilobytes on a Microsoft Windows operating system and was created on November 7, 2022, is filed electronically herewith and is incorporated herein by reference.

[0003] The present disclosure relates to the field of agricultural biotechnology, and more specifically, to methods and compositions for genome editing in plants. [Background technology]

[0004] Precise genome editing technology is a powerful tool for manipulating gene expression and function, and has the potential to improve important agronomic traits. There is a continuing need in the art to develop novel compositions and methods for effectively and efficiently editing the plant genomes of various crop plants to alter finiteness, increase yield, reduce lodging, and obtain other beneficial results. Summary of the Invention

[0005] Provided herein is a modified plant, plant seed, plant part, or plant cell comprising a modification that reduces the expression or activity of TFL1 or a homologue thereof compared to the expression or activity of TFL1 or a homologue thereof in an otherwise identical plant, plant seed, plant part, or plant cell lacking the modification. In some embodiments, the modification is present in at least one allele of the endogenous TFL1 gene or a homologue thereof. The TFL1 gene can be a TFL1b gene. In certain embodiments, the TFL1 gene or a homologue thereof encodes a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:2. In other embodiments, the modification is in a non-coding region of the TFL1 gene or a homologue thereof, including, but not limited to, a promoter, an intron, a 5'-untranslated region, a 3'-untranslated region, and any combination thereof. The plants, plant seeds, plant parts, or plant cells provided herein can be, for example, from legumes, cotton plants, canola plants, corn plants, sorghum plants, rice plants, wheat plants, barley plants, tomato plants, or pepper plants, or other crops, ornamental plants, or other types of plant species. In further embodiments, the legume is a soybean plant, a kidney bean plant, a pea plant, a chickpea plant, an alfalfa plant, a peanut plant, a carob plant, a lentil plant, or a liquorice plant. In specific embodiments, the legume is a soybean plant. In some embodiments, the plant, plant seed, plant part, or plant cell is heterozygous for the modification, and in other embodiments, the plant, plant seed, plant part, or plant cell is homozygous for the modification. In certain embodiments, the plant, plant seed, plant part or plant cell is defined as comprising a first modification in a first allele of the TFL1 gene and a second modification in a second allele of the TFL1 gene, wherein the first modification and the second modification are different from each other.

[0006] The modified plants, plant seeds, plant parts, or plant cells provided herein may, in certain embodiments, comprise a modification that reduces the expression or activity of TFL1 or a homolog thereof, the modification comprising a deletion, an insertion, a substitution, an inversion, or any combination thereof. In some embodiments, for example, the modification is located about 100, 125, 150, 175, 200, 225, 250, 275, or 300 nucleotides or more from the 3' end of a sequence selected from the group consisting of SEQ ID NOs: 4, 67-77, 79, and 81. In other embodiments, the modification in the promoter of at least one allele of the TFL1b gene is comprised within the genomic region between nucleotides 1237 and 1570 of the reference sequence of SEQ ID NO: 4. In some embodiments, the modification comprises a deletion of at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 125, or at least about 150 contiguous nucleotides. The plant, plant seed, plant part, or plant cell can also comprise, for example, a modification in at least one allele of the promoter of the TFL1b gene, the modification comprising a deletion of 30 base pairs from nucleotide 1539 to nucleotide 1568 compared to the reference sequence of SEQ ID NO:4; a deletion of 388 base pairs from nucleotide 1217 to nucleotide 1604 compared to the reference sequence of SEQ ID NO:4; a deletion of 112 base pairs from nucleotide 1518 to nucleotide 1629 compared to the reference sequence of SEQ ID NO:4; a 272 base pair deletion from nucleotide 951 to nucleotide 1222 compared to the reference sequence SEQ ID NO:4; a 44 base pair deletion from nucleotide 1364 to nucleotide 1407 compared to the reference sequence SEQ ID NO:4; a 150 base pair deletion from nucleotide 1367 to nucleotide 1516 compared to the reference sequence SEQ ID NO:4; a 1053 base pair deletion from nucleotide 754 to nucleotide 1806 compared to the reference sequence SEQ ID NO:4; a 104 base pair deletion from nucleotide 1216 to nucleotide 1319 compared to the reference sequence SEQ ID NO:4;a deletion of 807 base pairs from nucleotide 1159 to nucleotide 1965 compared to the reference sequence of SEQ ID NO:4; a deletion of 455 base pairs from nucleotide 760 to nucleotide 1214 compared to the reference sequence of SEQ ID NO:4; a deletion of 90 base pairs from nucleotide 939 to nucleotide 1028 compared to the reference sequence of SEQ ID NO:4; an inversion of 37 base pairs in which the sequence from nucleotide 1029 to nucleotide 106 of SEQ ID NO:4 has been deleted, inverted and reinserted in the same position; a 599 base pair deletion from nucleotide 952 to nucleotide 1552 compared to the reference sequence of SEQ ID NO:4; a 601 base pair deletion from nucleotide 952 to nucleotide 1552 compared to the reference sequence of SEQ ID NO:4; a 132 base pair deletion from nucleotide 1677 to nucleotide 1808 compared to the reference sequence of SEQ ID NO:4; a 35 base pair deletion from nucleotide 1524 to nucleotide 1558 compared to the reference sequence of SEQ ID NO:4; a 930 base pair deletion from nucleotide 476 to nucleotide 1405 compared to the reference sequence of SEQ ID NO:4; a 1 base pair deletion from nucleotide 1365 to nucleotide 1559 compared to the reference sequence of SEQ ID NO:4 a deletion of 95 base pairs; a deletion of 633 base pairs from nucleotide 928 to nucleotide 1560 compared to the reference sequence of SEQ ID NO:4; a deletion of 1221 base pairs from nucleotide 593 to nucleotide 1813 compared to the reference sequence of SEQ ID NO:4; a deletion of 5 base pairs from nucleotide 1552 to nucleotide 1556 compared to the reference sequence of SEQ ID NO:4; a deletion of 29 base pairs from nucleotide 1537 to nucleotide 1565 compared to the reference sequence of SEQ ID NO:4; a deletion of 38 base pairs from nucleotide 1209 to nucleotide 1246 compared to the reference sequence of SEQ ID NO:4 a 49 base pair deletion from nucleotide 1552 to nucleotide 1600 compared to the reference sequence of SEQ ID NO:4; a 49 base pair deletion from nucleotide 1553 to nucleotide 1601 compared to the reference sequence of SEQ ID NO:4; an 11 base pair deletion from nucleotide 1368 to nucleotide 1378 compared to the reference sequence of SEQ ID NO:4; a 28 base pair deletion from nucleotide 1368 to nucleotide 1395 compared to the reference sequence of SEQ ID NO:4; a 41 base pair deletion from nucleotide 1344 to nucleotide 1384 compared to the reference sequence of SEQ ID NO:4;a 235 base pair deletion from nucleotide 1219 to nucleotide 1453 compared to the reference sequence of SEQ ID NO:4; a 13 base pair deletion from nucleotide 1215 to nucleotide 1227 compared to the reference sequence of SEQ ID NO:4; a 7 base pair deletion from nucleotide 1370 to nucleotide 1376 compared to the reference sequence of SEQ ID NO:4; a 337 base pair deletion from nucleotide 1220 to nucleotide 1556 compared to the reference sequence of SEQ ID NO:4; a 161 base pair deletion from nucleotide 1216 to nucleotide 1376 compared to the reference sequence of SEQ ID NO:4 a 17 base pair deletion from nucleotide 1368 to nucleotide 1384 compared to the reference sequence of SEQ ID NO:4; a 283 base pair deletion from nucleotide 1366 to nucleotide 1648 compared to the reference sequence of SEQ ID NO:4; a 120 base pair deletion from nucleotide 1370 to nucleotide 1489 compared to the reference sequence of SEQ ID NO:4; a 75 base pair deletion from nucleotide 1541 to nucleotide 1615 compared to the reference sequence of SEQ ID NO:4; a 9 base pair deletion from nucleotide 1367 to nucleotide 1375 compared to the reference sequence of SEQ ID NO:4; a 51 base pair deletion from nucleotide 551 to nucleotide 1601; a 7 base pair deletion from nucleotide 1371 to nucleotide 1377 compared to the reference sequence of SEQ ID NO:4; a 186 base pair deletion from nucleotide 1368 to nucleotide 1553 compared to the reference sequence of SEQ ID NO:4; a 14 base pair deletion from nucleotide 1365 to nucleotide 1378 compared to the reference sequence of SEQ ID NO:4; a 16 base pair deletion from nucleotide 1365 to nucleotide 1380 compared to the reference sequence of SEQ ID NO:4; a 43 base pair deletion from nucleotide 1367 to nucleotide 1376 compared to the reference sequence of SEQ ID NO:4; a 10 base pair deletion from nucleotide 1552 to nucleotide 1682 compared to the reference sequence of SEQ ID NO:4; a 438 base pair deletion from nucleotide 954 to nucleotide 1391 compared to the reference sequence of SEQ ID NO:4; a 26 base pair deletion from nucleotide 1535 to nucleotide 1560 compared to the reference sequence of SEQ ID NO:4; a 111 base pair deletion from nucleotide 1534 to nucleotide 1644 compared to the reference sequence of SEQ ID NO:4;a 663 base pair deletion from nucleotide 937 to nucleotide 1599 compared to the reference sequence of SEQ ID NO:4; a 98 base pair deletion from nucleotide 1551 to nucleotide 1648 compared to the reference sequence of SEQ ID NO:4; a 34 base pair deletion from nucleotide 1526 to nucleotide 1559 compared to the reference sequence of SEQ ID NO:4; a 79 base pair deletion from nucleotide 1528 to nucleotide 1606 compared to the reference sequence of SEQ ID NO:4; a 61 base pair deletion from nucleotide 1542 to nucleotide 1602 compared to the reference sequence of SEQ ID NO:4 a deletion of 381 base pairs from nucleotide 1214 to nucleotide 1594 compared to the reference sequence of SEQ ID NO:4; a deletion of 187 base pairs from nucleotide 1368 to nucleotide 1554 compared to the reference sequence of SEQ ID NO:4; a deletion of 109 base pairs from nucleotide 1369 to nucleotide 1477 compared to the reference sequence of SEQ ID NO:4; a deletion of 5 base pairs from nucleotide 1550 to nucleotide 1554 compared to the reference sequence of SEQ ID NO:4; a deletion of 1267 base pairs from nucleotide 734 to nucleotide 2000 compared to the reference sequence of SEQ ID NO:4; a 190 base pair deletion from nucleotide 71 to nucleotide 1560; a 36 base pair deletion from nucleotide 1537 to nucleotide 1572 compared to the reference sequence of SEQ ID NO:4; a 46 base pair deletion from nucleotide 1541 to nucleotide 1586 compared to the reference sequence of SEQ ID NO:4; a 5 base pair deletion from nucleotide 1552 to nucleotide 1556 compared to the reference sequence of SEQ ID NO:4; a 955 base pair deletion from nucleotide 669 to nucleotide 1623 compared to the reference sequence of SEQ ID NO:4; a 38 base pair deletion from nucleotide 1369 to nucleotide 1477 compared to the reference sequence of SEQ ID NO:4; a 109 base pair deletion from nucleotide 1540 to nucleotide 1554 compared to the reference sequence of SEQ ID NO:4; a 15 base pair deletion from nucleotide 1217 to nucleotide 1674 compared to the reference sequence of SEQ ID NO:4; a 81 base pair deletion from nucleotide 1546 to nucleotide 1626 compared to the reference sequence of SEQ ID NO:4; a 89 base pair deletion from nucleotide 1547 to nucleotide 1635 compared to the reference sequence of SEQ ID NO:4;and any combination thereof. In some embodiments, the plant, plant seed, plant part, or plant cell comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128. The plants, plant seeds, plant parts, or plant cells provided herein can also include a chromosomal sequence of a TFL1b gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:4, e.g., in regions other than deletions, insertions, substitutions, or inversions;

[0007] Also provided herein are modified plants or seeds, plant parts, or cells thereof, comprising a modification that changes the finite phenotype of the plant compared to an otherwise identical plant lacking the modification. In certain embodiments, the modification increases the finiteness of the plant compared to the finiteness of an otherwise identical plant lacking the modification. In some embodiments, the modified plant reaches a terminal flowering date earlier, exhibits a reduced lodging rate, exhibits substantially the same or increased yield, or exhibits a lower susceptibility to fungal disease, or any possible combination thereof, compared to an otherwise identical plant lacking the modification.

[0008] In certain embodiments, a polynucleotide is provided comprising a sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128. Also provided is a guide RNA comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29.

[0009] Further disclosed herein is a method for producing a plant comprising a modified TFL1 gene, the method comprising: a) introducing a modification into at least one target site in an endogenous TFL1 gene or a homologue thereof in a plant cell; b) identifying and selecting one or more plant cells of step (a) comprising the modification in said TFL1 gene or a homologue thereof; and c) regenerating at least one plant from at least one or more cells selected in step (b). In some embodiments, the target site is located in a non-coding region of the endogenous TFL1 gene or a homologue thereof. In other embodiments, the non-coding region is selected from the group consisting of a promoter, an intron, a 5'-untranslated region, a 3'-untranslated region, and any combination thereof. In a further embodiment, the non-coding region is a promoter. In yet another embodiment, the modification is facilitated by the presence of at least one site-specific genome modification enzyme in said plant cell. Non-limiting examples of such enzymes include RNA-guided nucleases, zinc finger nucleases, meganucleases, TALE nucleases, recombinases, transposases, and any combination thereof. Examples of RNA-guided nucleases include Cas nucleases, Cpf1 nucleases, or any variants thereof. Some site-specific genome modification enzymes that can be used according to the present disclosure generate at least one strand break at the target site. The methods disclosed herein can be used to generate any modification according to the present disclosure, including, for example, substitutions, insertions, inversions, deletions, duplications, and combinations thereof. In some embodiments the modification is a deletion, and the deletion comprises a region of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 contiguous nucleotides.

[0010] The methods provided herein may be used, for example, to produce plants with desired phenotypes. Non-limiting examples of such phenotypes include an earlier terminal flowering date, a lower lodging rate, substantially the same or an increased yield, a lower susceptibility to fungal disease, and an increased finitude, or any possible combination thereof. In specific embodiments, such phenotypes may be defined as present in a plant when compared to an otherwise identical plant lacking the phenotypic modification according to the present disclosure.

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0012] [Figure 1] The relative positions of the gRNAs on the approximately 2 kb promoter region upstream of the transcription start site (tis) of the GmTFL1b gene are shown. [Diagram 2] Unique, non-overlapping homozygous deletions are indicated. An asterisk (*) indicates small deletions of less than 20 base pairs. The allele names omit the prefix "AL." [Diagram 3] Figure 1 shows the relative expression levels of the GmTFL1b gene in shoot tips and leaves of plants containing various Dt1 alleles generated by promoter editing. Expression of GmTFL1b in leaf tissue was negligible in all samples tested. [Figure 4] Images showing the terminal raceme phenotype are shown. The photograph was taken within 2 days of terminal flowering of wild type plants. [Diagram 5]Pairwise comparison of the soybean TFL1b polypeptide sequence with the polypeptide sequences of putative homologues from related plant species identified by BLAST searches is shown. The decimal point numbers in the upper right triangle of the table indicate the percentage of identical residues at the aligned positions relative to the aligned positions that overlap between the two sequences. The integers in the lower left triangle indicate the number of residue variations between the two sequences at the aligned positions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] A brief description of the sequence SEQ ID NO:1 is the polynucleotide coding sequence of the Glycine max TFL1b (GmTFL1b) gene.

[0014] SEQ ID NO:2 is the amino acid sequence of the TFL1b protein (encoded by SEQ ID NO:1).

[0015] SEQ ID NO:3 is the polynucleotide sequence of the GmTFL1b gene, including the 5' and 3' untranslated regions (UTRs) and introns.

[0016] SEQ ID NO:4 is the polynucleotide sequence of the 2 kb promoter region upstream of the transcription start site (tis) of the GmTFL1b gene, which immediately precedes SEQ ID NO:3 in wild-type soybean plants.

[0017] SEQ ID NO:5 is the polynucleotide sequence of a consensus scaffold that matches the Cpf1 gene.

[0018] SEQ ID NO:6 is the polynucleotide sequence of the Dahlia mosaic virus FLT promoter.

[0019] SEQ ID NO:7 is a polynucleotide sequence encoding the Lachnospiraceae Cpf1 RNA-guided endonuclease enzyme codon-optimized for rice.

[0020] SEQ ID NO:8 is the polynucleotide sequence of a nuclear localization signal from Solanum lycopersicum.

[0021] SEQ ID NO:9 is the polynucleotide sequence of the Medicago truncatula ubiquitin promoter.

[0022] SEQ ID NO:10 is a polynucleotide sequence encoding the Lachnospiraceae Cpf1 RNA-guided endonuclease enzyme that has been codon-optimized for maize.

[0023] SEQ ID NO:11 is the polynucleotide sequence of the soybean RNA polymerase III (Pol3) promoter.

[0024] SEQ ID NO:12 is the polynucleotide sequence of the soybean 7SL_CR10 promoter.

[0025] SEQ ID NOs: 13 to 29 are polynucleotide sequences of spacer sequences in a guide RNA (gRNA) used to edit the promoter region of the GmTFL1b gene.

[0026] SEQ ID NOs:30 to 46 and 82 to 128 are polynucleotide sequences of alleles of the GmTFL1b promoter having various deletions (and, in the case of SEQ ID NO:40, an inversion) compared to SEQ ID NO:4.

[0027] SEQ ID NO: 47 is the polynucleotide sequence of the 5'UTR of the GmTFL1b gene.

[0028] SEQ ID NOs: 48, 49 and 50 are the polynucleotide sequences of the first, second and third introns of the GmTFL1b gene, respectively.

[0029] SEQ ID NO:51 is the polynucleotide sequence of the 3'UTR of the GmTFL1b gene.

[0030] SEQ ID NOs:52-66, 78, and 80 are the amino acid sequences of TFL1b protein homologues found in Medicago truncatula (SEQ ID NO:52), Cajanus cajan (SEQ ID NO:53), Pisum sativum (SEQ ID NO:54), Arabidopsis thaliana (SEQ ID NO:55), Brassica napus (SEQ ID NO:56), Gossypium hirsutum (SEQ ID NO:57), Capsicum annuum (SEQ ID NO:58), Nicotiana tabacum (SEQ ID NO:59), Solanum tuberosum (SEQ ID NO:60), Solanum lycopersicum (SEQ ID NO:61), Zea Mays (SEQ ID NO:62), Oryza sativa (SEQ ID NO:63), Hordeum vulgare (SEQ ID NO:64), Triticum aestivum (SEQ ID NO:65), Sorghum bicolor (SEQ ID NO:66), Arachis Hypogaea (SEQ ID NO:78), and Cicer arietinum (SEQ ID NO:80), respectively.

[0031] SEQ ID NOs:67-77, 79, and 81 are polynucleotide sequences of approximately 2 kb of the promoter region upstream of the transcription start site (tis) of the TFL1b gene homologue found in Zea mays (SEQ ID NO:67), Sorghum bicolor (SEQ ID NO:68), Oryza sativa (SEQ ID NO:69), Triticum aestivum (SEQ ID NO:70), Hordeum vulgare (SEQ ID NO:71), Solanum lycopersicum (SEQ ID NO:72), Gossypium hirsutum (SEQ ID NO:73), Capsicum annuum (SEQ ID NO:74), Brassica napus (SEQ ID NO:75), Arabidopsis thaliana (SEQ ID NO:76), Medicago truncatula (SEQ ID NO:77), Arachis hypogaea (SEQ ID NO:79), and Cicer arietinum (SEQ ID NO:81), respectively.

[0032] Stem growth habit is an important agronomic trait that directly affects plant characteristics such as plant height, flowering time and duration, node production, and root architecture in soybean (Glycine max). Plant height is a particularly important agronomic trait in soybean and other crops because it can directly affect yield potential and lodging resistance. Plant height is influenced by the timing of the vegetative to reproductive phase transition at the shoot apical meristem. In soybean, growth habit at both the shoot apex and branch tips and the timing of terminal differentiation of the stem tip are controlled by the Dt1 (indeterminate growth 1) locus. The Dt1 locus also affects other related traits such as branch density, stem pod density, stem node number, number of three seeds per pod, and total seed number. Wild-type expression of Dt1 specifies indeterminate growth and has incomplete dominance over the dt1 (determinate growth 1) allele that causes determinate growth. Expression of the gene TERMINAL FLOWER 1b (GmTFL1b) at the Dt1 locus in the shoot tip protects the apical meristem from terminal differentiation, which is induced by FT2a, the soybean FLOWERING LOCUS T orthologue.

[0033] For example, the majority of commercially grown soybean varieties are classified as having one of two stem growth habits: determinate or determinate. Determinate and determinate soybean plants are similar in their development during the vegetative growth phase, but there are significant differences in their stem growth habits during the reproductive / flowering phase. Determinate soybean varieties produce plants that grow in height from the tip of the stem over a period of several weeks, but at the same time, flowering begins low on the stem while the plant is still in the vegetative growth phase. In contrast, determinate soybean varieties produce plants that complete growth in main stem height at the beginning of the reproductive phase (R1 phase) and at approximately the same time produce all the flowers that the plant will produce. As a result, determinate plants are only about 1 / 2 to about 2 / 3 the height of determinate plants.

[0034] In general, determinate soybean varieties have higher yield potential compared to determinate varieties, but suffer from a higher top-heavy stem that is more prone to lodging. However, because of its important role in development, the Dt1 locus was fixed early in soybean domestication, resulting in an overall lack of diversity at the locus. This has hindered efforts to develop varieties with new determinate phenotypes. Furthermore, because plant-to-plant competition in soybean cultivation favors tall plants with greater flexibility, it is highly unlikely that shorter, more compact semi-determinate plants with varying degrees of determinateness will be selected, even in mutagenesis screens.

[0035] The present disclosure represents a significant advance in the art in that it provides engineered alleles that confer novel intermediate phenotypes (semi-determinate) between the current determinate and determinate growth habits in soybeans and other crops, as well as methods for their production, which improve key traits, reduce crop lodging, and increase productivity per seedling and field. The methods and compositions disclosed herein provide an opportunity to create diversity that cannot be selected from traditional plant breeding or random mutagenesis. Thus, provided herein are methods and compositions for modifying plant determinateness that can be used to achieve such benefits, including the development of semi-determinate plants that provide unique benefits to growers, even though the market to date has consisted mainly of determinate and determinate varieties, for example in crops such as soybeans.

[0036] To produce soybeans and other plants with novel growth habit phenotypes, the present disclosure provides, in certain embodiments, methods and compositions for creating novel alleles at the Dt1 locus via editing of the TFL1b gene promoter. For example, the promoter region upstream of the TFL1b gene was modified as disclosed herein by co-expressing eight guide RNAs targeting approximately 1.2 kb of the sequence. Edited individuals containing a series of deletions ranging from 30 to 1746 bp were selected and evaluated. Surprisingly, the results showed that edited dt1 lines could be created that represent a series of alleles at the Dt1 locus with a spectrum of distinct determinant phenotypes, ranging from determinant to hyperdeterminant. Alleles were generated that confer terminal flowering dates ranging from 3 weeks before to nearly 1 week after the terminal flowering date of the indeterminant background cultivar, enabling for the first time the ability to engineer optimal degrees of semideterminantity. Thus, the present disclosure represents a significant advance in the art in that it enables the production of novel engineered alleles in soybean and other crops that confer novel semi-determinant phenotypes that have the potential to protect yield by reducing lodging, thereby increasing productivity per plant.

[0037] I. Genome Editing In certain embodiments, the present disclosure provides plants, plant parts, plant cells and seeds that are created through genome modification using site-specific integration or genome editing. Genome editing can be used to create one or more edit(s) or mutation(s) at a desired target site in the genome of a plant, such as changing the expression and / or activity of one or more genes or integrating an insertion sequence or transgene at a desired location in the plant genome. Any site or locus in the genome of a plant can potentially be selected for genome editing (or gene editing) or site-directed integration of a transgene, construct or transcribable DNA sequence. As used herein, a "target site" for genome editing or site-directed integration refers to the location of a polynucleotide sequence in a plant genome that is bound and cleaved by a site-specific nuclease to introduce a double-strand break (DSB) or a single-stranded nick into the nucleic acid backbone and / or its complementary DNA strand of the polynucleotide sequence in the plant genome. A target site may comprise, for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. A "target site" for an RNA-guided nuclease may comprise a sequence of either the complementary strand of a double-stranded nucleic acid (DNA) molecule or a chromosome at the target site. A site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (such as, but not limited to, a CRISPR RNA (crRNA) or a single guide RNA (sgRNA) as further described herein). A non-coding guide RNA provided herein may be complementary to a target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or a chromosome at the target site). It should be understood that complete identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site.For example, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight mismatches (or more) between the target site and the non-coding RNA may be tolerated. "Target site" also refers to the location of a polynucleotide sequence in a plant genome that is bound and cleaved by any other site-specific nuclease that may not be induced by a non-coding RNA molecule, such as, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and introduces a DSB or single-stranded nick in the polynucleotide sequence and / or its complementary DNA strand. As used herein, "target region" or "targeted region" refers to a sequence or region of a polynucleotide that is flanked by two or more target sites. Without limitation, in some embodiments, the target region may be mutated, deleted, inserted, or inverted. As used herein, "adjacent" when used to describe a target region of a polynucleotide sequence or molecule refers to two or more target sites in the polynucleotide sequence or molecule that surround the target region, one target site on each side of the target region.

[0038] As used herein, "targeted genome editing technology" refers to any method, protocol, or technique that allows for precise and / or targeted editing of specific locations within a plant's genome (i.e., editing is mostly or completely non-random) using site-specific nucleases, such as meganucleases, zinc finger nucleases (ZFNs), RNA-guided endonucleases (e.g., CRISPR / Cas9 systems), TALE (transcription activator-like effector) endonucleases (TALENs), recombinases, or transposases. As used herein, "editing" or "genome editing" refers to generating targeted mutations, deletions, inversions or substitutions of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000 or at least 25,000 nucleotides of an endogenous plant genomic nucleic acid sequence. As used herein, "editing" or "genome editing" can also include targeted insertion or site-directed integration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 10,000 or at least 25,000 nucleotides into the endogenous plant genome of a plant.The singular "edit" or "genomic edit" refers to one such targeted mutation, deletion, inversion, substitution or insertion, while the plural "edits" or "genomic edits" refers to two or more targeted mutation(s), deletion(s), inversion(s), substitution(s) and / or insertion(s), each "edit" being introduced via targeted genome editing technology.

[0039] According to some embodiments, the site-specific nuclease may be co-delivered with a donor template molecule that serves as a template for making the desired edit, mutation or insertion into the genome at the desired target site via repair of the double-stranded break (DSB) or nick created by the site-specific nuclease. According to some embodiments, the site-specific nuclease may be co-delivered with a DNA molecule that includes a selectable or screenable marker gene.

[0040] The site-specific nuclease provided herein may be selected from the group consisting of zinc finger nuclease (ZFN), TALE endonuclease (TALEN), meganuclease, RNA-guided endonuclease, recombinase, transposase, or any combination thereof. See, for example, Khandagale et al. (Plant Biotechnol Rep10:327-343,2016) and Gaj et al. (Trends Biotechnol.31(7):397-405,2013). Zinc finger nucleases are synthetic proteins consisting of an engineered zinc finger DNA binding domain fused to a cleavage domain (or half of a cleavage domain) that may be derived from a restriction enzyme (e.g., FokI). The DNA binding domain may be canonical (C2H2) or non-canonical (e.g., C3H or C4). A DNA-binding domain may contain one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site, but typically consists of 3-4 (or more) zinc fingers. Multiple zinc fingers in a DNA-binding domain may be separated by a linker sequence(s). ZFNs can be designed to cleave almost any span of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., from FokI nuclease) fused to a DNA-binding domain containing a zinc finger array engineered to bind the target site DNA sequence. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger alpha helix, which contribute to site-specific binding to the target site, can be altered and customized to fit a particular target sequence. Other amino acids can form consensus scaffolds to generate ZFNs with different sequence specificities.

[0041] Methods and rules for designing ZFNs to target and bind to specific target sequences are known in the art. See, for example, U.S. Patent Application Publication Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain may require dimerization to cleave DNA, so two ZFNs with C-terminal regions are required to bind opposite DNA strands at the cleavage site (separated by 5-7 bp). If the two ZF binding sites are palindromic, the ZFN monomers can cleave the target site. ZFN, as used herein, is broad and includes monomeric ZFNs that can cleave double-stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to function together to cleave DNA at the same site. Because the DNA-binding specificity of zinc finger domains can be re-engineered using one of a variety of methods, customized ZFNs can theoretically be constructed to target almost any target sequence (e.g., at or near a gene in a plant genome). Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

[0042] Transcription Activator-Like Effectors (TALEs) can be engineered to bind virtually any DNA sequence, for example at or near the genomic locus of a plant gene. TALEs have a central DNA-binding domain composed of 13-28 repeating monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeating variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of the RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and the RVDs can be adjusted to recognize stretches of DNA bases. This simple relationship between amino acid sequence and DNA recognition makes it possible to engineer specific DNA-binding domains by selecting combinations of repeating segments containing appropriate RVDs.

[0043] TALEN is an artificial restriction enzyme generated by fusing a TALE DNA binding domain to a nuclease domain. In some embodiments, the nuclease is selected from the group consisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051 and Pept071. When each member of a TALEN pair binds to a DNA site adjacent to a target site, the FokI monomer dimerizes and causes double-stranded DNA cleavage at the target site. The term TALEN as used herein broadly encompasses monomeric TALENs that can cleave double-stranded DNA without assistance from another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that function together to cleave DNA at the same site.

[0044] Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer and requires two constructs with unique DNA binding domains for sites in the target genome in the appropriate orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites, are parameters to achieve high levels of activity. The cleavage domains of PvuII, MutH and TevI are useful alternatives to FokI and variants of FokI for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to TALEs (Yank et al., PLoS One 8:e82539, 2013). MutH is capable of introducing specific nicks into strands in DNA (Gabsalilow et al., Nucleic Acids Research. 41:e83, 2013). TevI introduces a double-stranded break in DNA at the targeted site (Beurdeley et al., Nature Communications 4:1762, 2013).

[0045] The relationship between the amino acid sequence of the TALE binding domain and DNA recognition allows for designable proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods for designing TALE constructs are known to those skilled in the art. See Doyle et al. (Nucleic Acids Research 40:W117-122, 2012), Cermak et al. (Nucleic Acids Research 39:e82, 2011) and tale-nt.cac.cornell.edu / about. In another aspect, the TALENs provided herein are capable of generating targeted DSBs.

[0046] The site-specific nuclease can be a meganuclease. Meganucleases, usually identified in microorganisms such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (>14 bp), resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases usually have extended DNA recognition sequences (e.g., 14-40 bp). Engineering meganucleases can be more challenging than ZFNs and TALENs, as the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening are used to generate novel meganuclease variants that recognize unique sequences and retain improved nuclease activity.

[0047] The site-specific nuclease may be an RNA-guided nuclease. According to some embodiments, the RNA-guided nuclease is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr 1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, and any homologs or modified versions thereof, and Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), and any homologs or modified versions thereof). According to some embodiments, the RNA-guided endonuclease is a Cas9 or Cpf1 enzyme. The RNA-guided nuclease may be delivered as a protein with or without a guide RNA, or the guide RNA may be delivered as a ribonucleoprotein complexed with an RNA-guided nuclease enzyme.

[0048] For RNA-guided endonucleases, a guide RNA molecule may be further provided to direct the endonuclease to a target site in the genome of the plant through base pairing or hybridization, resulting in a DSB or nick at or near the target site. The guide RNA may be transformed or introduced into the plant cell or tissue as a gRNA nucleic acid molecule or a recombinant DNA molecule, construct or vector that includes a transcribable DNA sequence encoding the guide RNA operably linked to a promoter. As understood in the art, the guide RNA may include, for example, a CRISPR RNA (crRNA), a single-stranded guide RNA (sgRNA), or any other RNA molecule that can guide or direct the endonuclease to a specific target site in the genome. Cas9, the prototype CRISPR-associated protein from Streptococcus pyogenes, naturally binds to two RNAs, a CRISPR RNA (crRNA) guide and a trans-acting CRISPR RNA (tracrRNA), to assemble a CRISPR ribonucleoprotein (crRNP). A "single-stranded guide RNA" (or "sgRNA") is an RNA molecule that includes a crRNA covalently linked to a tracrRNA by a linker sequence and can be expressed as a single RNA transcript or molecule. A guide RNA includes a guide or targeting sequence (also referred to herein as a "spacer sequence") that is identical or complementary to a target site in a plant genome, such as at or near a gene. A guide RNA is typically a non-coding RNA molecule that does not code for a protein. The guide sequence of a guide RNA may be at least 10 nucleotides long, such as, for example, 12-40 nucleotides long, 12-30 nucleotides long, 12-20 nucleotides long, 12-35 nucleotides long, 12-30 nucleotides long, 15-30 nucleotides long, 17-30 nucleotides long, or 17-25 nucleotides long, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides long.A guide sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical to or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more contiguous nucleotides of the DNA sequence at the target site in the genome.

[0049] As described above, the target gene of genome editing can be any of the TERMINAL FLOWER1-like genes described herein for suppression, including the soybean TERMINAL FLOWER1b (GmTFL1b) gene. With regard to mutation of knockdown of TFL1 gene by genome editing, RNA-guided endonuclease is targeted to the promoter and / or enhancer sequence of TFL1 gene, or upstream or downstream sequence such as intron, 5'UTR and / or 3'UTR sequence, to mutate one or more promoter and / or regulatory sequences of TFL1 gene and affect or reduce its expression level. For knockdown of GmTFL1b gene in soybean, a guide RNA may be used that comprises a guide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of SEQ ID NO: 3 or 4 or a sequence complementary thereto (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more consecutive nucleotides of SEQ ID NO: 3 or 4 or a sequence complementary thereto), although alternative splicing and different exon / intron boundaries may occur. As used herein, the term "contiguous" with respect to a polynucleotide or protein sequence means that there are no deletions or gaps in the sequence.

[0050] As used herein, with respect to a given sequence, "complement," "complementary sequence," and "reverse complement" are used interchangeably. All three terms refer to the reverse complementary sequence of a nucleotide sequence, i.e., the sequence complementary to a given sequence in reverse order of nucleotides.

[0051] As used herein, the term "antisense" refers to a DNA or RNA sequence that is complementary to a specific DNA or RNA sequence. Antisense RNA molecules are single-stranded nucleic acids that can combine with a sense RNA strand, sequence, or mRNA to form a duplex due to sequence complementarity. The term "antisense strand" refers to a nucleic acid strand that is complementary to the "sense" strand. The "sense strand" of a gene or locus is a strand of DNA or RNA that has the same sequence as the RNA molecule transcribed from the gene or locus (except for uracil in RNA and thymine in DNA).

[0052] A protospacer adjacent motif (PAM), as known in the art, may be present in the genome immediately adjacent to and upstream of the 5' end of the genomic target site sequence that is complementary to the targeting sequence of the guide RNA, i.e., directly downstream (3') of the sense (+) strand of the genomic target site (relative to the targeting sequence of the guide RNA). See, for example, Wu et al. (Quant Biol. 2(2):59-70 (2014)). The genomic PAM sequence on the sense (+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may include 5'-NGG-3'. However, in general, the corresponding sequence of the guide RNA (i.e., directly downstream (3') of the targeting sequence of the guide RNA) may not be complementary to the genomic PAM sequence.

[0053] In some embodiments, the site-specific nuclease is a recombinase. Non-limiting examples of recombinases that can be used include serine recombinases attached to DNA recognition motifs, tyrosine recombinases attached to DNA recognition motifs, or any recombinase enzyme known in the art attached to DNA recognition motifs. In certain embodiments, the site-specific nuclease is a recombinase or transposase, which can be a DNA transposase or recombinase attached or fused to a DNA binding domain. Non-limiting examples of recombinases include tyrosine recombinases selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase attached to a DNA recognition motif provided herein. In one aspect of the present disclosure, the Cre recombinase or Gin recombinase provided herein is tethered to a zinc finger DNA binding domain, a TALE DNA binding domain, or a Cas9 nuclease. In another embodiment, a serine recombinase selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase can be attached to a DNA recognition motif provided herein. In yet another embodiment, a DNA transposase selected from the group consisting of TALE-piggyBac and TALE mutagen can be attached to a DNA binding domain provided herein.

[0054] Site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases and TALENs, are not RNA-guided, but instead rely on their protein structure to determine their target site to cause DSB or nick, or they are fused, tethered or attached to a DNA-binding protein domain or motif. The protein structure of the site-specific nuclease (or the fused / attached / tethered DNA-binding domain) can direct the site-specific nuclease to the target site. According to many of these embodiments, non-RNA-guided site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases and TALENs, can be designed, engineered and constructed according to known methods to target and bind to a target site at or near the genomic locus of an endogenous gene of a plant, and create a DSB or nick at such genomic locus. DSBs or nicks created by non-RNA-guided site-specific nucleases can lead to knockdown of gene expression through repair of the DSB or nick, which can result in mutation or insertion of sequences at the site of the DSB or nick by cellular repair mechanisms that can be induced by donor template molecules.

[0055] As used herein, a "donor molecule," "donor template," or "donor template molecule" (collectively "donor template"), which may be a donor template or sequence of a recombinant polynucleotide, DNA, or RNA, is defined as a nucleic acid molecule having a homologous nucleic acid template or sequence (e.g., a homologous sequence) and / or an insertion sequence for site-directed targeted insertion or recombination into the genome of a plant cell via repair of a nick or DSB in the genome of the plant cell. A donor template may be a separate DNA molecule that contains one or more homologous sequence(s) and / or an insertion sequence for targeted integration, or a donor template may be a sequence portion (i.e., a donor template region) of a DNA molecule that further contains one or more other expression cassettes, genes / transgenes, and / or transcribable DNA sequences. For example, a "donor template" may be used for site-directed integration of a transgene or construct, or as a template to introduce a mutation, such as an insertion, deletion, substitution, etc., into a target site in the genome of a plant. The targeted genome editing techniques provided herein may include the use of one or more, two or more, three or more, four or more, or five or more donor molecules or templates. The donor templates provided herein may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten gene(s) or transgene(s) and / or transcribable DNA sequence(s). Alternatively, the donor template may not include any genes, transgenes, or transcribable DNA sequences.

[0056] Without being limited thereto, the gene / transgene or transcribable DNA sequence of the donor template may include, for example, insecticide resistance genes, herbicide resistance genes, nitrogen utilization efficiency genes, water utilization efficiency genes, yield improvement genes, nutritional quality genes, DNA binding genes, selectable marker genes, RNAi or suppression constructs, site-specific genome modification enzyme genes, single guide RNA of CRISPR / Cas9 system, geminivirus-based expression cassette, or plant virus expression vector system. According to other embodiments, the insert sequence of the donor template may include a transcribable DNA sequence that codes for a protein or a non-coding RNA molecule that can target an endogenous gene for suppression. The donor template may include a promoter (such as a constitutive promoter, a tissue-specific or tissue-preferred promoter, a developmental stage promoter, or an inducible promoter) operably linked to the coding sequence, gene, or transcribable DNA sequence. The donor template may include a leader, enhancer, promoter, transcription initiation site, 5'-UTR, one or more exon(s), one or more intron(s), transcription termination site, region or sequence, 3'-UTR, and / or polyadenylation signal, each of which may be operably linked to a coding sequence, gene (or transgene), or transcribable DNA sequence encoding a non-coding RNA, guide RNA, mRNA, and / or protein. The donor template may be a single- or double-stranded DNA or RNA molecule, or a plasmid.

[0057] The "insertion sequence" of the donor template is a sequence designed for targeted insertion into the genome of a plant cell, which can be any suitable length. For example, the insertion sequence of the donor template can be between 2 and 50,000, between 2 and 10,000, between 2 and 5000, between 2 and 1000, between 2 and 500, between 2 and 250, between 2 and 100, between 2 and 50, between 2 and 30, between 15 and 50, between 15 and 100, between 15 and 500, between 15 and 1000, between 15 and 5000, between 18 and 30, between 18 and 26, between 20 and 26, between 20 and 50, between 20 and 100, between 20 and 250, between 20 and 50 ... The length may be between 1000, 20-5000, 20-10,000, 50-250, 50-500, 50-1000, 50-5000, 50-10,000, 100-250, 100-500, 100-1000, 100-5000, 100-10000, 250-500, 250-1000, 250-5000, or 250-10,000 nucleotides or base pairs. The donor template also has at least one homologous sequence or arm (such as two homologous arms) to direct integration of the mutation or insertion sequence into the target site in the genome of the plant via homologous recombination, where the homologous sequence or arm(s) are identical or complementary or have a percent identity or percent complementarity to a sequence at or near the target site in the genome of the plant. When the donor template includes a homologous arm(s) and an insertion sequence, the homologous arm(s) will be adjacent to or surround the insertion sequence of the donor template.Each homology arm is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% identical or complementary to at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 500, at least 1000, at least 2500, or at least 5000 nucleotides of a target DNA sequence in the genome of the plant.

[0058] Any method known in the art for site-directed integration can be used with the present disclosure.In the presence of donor template molecule with insertion sequence, DSB or nick can be repaired by homologous recombination between donor template and homologous arm(s) of plant genome or by non-homologous end joining (NHEJ), resulting in site-directed integration of insertion sequence into plant genome to generate targeted insertion event at the site of DSB or nick.Therefore, if transgene, transcribable DNA sequence, construct or sequence is located in the insertion sequence of donor template, site-specific insertion or integration of transgene, transcribable DNA sequence, construct or sequence can be achieved.

[0059] Any method known in the art for suppressing a target gene can be used to suppress the TFL1 gene according to the embodiments of the present disclosure, including expression of antisense RNA, double-stranded RNA (dsRNA) or inverted repeat RNA sequences, or through co-suppression or RNA interference (RNAi) by expression of small interfering RNA (siRNA), short hairpin RNA (shRNA), trans-acting siRNA (ta-siRNA) or microRNA (miRNA). Additionally, sense and / or antisense RNA molecules targeted to coding and / or non-coding genomic sequences or regions within or near the TFL1 gene can be used to cause gene silencing. Thus, any of these methods can be used for targeted suppression of the endogenous TFL1 gene in a tissue-specific or tissue-preferential manner. See, for example, U.S. Patent Application Publication Nos. 2009 / 0070898, 2011 / 0296555, and 2011 / 0035839.

[0060] The introduction of DSBs or nicks can also be used to introduce targeted mutations in the genome of plants. According to this approach, mutations (such as deletions, insertions, inversions and / or substitutions) can be introduced at the target site via incomplete repair of DSBs or nicks to produce knockouts or knockdowns of genes. Such mutations can be generated by incomplete repair of targeted loci without the use of donor template molecules. A "knockout" of a gene can be achieved by inducing a DSB or nick at or near the endogenous locus of the gene, which results in non-expression of the protein or expression of a non-functional protein, while a "knockdown" of a gene can be achieved in a similar manner by inducing a DSB or nick at or near the endogenous locus of the gene that is incompletely repaired at a site that does not affect the coding sequence of the gene in a manner that abolishes the function of the encoded protein. For example, the site of the DSB or nick in the endogenous locus can be located upstream or in the 5' region of the gene (e.g., promoter and / or enhancer sequences) to affect or reduce the level of expression.

[0061] Similarly, such targeted knockout or knockdown mutations of genes can be generated by donor template molecules that direct specific or desired mutations at or near the target site through repair of DSBs or nicks. The donor template molecule can include a homologous sequence, with or without an insertion sequence, that includes one or more mutations (such as one or more deletions, insertions, inversions and / or substitutions) at or near the site of the DSB or nick compared to the targeted genomic sequence. For example, targeted knockout or knockdown mutations of genes can be achieved by replacing, inserting, deleting or inverting at least a portion of a gene (such as by introducing a frameshift or premature stop codon into the coding sequence of the gene, or by disrupting the promoter sequence or the sequence of other non-coding regulatory elements of the gene). Deletion of a portion of a gene can also be introduced by creating a DSB or nick at two target sites, causing deletion of the intervening target region flanked by the target sites.

[0062] In one aspect, the disclosure provides a modified soybean plant, or plant part thereof, comprising a mutant allele of the TFL1 gene, wherein the mutant allele comprises at least one genomic modification comprising at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 125, at least 150, at least 175, or at least 200 contiguous nucleotides of the promoter region of the endogenous TFL1 gene. The promoter sequence of the soybean TFL1b gene comprises the sequence of SEQ ID NO: 4, which is a 2 kb polynucleotide sequence upstream of the transcription start site of the TFL1b gene. The genomic modification can be a deletion of a region comprising at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 125, at least 150, at least 175, or at least 200 consecutive nucleotides within the sequence of SEQ ID NO:4. Such deletions in SEQ ID NO:4 can include a region spanning from nucleotide 1539 to nucleotide 1568, from nucleotide 1217 to nucleotide 1604, from nucleotide 1518 to nucleotide 1629, from nucleotide 951 to nucleotide 1222, from nucleotide 1364 to nucleotide 1407, from nucleotide 1367 to nucleotide 1516, from nucleotide 754 to nucleotide 1806, from nucleotide 1216 to nucleotide 1319, from nucleotide 1159 to nucleotide 1965, from nucleotide 760 to nucleotide 1214, from nucleotide 939 to nucleotide 1028, from nucleotide 1066 to nucleotide 1664, from nucleotide 952 to nucleotide 1552, from nucleotide 1677 to nucleotide 1808, from nucleotide 1524 to nucleotide 1558, from nucleotide 476 to nucleotide 1405, from nucleotide 1365 to nucleotide 1559, from nucleotide 928 to nucleotide 1560, or from nucleotide 593 to nucleotide 1813 of SEQ ID NO:4.

[0063] In one aspect, the genomic modification can be an inversion of a region of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 125, at least 150, at least 175, or at least 200 contiguous nucleotides within the sequence of SEQ ID NO: 4. Such an inversion can include the region from nucleotide 1029 to nucleotide 1065 of SEQ ID NO: 4 that has been removed, inverted, and reinserted into the same position in SEQ ID NO: 4. In one aspect, a mutant allele of the TFL1 gene can include two or more modifications in the promoter region of the endogenous TFL1 gene. Examples of such mutant alleles of the soybean TFL1b gene are disclosed herein and include, for example, an allele comprising two deletions in the sequence of SEQ ID NO:4, the first deletion spanning the region from nucleotide 952 to nucleotide 1552 of SEQ ID NO:4 and the second deletion spanning from nucleotide 1677 to nucleotide 1808 of SEQ ID NO:4; an allele comprising two deletions and an inversion within the sequence of SEQ ID NO:4, the first deletion spanning the region from nucleotide 939 to nucleotide 1028 of SEQ ID NO:4 and the second deletion spanning from nucleotide 1066 to nucleotide 1664 of SEQ ID NO:4; and an inversion comprising the region from nucleotide 1029 to nucleotide 1065 of SEQ ID NO:4 that has been removed, inverted, and reinserted at the same position in SEQ ID NO:4.

[0064] Other targeted modifications can be made to the promoter region to create novel alleles in the soybean TFL1b gene and its homologs. SEQ ID NOs: 67-77, 79, and 81 represent approximately 2 kb of polynucleotide sequence upstream of the transcription start site of the TFL1b gene homologs found in Zea mays (SEQ ID NO: 67), Sorghum bicolor (SEQ ID NO: 68), Oryza sativa (SEQ ID NO: 69), Triticum aestivum (SEQ ID NO: 70), Hordeum vulgare (SEQ ID NO: 71), Solanum lycopersicum (SEQ ID NO: 72), Gossypium hirsutum (SEQ ID NO: 73), Capsicum annuum (SEQ ID NO: 74), Brassica napus (SEQ ID NO: 75), Arabidopsis thaliana (SEQ ID NO: 76), Medicago truncatula (SEQ ID NO: 77), Arachis hypogaea (SEQ ID NO: 79), and Cicer arietinum (SEQ ID NO: 81), respectively. For example, one or more modification sites can be located about 200 or more than 200 nucleotides from the 3' end of the sequences of SEQ ID NOs: 4, 67-77, 79, and 81. In one aspect, one or more modifications can be made within the region of DNA spanning nucleotide position 1237 to nucleotide position 1570 of SEQ ID NO: 4 to create novel alleles in the soybean TFL1b gene.

[0065] In a further aspect, the disclosure provides a modified soybean plant or plant part thereof comprising a mutant allele of the TFL1 gene, the mutant allele comprising one or more junction sequences, the junction sequence being at least 30, at least 60, at least 100 nucleotides at the junction site. As used herein, a "junction" or "junction site" is the connection point between nucleotide sequences at the site of a deletion, insertion, substitution, or inversion. In the case of a deletion, the junction is the connection point of the deletion site of the sequences previously adjacent to the deletion. For example, in the case of a deletion of 30 base pairs from nucleotide 1539 to nucleotide 1568, as compared to SEQ ID NO: 4 of the reference sequence described herein, the junction would be between nucleotide 1538 to nucleotide 1569. In the case of an insertion, substitution, or inversion, the junction is the connection point between the inserted, inverted, or replaced sequence and the adjacent DNA sequence. In the case of an insertion, substitution or inversion, one junction is found at the 5' end of the insertion, substitution or inversion and another junction is found at the 3' end of the insertion, substitution or inversion. "Junction sequence" refers to a DNA sequence of any length that spans the junction. The junction sequence may include at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides or more.

[0066] II. Constructs for genome editing Recombinant DNA constructs and vectors are provided that include polynucleotide sequences encoding site-specific nucleases, such as zinc finger nucleases (ZFNs), meganucleases, RNA-guided endonucleases, TALE endonucleases (TALENs), recombinases or transposases, where the coding sequences are operably linked to a plant-expressible promoter. For RNA-guided endonucleases, recombinant DNA constructs and vectors are further provided that include polynucleotide sequences encoding guide RNAs, where the guide RNAs include guide sequences of sufficient length that have percent identity or complementarity to a target site in the genome of a plant, such as at or near the target TFL1 gene. The polynucleotide sequences of the recombinant DNA constructs and vectors that encode site-specific nucleases or guide RNAs can be operably linked to a plant-expressible promoter, such as an inducible promoter, a constitutive promoter, or a tissue-specific promoter.

[0067] As used herein, "gene" refers to a nucleic acid sequence that forms a genetic and functional unit and encodes one or more sequence-related RNA and / or polypeptide molecules. A gene generally comprises a coding region operably linked to appropriate regulatory sequences that regulate the expression of a gene product (e.g., a polypeptide or functional RNA). A gene can have various sequence elements, including, but not limited to, promoters, untranslated regions (UTRs), exons, introns, and other upstream or downstream regulatory sequences.

[0068] As used herein, "allele" refers to an alternative nucleic acid sequence of a gene or a particular locus (e.g., a nucleic acid sequence of a gene or locus that differs from other alleles of the same gene or locus). Such alleles can be considered (i) wild type, or (ii) mutant, if there is one or more mutations or edits in the nucleic acid sequence of the mutant allele compared to the wild type allele. A mutant or edited allele of a gene can have a reduced or eliminated level of activity or expression of the gene compared to the wild type allele. For example, a mutant or edited allele of the TFL1 gene can have a deletion in the promoter region upstream of the endogenous TFL1 gene. In the case of a diploid organism such as soybean, a first allele can occur on one chromosome and a second allele can occur at the same locus on a second homologous chromosome. If one allele at a locus on one chromosome of a plant is a mutant or edited allele and the other corresponding allele on the homologous chromosome of the plant is wild type, then the plant is described as heterozygous for the mutant or edited allele. However, if both alleles at a locus are mutant or edited alleles, the plant is described as homozygous for the mutant or edited allele. Plants that are homozygous for mutant or edited alleles at a locus may contain the same mutant or edited allele, or different mutant or edited alleles, in heteroallelic or biallelic cases.

[0069] As used herein, a "wild-type gene" or "wild-type allele" refers to a gene or allele having the most common sequence or genotype in a particular plant species, or another sequence or genotype that has only natural mutations, polymorphisms, or other silent mutations compared to the most common sequence or genotype that do not significantly affect the expression and activity of the gene or allele. Indeed, a "wild-type" gene or allele does not contain any mutations, polymorphisms, or other types of mutations that substantially affect the normal function, activity, expression, or phenotypic outcome of the gene or allele compared to the most common sequence or genotype.

[0070] In general, the term "variant" refers to a molecule that has some synthetic or naturally produced differences in its nucleotide or amino acid sequence compared to a reference (natural) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions, or any desired combination of such changes in the native polynucleotide or amino acid sequence.

[0071] As used herein, the term "expression" refers to the biosynthesis of a gene product, typically the transcription and / or translation of a nucleotide sequence, such as an endogenous gene, a heterologous gene, a transgene or an RNA and / or a protein coding sequence, in a cell, tissue, organ or organism, such as a plant, plant part or plant cell, tissue or organ.

[0072] The term "recombinant" with respect to a polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc., refers to a polynucleotide or protein molecule or sequence that is man-made and not normally found in nature and / or exists in a context in which it is not normally found in nature, and includes polynucleotide (DNA or RNA) molecules, proteins, constructs, etc. that contain a combination of two or more polynucleotide or protein sequences that do not occur in nature in the same manner without human intervention, e.g., a polynucleotide molecule, protein, construct, etc. that contains at least two polynucleotide or protein sequences that are operably linked but heterologous to each other. For example, the term "recombinant" can refer to any combination of two or more DNA or protein sequences in the same molecule (e.g., a plasmid, construct, vector, chromosome, protein, etc.), where such a combination is man-made and not normally found in nature. As used in this definition, the phrase "not normally found in nature" means not found in nature without introduction by humans. A recombinant polynucleotide or protein molecule, construct, etc. may include a polynucleotide or protein sequence(s) that is (i) separated from other polynucleotide or protein sequence(s) that are naturally adjacent to each other, and / or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequence(s) that are not naturally adjacent to each other. Such recombinant polynucleotide molecules, proteins, constructs, etc. may also refer to polynucleotide or protein molecules or sequences that have been genetically engineered and / or constructed outside of a cell. For example, a recombinant DNA molecule may include any engineered or artificial plasmid, vector, etc., and may encompass linear or circular DNA molecules. Such plasmids, vectors, etc. may contain various maintenance elements, including one or more transgenes or expression cassettes, in addition to origins of replication and selectable markers for prokaryotes, and possibly selectable marker genes for plants.The term "operably linked" refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene) such that the promoter or the like operates or functions to initiate, assist, influence, cause, and / or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in a particular cell(s), tissue(s), developmental stage(s), and / or condition(s).

[0073] Reference in this application to an "isolated DNA molecule" or "isolated polynucleotide" or equivalent terms or expressions is intended to mean that the DNA molecule or polynucleotide is present alone or in combination with other compositions, but not in its natural environment. For example, nucleic acid elements naturally found in the DNA of the genome of an organism, such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc., are not considered "isolated" as long as the elements are in the genome of the organism and in the location in the genome where the elements are found in nature. However, each of these elements and subportions of these elements are "isolated" within the scope of this disclosure as long as the elements are not in the genome of the organism and in the location in the genome where the elements are found in nature. Similarly, a nucleotide sequence encoding a protein or any naturally occurring variant of that protein will be an isolated nucleotide sequence as long as the nucleotide sequence is not in the DNA of an organism in which the sequence encoding the protein is found in nature. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring protein is considered isolated for the purposes of this disclosure. For purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, is considered to be an isolated nucleotide sequence, whether it is present in a plasmid or similar structure used to transform a cell, in the genome of the plant or bacteria, or present in detectable amounts in tissues, progeny, biological samples, or commercial products derived from the plant or bacteria.

[0074] As generally understood in the art, the term "promoter" may generally refer to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, and that supports or enhances the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters may be synthetically produced, modified, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may also include chimeric promoters that include a combination of two or more heterologous sequences. Promoters of the present disclosure may thus encompass variants of promoter sequences or fragments thereof that are similar in composition to, but not identical to, other promoter sequence(s) known or provided herein. Promoters provided herein, or variants or fragments thereof, may include "minimal promoters," which provide a basal level of transcription and are composed of a TATA box or equivalent sequence for recognition and binding of the RNA polymerase II complex to initiate transcription. Promoters can be classified according to various criteria regarding the pattern of expression of the associated coding or transcribable sequence or gene (including transgene) operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter that drives expression in all or most tissues of a plant is referred to as a "constitutive" promoter. A promoter that drives expression during a specific period or stage of development is referred to as a "developmental" promoter. A promoter that drives enhanced expression in a specific tissue of a plant compared to other plant tissues is referred to as a "tissue-promoted" or "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively higher or selective expression in a specific tissue(s) of a plant, but with lower levels of expression in other tissue(s) of the plant. A promoter that expresses in a specific tissue(s) of a plant, with little or no expression in other tissues of the plant, is referred to as a "tissue-specific" promoter.An "inducible" promoter is a promoter that initiates transcription in response to an environmental stimulus (such as cold, drought or light) or other stimulus (such as wounding or application of a chemical). Promoters can also be classified in terms of their origin, e.g., heterologous, homologous, chimeric, synthetic, etc.

[0075] As used herein, a "plant-expressible promoter" refers to a promoter that is capable of initiating, assisting, influencing, causing, and / or promoting the transcription and expression of an associated transcribable DNA sequence, coding sequence or gene in a plant cell or tissue.

[0076] The term "heterologous" refers to a promoter or other regulatory sequence with respect to an associated polynucleotide sequence (e.g., a transcribable DNA sequence or a coding sequence or gene) that is not naturally linked to such associated polynucleotide sequence in the absence of human introduction (e.g., the promoter or regulatory sequence has a different origin than the associated polynucleotide sequence and / or the promoter or regulatory sequence does not naturally occur in the plant species being transformed with the promoter or regulatory sequence).

[0077] As used herein, "endogenous gene" or "endogenous locus" refers to a gene or locus in its natural, original chromosomal location. As used herein, "endogenous TFL1 gene" refers to the TFL1 locus in its original chromosomal location.

[0078] As used herein, in the context of a protein-coding gene, "exon" refers to a segment of a DNA or RNA molecule that contains the information that codes for a protein or polypeptide sequence.

[0079] As used herein, an "intron" of a gene refers to a segment of a DNA or RNA molecule that does not contain any information encoding a protein or polypeptide and that is initially transcribed into an RNA sequence but is subsequently spliced ​​out of the mature RNA molecule.

[0080] As used herein, a "untranslated region (UTR)" of a gene refers to a segment of an RNA molecule or sequence (e.g., an mRNA molecule) expressed from a gene (or transgene), but does not include the exon and intron sequences of the RNA molecule. A "untranslated region (UTR)" also refers to a DNA segment of an RNA molecule, or a sequence that codes for such a UTR segment. An untranslated region can be a 5'-UTR or a 3'-UTR, depending on whether it is located at the 5' or 3' end of a DNA or RNA molecule or sequence relative to the coding region of the DNA or RNA molecule or sequence (i.e., upstream (5') or downstream (3') of the exon and intron sequences, respectively).

[0081] As used herein, "transcription termination sequence" refers to a nucleic acid sequence that contains a signal that triggers the release of a newly synthesized transcribed RNA molecule from the RNA polymerase complex, indicating the end of transcription of a gene or locus.

[0082] As used herein, "homolog" or "homologues" refers to proteins of a group of proteins that perform the same biological function, e.g., proteins that belong to the same TFL1-like protein family and provide a common enhanced trait in modified plants of the present disclosure. Homologs are expressed by homologous genes. With respect to homologous genes, homologs include orthologs, e.g., genes expressed in different species that have evolved from a common ancestral gene by speciation and encode proteins that retain the same function, but do not include paralogs, i.e., genes that are related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially created variants.

[0083] The term "percent identity", "% identity" or "percent identical" as used herein in the context of two or more nucleotide or protein sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a comparison window, (ii) determining the number of positions where the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to obtain the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percent identity" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing for gaps in their alignment), the "percent identity" to the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), then multiplied by 100%. When percentages of sequence identity are used in the context of proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity), and thus do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Sequences that have percent identity to a base sequence may indicate the activity of the base sequence.

[0084] The degeneracy of the genetic code allows for the possibility of replacing at least one base in the protein coding sequence of a gene with a different base without changing the amino acid sequence of the polypeptide produced from the gene. When optimally aligned, homologous proteins or their corresponding nucleotide sequences usually have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% identity over the entire length of the protein or its corresponding nucleotide sequence identified as being associated with conferring an altered finite sex phenotype when expressed in a plant cell. According to embodiments of the invention, the TFL1 gene or a homolog thereof encodes a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO: 2. Examples of homologs of soybean TFL1b protein (SEQ ID NO: 2) include, but are not limited to, the sequences of SEQ ID NOs: 52-66, 78, and 80.

[0085] Homologs are estimated from sequence similarity by comparison of protein sequences, for example, manually or using computer-based tools. Various pairwise or multiple sequence alignment algorithms and programs, such as ClustalW or Basic Local Alignment Search Tool® (BLAST), are known in the art, which can be used to compare sequence identity or similarity between two or more nucleotide or protein sequences with respect to optimal alignment of the sequences to calculate percent identity of the sequences. BLAST can also be used, for example, to search a query protein sequence of a base organism against a database of protein sequences of various organisms to find similar sequences. The generated summary expectation value (E-value) can be used to measure the level of sequence similarity. Because the protein hit with the lowest E-value of a particular organism is not necessarily an ortholog or the only ortholog, a reverse query is used to filter hit sequences with significant E-values ​​to identify orthologs. A reverse query involves searching a database of protein sequences of a base organism for significant hits. If the best hit of the reverse query is the query protein itself or a paralog of the query protein, the hit can be identified as an ortholog. The reverse query process further distinguishes orthologs from paralogs among all homologs, allowing inference of functional equivalence of genes.

[0086] As used herein with respect to two nucleotide sequences, the term "percent complementarity" or "percent complementary" is similar to the concept of percent identity, but refers to the percentage of nucleotides in the query sequence that optimally base-pair or hybridize with nucleotides in the subject sequence when the query and subject sequences are aligned linearly and optimally base-paired without secondary folded structures such as loops, stems, or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. "Percent complementarity" is calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully unfolded alignment (i.e., without folding or secondary structures) over a comparison window, (ii) determining the number of positions that base-pair between the two sequences over the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined based on known pairing of nucleotide bases such as GC, AT and AU by hydrogen bonding. When "percent complementarity" is calculated in relation to a reference sequence without specifying a specific comparison window, the percent identity is determined by dividing the complementary positions between two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally base paired (allowing mismatched or non-base paired nucleotides, but no folding or secondary structures), the "percent identity" to the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or divided by the total number of positions in the query sequence over the comparison window), then multiplied by 100%.

[0087] As used herein, a "fragment" of a polynucleotide refers to a sequence that comprises at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides or more of a DNA molecule or protein disclosed herein. Methods for generating such fragments from a starting promoter molecule are well known in the art. A fragment of a DNA molecule or protein may exhibit the activity of the DNA molecule or protein from which it is derived.

[0088] According to another aspect, the present disclosure provides a method for modifying a phenotype, such as increasing yield, altering finitude, or reducing lodging, of a plant, comprising: (a) modifying the genome of a plant cell by (i) identifying an endogenous gene of the plant that corresponds to a soybean TFL1 gene, such as the GmTFL1b gene described herein, and its homologs; (ii) modifying the promoter sequence of the endogenous gene in the plant cell via targeted mutagenesis to modify the expression level of the endogenous gene; and (b) regenerating or generating a plant from the plant cell. Various TFL1 genes and proteins from different plant species may be identified and considered as TFL1 homologs or orthologs for use in the present disclosure if they have similar nucleic acid and / or protein sequences and share conserved amino acid and / or structural domain(s) with at least one known TFL1 gene or protein.

[0089] The plant selectable marker transgene in the transformation vector or construct of the present disclosure can be used to aid in the selection of transformed cells or tissues by the presence of a selective agent, such as an antibiotic or herbicide, where the plant selectable marker transgene provides resistance or tolerance to the selective agent. Thus, the selective agent may bias or favor the survival, development, growth, proliferation, etc. of transformed cells expressing the plant selectable marker gene, such as increasing the proportion of transformed cells or tissues in the R0 plant. Commonly used plant selectable marker genes include those that confer resistance or tolerance to antibiotics (e.g., kanamycin and paromomycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aadA), and gentamicin (aac3 and aacC4)) or those that confer resistance or tolerance to herbicides (e.g., glufosinate (bar or pat), dicamba (DMO), and glyphosate (proA or EPSPS)). Plant screenable marker genes can also be used that provide the ability to visually screen for transformants, such as genes expressing luciferase or green fluorescent protein (GFP), or β-glucuronidase or the uidA gene (GUS), for which various chromogenic substrates are known. Plant transformation can also be performed in the absence of selection during one or more steps or stages of the culture, development or regeneration of transformed explants, tissues, plants and / or plant parts.

[0090] III. Transformation Methods Methods and compositions are provided for transforming plant cells, tissues or explants with recombinant DNA molecules or constructs that code for one or more molecules (e.g., guide RNA(s) and / or site-specific nucleases) required for targeted genome editing. Suitable methods for transforming host plant cells include virtually any method that can introduce DNA or RNA into cells (e.g., when recombinant DNA constructs are stably integrated into plant chromosomes, or when recombinant DNA constructs or RNA are transiently provided to plant cells), and are well known in the art. Two effective methods for cell transformation are bacteria-mediated transformation, such as Agrobacterium- or Rhizobium-mediated transformation, and microparticle- or particle gun-mediated transformation. Microparticle gun methods are shown, for example, in U.S. Pat. Nos. 5,550,318, 5,538,880, 6,160,208, and 6,399,861. Agrobacterium-mediated transformation methods are described, for example, in U.S. Patent No. 5,591,616. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, PEG-mediated transformation, and the like, are also known in the art.

[0091] Transformation of plant material is performed in tissue culture in a nutrient medium, such as a mixture of nutrients that allows the cells to grow in vitro. Recipient cell targets include, but are not limited to, meristematic cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gamete cells such as microspores and pollen. Calli may be initiated from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores, and the like. Cells containing transformed nuclei are propagated within the transformed plant. Any suitable method or technique for transformation of plant cells known in the art can be used in accordance with the methods of the present invention. In transformation, DNA is introduced into target plant cells, which typically comprise a small percentage of cells in any one transformation experiment. Marker genes are used to provide an effective system for identifying stably transformed cells by accepting and integrating recombinant DNA molecules into their genome.

[0092] As used herein, the terms "regeneration" and "regenerating" refer to the process of growing or developing a plant from one or more plant cells through one or more culture steps. Transformed or edited cells, tissues, or explants containing the insertion or editing of a DNA sequence can be grown, developed, or regenerated into transgenic plants in culture, plugs, or soil according to methods known in the art. Thus, certain embodiments of the present disclosure relate to methods and constructs for regenerating plants from cells with modified genomic DNA resulting from genome editing. The regenerated plants can then be used to propagate additional plants.

[0093] According to one aspect of the present disclosure, the regenerated plant, or its progeny plant, plant part, or seed, can be screened or selected based on a marker, trait, or phenotype due to editing or mutation, or due to site-specific integration of an insertion sequence, transgene, etc., in the generated or regenerated plant, or its progeny plant, plant part, or seed. If a given mutation, editing, trait, or phenotype is recessive, one or more generations or crosses (e.g., selfing) from the original R0 plant may be required to generate a plant homozygous for the editing or mutation so that the trait or phenotype can be observed. The progeny plant (such as a plant propagated from an R1 seed or a plant in a subsequent generation) can be tested for zygosity using any known zygosity assay (such as by using single nucleotide polymorphism (SNP) assays, DNA sequencing, thermal amplification or polymerase chain reaction (PCR), and / or Southern blotting, etc., that allows for the distinction between heterozygotes, homozygotes, and wild-type plants).

[0094] Methods and techniques are provided for screening and / or identifying cells or plants, etc., for the presence of a target editing or transgene, and for selecting cells or plants that contain a target editing or transgene, which can be based on one or more phenotypes or traits, or the presence or absence of a molecular marker or polynucleotide or protein sequence in a cell or plant. As used herein, "molecular techniques" refer to any method known in the art of molecular biology, biochemistry, genetics, plant biology, or biophysics that involves the use, manipulation, or analysis of nucleic acids, proteins, or lipids. Molecular techniques useful for detecting the presence of modified sequences in a genome include, but are not limited to, phenotypic screening; molecular marker techniques such as SNP analysis by TaqMan® or Illumina / Infinium technology; Southern blot; PCR; enzyme-linked immunosorbent assay (ELISA); and sequencing (e.g., Sanger, Illumina®, 454, Pac-Bio, Ion Torrent™). In one aspect, the detection methods provided herein include phenotypic screening. In another aspect, the detection methods provided herein include SNP analysis. In a further aspect, the detection method provided herein comprises Southern blot. In a further aspect, the detection method provided herein comprises PCR. In one aspect, the detection method provided herein comprises ELISA. In a further aspect, the detection method provided herein comprises determining a nucleic acid or protein sequence. Without being limited thereto, nucleic acids can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0095] Nucleic acids can be isolated using techniques conventional in the art. For example, nucleic acids can be isolated using any method, including but not limited to recombinant nucleic acid techniques and / or PCR. General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate nucleic acids. Isolated nucleic acids can also be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

[0096] Detection (e.g., of the amplification product, hybridization complex, or polypeptide) can be accomplished using a detectable label that can be attached or associated with the hybridization probe or antibody. The term "label" is intended to encompass the use of direct and indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Screening and selection of modified (e.g., edited) plants or plant cells can be performed by any methodology known to those skilled in the art of molecular biology. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for polynucleotide detection, Northern blots, RNase protection, primer extension, RT-PCR amplification for RNA transcript detection, Sanger sequencing, next-generation sequencing technologies (e.g., Illumina®, PacBio®, Ion Torrent™, etc.), enzyme assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis for detecting polypeptides, Western blots, immunoprecipitation, and enzyme-linked immunoassays. Other techniques, such as in situ hybridization, enzyme staining, and immunostaining, can also be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the referenced techniques are known in the art.

[0097] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide may be encoded by a polynucleotide provided herein. An example of a polypeptide is a protein. A protein provided herein may be encoded by a nucleic acid molecule provided herein. A polypeptide may be purified from a natural source (e.g., a biological sample) by known methods, such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide may also be purified, for example, by expressing a nucleic acid in an expression vector. Additionally, a purified polypeptide may be obtained by chemical synthesis. The degree of purity of a polypeptide may be measured using any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0098] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. The antibodies provided herein can be polyclonal or monoclonal. Antibodies having specific binding affinity for the polypeptides provided herein can be generated using methods well known in the art. The antibodies provided herein can be bound to a solid support such as a microtiter plate using methods well known in the art.

[0099] Plants that may be transformed with a transformation vector containing a recombinant DNA molecule or guide RNA can include a variety of flowering plants or angiosperms, which can be further defined as including a variety of dicotyledonous (dicot) or monocotyledonous (monocot) plant species. Dicotyledonous plants include the legumes (such as peas), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), sesame (Sesamum spp.), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea butatas), cassava (Manihot esculenta), coffee (Coffea spp.), tea (Camellia spp.), fruit trees such as apples (Malus spp.), plums, apricots, peaches, cherries, pears (Pyrus spp.), figs (Ficus carica), and other citrus fruits (Citrus spp.), cocoa (Theobroma spp.), and other plants such as peaches, pears (Pyrus spp.), figs (Ficus carica), and other plants such as figs (Ficus spp.). cacao), avocado (Persea americana), olive (Olea europaea), almond (Prunus amygdalus), walnut (Juglans spp.), strawberry (Fragaria spp.), watermelon (Citrullus lanatus), pepper (Capsicum spp.), beet (Beta vulgaris), grape (Vitis, Muscadinia), tomato (Lycopersicon esculentum, Solanum lycopersicum), cucumber (Cucumis sativus), as well as members of the Brassicaceae family, such as Arabidopsis thaliana and Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly Brassica species useful as a source of seed oil.Legumes and leguminous plants include peas (Pisum sativum), alfalfa (Medicago sativa), alfalfa (Medicago truncatula), pigeon peas (Cajanus cajan), guar (Cyamopsis tetragonoloba), carob (Ceratonia siliqua), fenugreek (Trigonella foenum-graecum), soybean (Glycine max), kidney bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), green bean (Vigna radiata), lima bean (Phaseolus lunatus), broad bean (Vicia faba), lentils (Lens culinaris or Lens esculenta), groundnuts (Arachis hypogaea), licorice (Glycyrrhiza glabra, and chickpea (Cicer arietinum). Monocotyledons include oil palms (Elaeis spp.), coconuts (Cocos spp.), bananas (Musa spp.), and cereal grains such as maize (Zea Mays), barley (Hordeum vulgare), sorghum (Sorghum bicolor), rice (Oryza sativa), and wheat (Triticum aestivum). Given that the present disclosure may be applied to a wide range of plant species, the present disclosure also applies to other plant structures similar to the legume pod, such as pods, siliques, fruits, nuts, tubers, etc.

[0100] IV. Genome-modified plants As used herein, "modified" in the context of a plant, plant seed, plant part, plant cell, and / or plant genome refers to a plant, plant seed, plant part, plant cell, and / or plant genome that contains an engineered change in the expression level and / or endogenous sequence of one or more genes of interest compared to a wild-type or control plant, plant seed, plant part, plant cell, and / or plant genome. Indeed, the term "modified" may further refer to a plant, plant seed, plant part, plant cell, and / or plant genome that has one or more deletions that affect the expression of the endogenous TFL1 gene introduced by chemical mutagenesis, transposon insertion or excision, or other known mutagenesis techniques, or introduced by genome editing. In one aspect, the modified plant, plant seed, plant part, plant cell, and / or plant genome can contain one or more transgenes. Thus, for clarity, modified plants, plant seeds, plant parts, plant cells and / or plant genomes include mutated, edited and / or transgenic plants, plant seeds, plant parts, plant cells and / or plant genomes having modified expression levels, expression patterns and / or sequences of the TFL1 gene compared to a wild-type or control plant, plant seed, plant part, plant cell and / or plant genome.

[0101] Modified plants, plant parts, seeds, etc. may have undergone mutagenesis, genome editing or site-specific integration, genetic transformation, or a combination thereof. Such "modified" plants, plant seeds, plant parts, and plant cells include plants, plant seeds, plant parts, and plant cells that are progeny of or are derived from "modified" plants, plant seeds, plant parts, and plant cells that carry molecular changes (e.g., changes in expression levels and / or activity) to the TFL1 gene. Modified seeds provided herein can give rise to modified plants provided herein. Modified plants, plant seeds, plant parts, plant cells, or plant genomes provided herein can include recombinant DNA constructs or vectors provided herein, or genome editing. A "modified plant product" can be any product made from modified plants, plant parts, plant cells, or plant chromosomes provided herein, or any part or component thereof.

[0102] The modified plant can be further crossed with itself or other plants to generate seeds and progeny of the modified plant. Modified plants can also be prepared by crossing a first plant containing the DNA sequence or construct or edit (e.g., a genomic deletion) with a second plant lacking the DNA sequence or construct or edit. For example, the DNA sequence or inversion can be introduced into a first plant line suitable for transformation or editing, which can then be crossed with a second plant line to introgress the DNA sequence or edit (e.g., the deletion) into the second plant line. The progeny of these crosses can be further backcrossed to a more desirable line multiple times, such as for 6-8 generations or backcrossing, to generate progeny plants having substantially the same genotype as the original parent line but for the introduction of the DNA sequence or edit. The modified plants, plant cells or seeds provided herein can be hybrid plants, plant cells or seeds. As used herein, a "hybrid" is created by crossing two plants from different varieties, strains, inbreds, or species such that the progeny contains genetic material from each parent. Those skilled in the art will recognize that higher order hybrids can be produced as well.

[0103] The modified plants, plant parts, plant cells, or seeds provided herein can be of an elite variety or line. "Elite variety" or "elite line" refers to a variety that has resulted from breeding and selection for superior agronomic performance.

[0104] As used herein, the term "control plant" (or similarly "control" plant seed, plant part, plant cell and / or plant genome) is used in comparison to a modified plant (or modified plant seed, plant part, plant cell and / or plant genome) and refers to a plant (or plant seed, plant part, plant cell and / or plant genome) that has the same or similar genetic background (e.g., same parent line, hybrid cross, inbred line, tester, etc.) as the modified plant (or plant seed, plant part, plant cell and / or plant genome), except for the genome edit(s) (e.g., deletion) that affects the TFL1 gene. For example, the control plant may be the same inbred line as the inbred line used to create the modified plant, or the control plant may be the product of a hybrid cross of the same inbred parent lines as the modified plant, except that the control plant is absent the transgenic event or genome edit(s) that affect the TFL1 gene. Similarly, an "unmodified control plant" refers to a plant that shares a substantially similar or essentially identical genetic background with a modified plant, but lacks one or more engineered changes (e.g., mutations or edits) to the genome of the modified plant. For purposes of comparison with a modified plant, plant seed, plant part, plant cell and / or plant genome, a "wild-type plant" (or similarly a "wild-type" plant seed, plant part, plant cell and / or plant genome) refers to a non-transgenic and non-genome-edited control plant, plant seed, plant part, plant cell and / or plant genome. As used herein, a "control" plant, plant seed, plant part, plant cell and / or plant genome can also be a plant, plant seed, plant part, plant cell and / or plant genome that has a similar (but not the same or identical) genetic background to the modified plant, plant seed, plant part, plant cell and / or plant genome, if deemed sufficiently similar for comparison of the characteristic or trait to be analyzed.

[0105] As used herein, the terms "suppress," "suppress," "inhibit," "inhibiting," "inhibiting," "knockout," "knockdown," and "downregulation" refer to the lowering, reduction, or elimination of the expression level of an mRNA and / or protein encoded by a target gene in a plant, plant cell, or plant tissue at one or more stage(s) of plant development compared to the expression level of such target mRNA and / or protein in a wild-type or control plant, cell, or tissue at the same stage(s) of plant development. According to some embodiments, modified plants are provided having a TFL1 gene expression level that is at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% reduced in at least one plant tissue compared to a control plant. According to further embodiments, modified plants are provided having TFL1 gene expression levels that are reduced by 5% to 20%, 5% to 25%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 75%, 5% to 80%, 5% to 90%, 5% to 100%, 75% to 100%, 50% to 100%, 50% to 90%, 50% to 75%, 25% to 75%, 30% to 80%, or 10% to 75% in at least one plant tissue compared to a control plant.

[0106] According to some embodiments, modified plants are provided having TFL1 mRNA levels that are reduced by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% in at least one plant tissue compared to a control plant. According to some embodiments, modified plants are provided having TFL1 mRNA expression levels that are reduced by 5% to 20%, 5% to 25%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 75%, 5% to 80%, 5% to 90%, 5% to 100%, 75% to 100%, 50% to 100%, 50% to 90%, 50% to 75%, 25% to 75%, 30% to 80%, or 10% to 75% in at least one plant tissue compared to a control plant. According to some embodiments, modified plants are provided having TFL1 protein expression levels that are reduced by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% in at least one plant tissue compared to a control plant. According to some embodiments, modified plants are provided having TFL1 protein expression levels that are reduced by 5% to 20%, 5% to 25%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 75%, 5% to 80%, 5% to 90%, 5% to 100%, 75% to 100%, 50% to 100%, 50% to 90%, 50% to 75%, 25% to 75%, 30% to 80%, or 10% to 75% in at least one plant tissue compared to a control plant.

[0107] The present disclosure relates to plants with improved economically important characteristics, including, but not limited to, increased yield, increased finitude, reduced time to reach terminal flowering date, reduced lodging rate, and reduced susceptibility to fungal diseases. More specifically, the present disclosure relates to modified plants comprising genome editing or mutations as described herein, where the plants have increased yield compared to control plants. Many plants of the present disclosure have shown increased yield or improved yield trait components compared to control plants. Yield can be defined as a measurable economically valuable output obtained from a crop. Yield can be defined in terms of quantity and / or quality. For example, soybean yield can include pods per plant, pods per acre, seeds per plant, seeds per pod, weight per seed, weight per pod, pods per node, number of nodes, and number of internodes per plant. Yield can be directly dependent on several factors, e.g. number and size of organs, plant architecture (number of branches, plant biomass, etc.), flowering time and duration, grain filling period. Root structure and development, photosynthetic efficiency, nutrient uptake, stress resistance, early vigor, delayed senescence, functional green leaf retention phenotype can be important factors determining yield. Therefore, optimizing the above factors can contribute to increasing crop yield.

[0108] Modified plants comprising or derived from plant cells transformed with the recombinant DNA of the present disclosure can be further enhanced with stacked traits, such as modified crop plants having enhanced traits resulting from expression of the DNA disclosed herein in combination with one or more genes of agronomic interest that provide beneficial agronomic traits to the crop plant, such as herbicide and / or pest resistance traits. For example, traits imparted by the recombinant DNA constructs of the present disclosure can be stacked with other traits of agronomic interest, such as traits that provide insect resistance, such as using genes from Bacillus thuringensis, to provide resistance to lepidopteran, coleopteran, homopteran, hemipteran and other insects, or improved quality traits, such as improved nutritional value. Molecules and methods for conferring insect / nematode / virus resistance are disclosed in U.S. Patent Nos. 5,250,515, 5,880,275, 6,506,599, 5,986,175, and U.S. Patent Application Publication No. 2003 / 0150017A1.

[0109] Herbicides for which transgenic plant tolerance has been demonstrated and to which the methods and compositions of the present disclosure can be applied include, but are not limited to, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil, norflurazon, 2,4-D (2,4-dichlorophenoxy)acetic acid, aryloxyphenoxypropionates, p-hydroxyphenylpyruvate dioxygenase inhibitors (HPPD), and protoporphyrinogen oxidase inhibitor (PPO) herbicides.Polynucleotide molecules encoding proteins involved in herbicide resistance known in the art include polynucleotide molecules encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as disclosed in U.S. Pat. Nos. 5,094,945, 5,627,061, 5,633,435, and 6,040,497 for conferring glyphosate tolerance; glyphosate oxidoreductase (GOX) as disclosed in U.S. Pat. No. 5,463,175 for conferring glyphosate tolerance. and polynucleotide molecules encoding glyphosate-N-acetyltransferase (GAT) as disclosed in U.S. Patent Application Publication No. 2003 / 0083480A1; dicamba monooxygenase as disclosed in U.S. Patent Application Publication No. 2003 / 0135879A1 for conferring dicamba resistance; polynucleotide molecules encoding bromoxynil nitrilase (Bxn) as disclosed in U.S. Patent No. 4,810,648 for conferring bromoxynil resistance; polynucleotide molecules encoding phytoene desaturase (crtI) as described by Sathasivan et al. (Nucl. Acids Res. 18:2188-2193, 1990) for conferring resistance to sulfonylurea herbicides; polynucleotide molecules encoding acetohydroxyacid synthase (AHAS, also known as ALS) as described by DeBlock et al. (EMBO) for conferring resistance to glufosinate and bialaphos. J. 6:2513-2519, 1987) for conferring N-aminomethylphosphonic acid tolerance; and polynucleotide molecules disclosed in U.S. Patent Application Publication No. 2003 / 010609A1 for conferring pyridine herbicide tolerance.Molecules and methods for conferring tolerance to multiple herbicides, such as glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides, are disclosed in US Pat. No. 6,376,754 and US Patent Application Publication No. 2002 / 0112260.

[0110] Genetic elements, methods, and transgenes that confer fungal disease resistance may also be used in the present disclosure (U.S. Pat. Nos. 6,653,280, 6,573,361, 6,506,962, 6,316,407, 6,215,048, 5,516,671, 5,773,696, 6,121,436, 6,316,407, 6,506,962). Soybean diseases caused by fungi include Phakopsora pachyrhizi, Phakopsora meibomiae (soybean rust), Colletotrichum truncatum, Colletotrichum dematium var. truncatum, Glomerella glycines (soybean anthracnose), Phytophthora sojae (Phytophthora root and stem rot), Sclerotinia sclerotiorum (Sclerotinia stem rot), Fusarium solani f.sp. glycines (sudden death syndrome), Fusarium spp. (Fusarium root rot), Macrophomina phaseolina (charcoal rot), Septoria glycines (brown spot), Pythium aphanidermatum, Pythium debaryanum, Pythium irregulare, Pythium ultimum, Pythium myriotylum, Pythium torulosum (Pythium seed rot), Diaporthe phaseolorum var. sojae (pod blight), Phomopsis longicola (stem blight), Phomopsis spp. (Phomopsis seed rot), Peronospora manshurica (downy mildew), Rhizoctonia solani (Rhizoctonia root and stem rot, Rhizoctonia leaf blight), Phialophora gregata (leaf spot), Diaporthe Phaseolorum var.caulivora (branch blight), Cercospora kikuchii (purpura), Alternaria sp.(brown ring blight), Cercospora sojina (spot disease), Sclerotium rolfsii (white silk blight), Arkoola nigra (black leaf blight), Thielaviopsis basicola (black root rot), Choanephora infundibulifera, Choanephora trispora (Choanephora leaf blight), Leptosphaerulina trifolii (Leptosphaerulina spot), Mycoleptodiscus terrestris (Mycoleptodiscus root rot), Neocosmospora vasinfecta (Neocosmospora stem rot), Phyllosticta sojicola (Phylosticta spot), Pyrenochaeta glycines (Pyrenochaeta spot), Cylindrocladium crotalariae, Dactuliochaeta glycines, Spaceloma glycines (red mold disease), Stemphylium botryosum (Stemphylium leaf blight), Corynesspora cassiicola (brown ring spot disease), Nematospora coryli (grain spot disease), and Phymatotrichum omnivorum (cotton root rot).

[0111] V. Definition The following definitions are provided to define and clarify the meaning of these terms with reference to the relevant embodiments of the present disclosure as used herein, and to guide those of skill in the art in understanding the present disclosure. Unless otherwise specified, terms are to be understood in accordance with their conventional meaning and usage in the relevant art, particularly in the fields of molecular biology and plant transformation.

[0112] When introducing an element of the disclosure or embodiment(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there is one or more of the element.

[0113] The term "and / or," when used in connection with a list of two or more items, means any one of the items, any combination of the items, or all of the items with which the term is associated.

[0114] The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. For example, any method that "comprises," "having," or "includes" one or more steps is not limited to retaining only those one or more steps, but may also include other unrecited steps. Similarly, any composition or device that "comprises," "having," or "includes" one or more features is not limited to retaining only those one or more features, but may also include other unrecited features.

[0115] As used herein, a "plant" includes a whole plant, an explant, a plant part, a seedling, or a plantlet at any stage of regeneration or development.

[0116] As used herein, a "plant part" can refer to any organ or intact tissue of a plant, such as a meristem, shoot organ / structure (e.g., leaf, stem or node), root, flower or floral organ / structure (e.g., bract, sepal, petal, stamen, carpel, anther, and ovule), seed, embryo, endosperm, seed coat, fruit, mature ovary, propagule, or other plant tissue (e.g., vascular tissue, epidermal tissue, ground tissue, etc.) or part thereof. Plant parts of the present disclosure may be viable, non-viable, regenerable, and / or non-regenerable. A "propagule" includes any plant part that can develop into a whole plant.

[0117] An "embryo" is the part of a plant seed that consists of precursor tissues (e.g., meristems) that can develop into all or part of an adult plant. "Embryo" can also include parts of a plant embryo.

[0118] "Meristem" or "meristematic tissue" includes undifferentiated or meristematic cells that are capable of differentiating to produce one or more types of plant parts, tissues or structures (e.g., all or part of a shoot, stem, root, leaf, seed, etc.).

[0119] As used herein, "determinate growth habit" refers to the cessation of vegetative growth after the main stem terminates with a cluster of mature pods. Determinate soybean varieties begin to flower when all or most of the nodes on the main stem are developed. They usually have elongated racemes that may be several centimeters long and may bear numerous flowers. As used herein, "determinate growth habit" refers to the simultaneous development of leaves and flowers throughout part of the reproductive period, with one to three pods at the terminal apex. Determinate soybean varieties, when grown at their adapted latitude, flower when about half of the nodes on the main stem are developed. They have short racemes, few flowers, and only a few flowers at the terminal nodes. "Semi-determinate" soybean varieties also flower when about half of the nodes on the main stem are developed, but the development and flowering of the nodes on the main stem ceases more abruptly than in determinate varieties. The racemes are short and have few flowers except for the terminal flowers, which are several times more numerous than the flowers lower on the plant.

[0120] As used herein, "lodging" refers to the bending of the stem near ground level of a cereal crop plant. Lodging is rated on a scale of 1 to 9. Generally, a score of 1 indicates an upright plant. A score of 5 indicates the plant is leaning at a 45 degree angle to the ground, and a score of 9 indicates the plant is lying on the ground. Table 4 shows the lodging scale used herein to rate control and modified soybean plants.

[0121] As used herein, "terminal flowering date" refers to the date when 50% of the plants in a uniform field plot have one open flower at the terminal node of the main stem.

[0122] As used herein, the "vegetative phase" of plant development is the period of growth between germination and flowering. The stages of the vegetative phase of soybean are as follows: VE (emergence), VC (cotyledon stage), V1 (first trifoliate leaf), V2 (second trifoliate leaf), V3 (third trifoliate leaf), V(n) (nth trifoliate leaf), V6 (soon to flower). As used herein, the "reproductive phase" of plant development is the period from flowering to the end of harvest. The stages of the reproductive phase of soybean are as follows: R1 (initiation of flowering, first flower), R2 (full bloom, flower at top 2 nodes), R3 (first pod, 3 / 16 inch pod at top 4 nodes), R4 (full pod, 3 / 4 inch pod at top 4 nodes), R5 (1 / 8 inch seed at top 4 nodes), R6 (full size seed at top 4 nodes), R7 (beginning to mature, one mature pod), R8 (fully mature, 95% of the pods on the plant have reached mature color). The vegetative and reproductive phases of soybean are well known to those skilled in the art, and numerous publications describing these phases can be found on the World Wide Web and elsewhere (e.g., North Dakota State University publication A-1174, June 1999, Reviewed and Reprinted August 2004).

[0123] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of exemplary language (e.g., "etc.") provided in any and all examples or with respect to specific embodiments herein is intended merely to illustrate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise stated in the claims.

[0124] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the spirit and scope of the present disclosure as further defined in the appended claims. Moreover, it should be recognized that all examples in the present disclosure, including those below, are provided as non-limiting examples. EXAMPLES

[0125] Example 1. Design of gene editing constructs. Targeted mutagenesis of the determinant locus using gene editing provides an opportunity to create alleles that confer different determinant phenotypes, resulting in diversity in soybean plant architecture and growth habits. Specific endogenous expression of the GmTFL1b gene, a dominant allele of the soybean Dt1 locus, in the stem tip protects the apical meristem from terminal differentiation, resulting in determinantism. The level of determinantism can be regulated by altering the expression level of the GmTFL1b gene through modification of the promoter region of the GmTFL1b gene. The coding sequence of the GmTFL1b gene is provided as SEQ ID NO:1, the amino acid sequence of the TFL1b protein is provided as SEQ ID NO:2, and the cDNA sequence of the GmTFL1b gene, including the 5'UTR and 3'UTR, is provided as SEQ ID NO:3.

[0126] The upstream promoter region of the GmTFL1b gene was targeted for mutagenesis using gene editing. Four gene editing constructs for plant transformation were designed using multiple guide RNAs (gRNAs) targeting various positions within the 2 kb promoter region (SEQ ID NO: 4) upstream of the transcription start site (tis) of the GmTFL1b gene, as shown in Figure 1.

[0127] Each of the plant transformation vector constructs was designed to create double-stranded breaks (DSBs) at multiple locations targeted by the gRNA, allowing for small deletions at the DSB sites as well as deletion of large segments between the DSB sites.

[0128] In this example, each genome editing construct generally contained 2-3 functional regions or cassettes related to gene editing and generation of DSBs in the GmTFL1b gene promoter region: expression of Cpf1 protein, expression of 2-4 gRNAs targeting the GmTFL1b gene promoter region, and optionally expression of 4 more gRNAs targeting the GmTFL1b gene promoter region. Each gRNA unit contains a common scaffold that matches the Cpf1 gene (SEQ ID NO: 5) and a unique spacer / targeting sequence complementary to its intended target site as listed in Table 1. The DNA sequences encoding the gRNA spacers and the intended target sites are listed in Table 1. [Table 1-1] [Table 1-2]

[0129] The Cpf1 expression cassette in the editing construct pM552 contained the Dahlia mosaic virus FLT promoter (SEQ ID NO:6) operably linked to a sequence encoding the rice-codon-optimized Lachnospiraceae bacterium Cpf1 RNA-guided endonuclease enzyme (SEQ ID NO:7) flanked on either side by a copy of a nuclear localization signal (SEQ ID NO:8). See, e.g., Gao et al., (Nature Biotechnol.35(8):789-792,2017). The Cpf1 expression cassette in the other four constructs shown in Table 1 contained the Medicago truncatula ubiquitin promoter (SEQ ID NO:9) operably linked to a maize-codon-optimized sequence encoding the Lachnospiraceae bacterium Cpf1 RNA-guided endonuclease enzyme (SEQ ID NO:10) flanked on either side by a copy of a nuclear localization signal (SEQ ID NO:8).

[0130] One type of gRNA expression cassette present in all constructs contained sequences encoding two to four gRNAs operably linked to the soybean RNA polymerase III (Pol3) promoter (SEQ ID NO: 11). The spacer sequences listed in Table 1 targeted alternative cleavage sites in the promoter region of GmTFL1b. An additional type of gRNA expression cassette, present only in pM552, contained sequences encoding another four guide RNAs operably linked to the soybean 7SL_CR10 promoter (SEQ ID NO: 12). The spacer sequences listed in Table 1 targeted alternative cleavage sites in the promoter region of GmTFL1b.

[0131] Specifically, gene editing constructs for plant transformation are designed to contain a gRNA that targets a region of DNA spanning nucleotide positions 1237 to 1570 of SEQ ID NO:4 of the reference sequence.

[0132] Example 2. Editing in the promoter region of GmTFL1b and confirmation of zygosity of genome-edited plants. Inbred wild type soybean lines were transformed via Agrobacterium-mediated transformation with the pM552 vector described in Example 1 above. Transformed plant tissues were grown to generate mature R0 plants. R0 plants with one or more unique genome edits were self-crossed to generate R1 plants. R1 plants homozygous for the allele containing the edited GmTFL1b promoter sequence and lacking the edited T-DNA sequence were self-crossed to generate R2 plants.

[0133] To determine the edits made to the GmTFL1b promoter region, amplicon sequencing technology was used to generate mutant sequences of the 2 kb promoter region for comparison to the wild-type sequence. Amplicon sequencing involves the creation of one or more unique PCR products spanning the entire genomic region of interest for next-generation sequencing. Sequence data from each sample is then mapped to a reference sequence to identify consensus sequence differences. To provide broad coverage of mutations in the target promoter region, plants with unique deletions ranging from 30 to 1221 base pairs (bp) in length were selected. Individual R1 plants, created by selfing R0 plants with one or more edits, were assayed for the nature of the edits and the zygosity of the edited variants or alleles and are listed in Table 2. All edited plants listed in Table 2 were created using the pM552 construct transformation vector. In Table 2, "R0 Event" is the R0 plant identifier, "Allele Name" is the unique allele identifier, and null (WT) corresponds to an inbred soybean plant that was not edited. "Causal lesion" indicates the start and end coordinates of the deleted segment of the promoter, starting from the 5' end of the 2 kb promoter sequence (SEQ ID NO: 4). "R1 zygosity" indicates the zygosity of the R1 plant as homozygous, heterozygous, undetermined (ND), or lethal (in the case of an edit that resulted in a lethal phenotype). The AL430a allele contains three modifications to the endogenous GmTFL1b gene: two deletions (a segment starting at position 939 and ending at position 1028 of SEQ ID NO: 4, and a segment starting at position 1066 and ending at position 1664 of SEQ ID NO: 4) and an inversion mutation (a segment starting at position 1065 and ending at position 1029 of SEQ ID NO: 4 that was inverted and reinserted into the deleted region). [Table 2]

[0134] The homozygous edits in Table 2 above are further shown in FIG. 2, aligned with the gRNA spacers shown in FIG. 1.

[0135] Similarly, soybean plants of maturity group 3.5 were used to generate edited plants using transformation vectors of the pM205, pM206 and pM207 constructs. Individual R1 plants, generated by selfing an R0 plant with one or more edits, were assayed for the nature of the edits and the zygosity of the edited variants or alleles and are listed in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

[0136] Similarly, soybean plants of maturity group 5.9 were used to generate edited plants using transformation vectors of the pM204 and pM205 constructs. Individual R1 plants, generated by selfing an R0 plant with one or more edits, were assayed for the nature of the edits and the zygosity of the edited variants or alleles and are listed in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0137] Example 3. Expression levels of the GmTFL1b gene in modified soybean plants. R2 plants carrying homozygous alleles of the GmTFL1b promoter mutant (Dt1 allele) and wild-type control plants were selected and grown in a controlled environment for sampling to assess differences in expression of the target genes. Plants were sampled at the VC phase, defined as the period after the budding phase but before the V1 phase, when the cotyledons and unifolia are fully unfolded. Unifolia leaf and apical tissues were collected on dry ice and subjected to gene expression analysis by TaqMan assay. Ten biological replicates were measured per allele entry.

[0138] Table 5 and Figure 3 show the relative expression levels of GmTFL1b gene in leaf and top tissues at VC phase of edited soybean plants with various homozygous Dt1 alleles. Edited plants were generated using the editing construct pM552. As shown in Figure 3, the expression of GmTFL1b in leaf tissues was only faint in all samples tested. [Table 5]

[0139] Because the promoter was mutated by genome editing, most of the Dt1 alleles caused a decrease in the expression levels of GmTFL1b compared to wild-type plants with the endogenous promoter. However, the mutant alleles AL398 and AL447b showed an increase in gene expression levels. Expression of TFL1 was significantly reduced in all but two of the samples tested.

[0140] Example 4. Evaluation of terminal flowering date and maturity date phenotypes of plants containing edited alleles. To test the phenotype of the edited plants, R2 plants carrying homozygous alleles of the GmTFL1b promoter mutant (Dt1 allele) and wild-type control plants were selected and grown in covered nurseries and in cultivated fields under standard agronomic practices.

[0141] All edited plants showed at least as good budding as the wild type, even those with a nearly determinate AL430a allele. All edited plants initiated flowering similarly to the wild-type determinate controls. No detectable changes in flowering were observed in the edited plants, indicating that the stem growth habit traits controlled by the Dt1 locus do not interact with genes controlling flowering.

[0142] As observed in covered nurseries, plants with the edited Dt1 allele displayed a range of determinate phenotypes from nearly determinate to more determinate than the wild type (extreme determinate), and the semi-determinate behavior of several different cultivars was first evidenced by different terminal flowering dates, as shown in Figure 4. The terminal flowering date is defined as the date when one open flower is present at the terminal node of the main stem of 50% of the plants in a uniform field plot. The fields for this evaluation consisted of two rows of 5-ft long furrows with 30-inch spacing between the furrows, with approximately 80 seeds sown per field and a 1.5-ft alley in the furrows.

[0143] Lodging in soybean fields is assessed through multiple observations beginning at the R6 growth phase using a rating scale to determine the portion of each field that exhibits stem lodging. Observations continue until senescence at the late R7 growth phase. Fields are assessed for lodging using the scale in Table 6. [Table 6]

[0144] Terminal flowering dates, maturity dates, and lodging ratings were determined for wild-type and homozygous edited allele plants in the observation fields. Terminal flowering dates are calculated here as the number of calendar days from January 1 of the test year. Maturity dates are defined as the date when 95% of the pods in the field reached mature pod color. If the exact maturity was not confirmed, a ">" or "<" symbol is displayed to indicate the start of maturity date was earlier or later than the stated date. Terminal flowering dates and maturity dates were determined by successive observations of the experimental fields until all fields reached the growth phase. Growth phase scoring was performed at the field level (containing approximately 15 consecutive plants) in a predefined 20-inch inner section of a uniform, undamaged field. A field is considered to have reached the growth phase in question if 50% or more of the plants in the measured field section are in the growth phase. Changes in determinacy can be quantified in days from planting to terminal flowering (DOTF). In fields with a given planting date that is the same for all plants in a study, the terminal flowering date and other phenological characteristics can be recorded by calendar day of the year to easily calculate differences from the control. From observations of plants in a field environment, edited alleles of the GmTFL1b gene promoter region resulted in diverse terminal flowering dates, ranging from 22 days earlier to 4 days later than wild-type determinacy plants, as shown in Table 7. Edited plants were created using the pM552 editing construct. [Table 7]

[0145] As shown in Table 7, the change in terminal flowering date was not accompanied by a delay in the R8 maturity date in most edited plants, except for plants with alleles AL398, AL391b, and AL447b, all of which had later terminal flowering dates than unedited wild-type plants and were more indeterminate than wild-type plants.

[0146] Plants with stronger finiteness or earlier terminal flowering dates are expected to produce structural features that create plants that are more resistant to lodging. Indeed, as shown in Table 7, there is a clear trend for plants with earlier terminal flowering dates to have reduced lodging. Genome editing of the promoter region of the GmTFL1b gene created plants with different terminal flowering dates corresponding to different levels of semi-effectiveness, leading to a reduction or elimination of the risk of lodging. Lodging is a cause of loss of harvest value, especially for tall elite soybean varieties. Reduced lodging may increase soybean harvest value and yield. Reduced lodging and improved standing may improve accessibility to arable land at all points after canopy closure, providing benefits such as effective fertilization and pest control.

[0147] The average relative expression levels of the GmTFL1b gene are copied from Table 5 to Table 7. Because the endogenous GmTFL1b gene in indeterminate soybeans is a dominant allele for the indeterminate trait, a decrease in the expression level of the GmTFL1b gene results in a decrease in the level of indeterminateness (or an increase in determinateness). As shown in Table 7, a lower average relative expression level tends to correspond to an earlier terminal flowering date or stronger determinateness. This correspondence indicates that the expression level of the GmTFL1b gene can be used as a predictor of the terminal flowering date or determinateness level of edited plants.

[0148] Differences in relative expression levels correlate not only with loss-of-function mutants but also with gain-of-function mutants in indeterminateness. Gain-of-function mutants show extreme indeterminateness compared to wild-type plants and increased expression consistent with the creation of a hypermorphic allele by genome editing of the promoter region of the GmTFL1b gene.

[0149] Example 5. Potential yield estimates derived from pod number per plant. As shown in Example 4, edited soybean plants with an early terminal flowering date phenotype tended to have improved lodging and standing characteristics. However, an early terminal flowering date may also result in loss of yield due to premature termination of the main stem and branches, and the number of productive nodes on the plant is reduced to an extent that cannot offset the improvement in node efficiency achieved by early assertion of apical dominance and complete dedication of resources to reproductive development. It is noteworthy that a terminal flowering date significantly earlier than the wild type can be maintained while retaining the productivity of finite apical nodes without substantially changing the height of the plant (and perhaps the total number of nodes). This means that the introduction of the semi-finite trait may not reduce pod yield per plant, while still benefiting from improved standing.

[0150] Pod number per plant is determined at the field level for a given 20-inch inner section (containing approximately 15 consecutive plants) of uniform, undamaged fields. At R6 phase or later, all plants in a given section of each field are cut and packaged to ensure that no pods are lost. Pods from each bunch are systematically counted and recorded. Pod number per entry (allele) is reported as the average of all plants counted per entry. Plants containing edited alleles AL391a and AL411a, which exhibited optimal lodging characteristics (see Table 7), as well as wild-type adventitious plants, were evaluated for pod number per plant and preliminary results are shown in Table 8 below. [Table 8]

[0151] Edited plants were generated using the pM552 editing construct. The number of pods per plant is comparable among the three plant groups within the margin of error. The edited semi-determinate plants showed improved standing ability without losing pods. In fact, the average number of pods in the edited semi-determinate plants was higher than in the wild-type determinate plants, suggesting that the introduction of semi-determinateness may result in improved productivity along with reduced yield losses due to reduced lodging.

[0152] Because the Dt1 locus was fixed early in soybean domestication, these edited alleles may be replicated in indeterminate soybean germplasm, conferring semi-determinateness and potentially resulting in traits in cultivated lines. Significant value may be generated from improved standing and productivity across soybean fields, especially in high-yielding environments. Additionally, the more compact plant species resulting from the semi-determinate trait may promote growth in narrower rows and at higher densities. Under these intensive agricultural configurations, compact, upright plant species may improve airflow under the canopy, thereby reducing humidity, which may reduce susceptibility to fungal diseases, including reduced susceptibility to economically important white mold and sudden death syndrome (SDS).

[0153] Example 6. Gene editing of TFL1 homologues. For sequence comparison based on total yield and diversity, a subset of plant species relevant to agriculture was selected, including members of the Solanaceae, Brassicaceae, and Legume families, as well as cotton and monocotyledonous cereals. To identify hit polypeptide sequences from each species with the highest identity, the BLASTP program from NCBI was used, using the soybean TFL1b (Dt1) polypeptide sequence as the query sequence and the Nonredundant Sequence Collection database as the search set. The identified sequences were aligned by a CLUSTAL type algorithm in CLC Workbench (default parameters for very accurate alignment). Pairwise comparisons are provided in tabular form (Figure 5).

[0154] The polynucleotide sequences of the 2 kb promoter region upstream of the transcription start site (tis) of the TFL1b gene homologues found in Zea mays, Sorghum bicolor, Oryza sativa, Triticum aestivum, Hordeum vulgare, Solanum lycopersicum, Gossypium hirsutum, Capsicum annuum, Brassica napus, Arabidopsis thaliana, Medicago truncatula, Arachis hypogaea, and Cicer arietinum are provided herein as SEQ ID NOs: 67-77, 79, and 81, respectively. Procedures similar to those described above in Example 1 can be used to generate plants with modified finite phenotypes in these additional plant species or other plant species.

[0155] Following a process similar to that described above in Example 1, about 10 editing guide RNAs can be designed approximately equally spaced across the ~2 kb promoter region of TFL1b homologues, following common gRNA design constraints (see FIG. 1). The ~200 nucleotide basal promoter region located at the 3' end of the sequences of SEQ ID NOs: 67-77, 79 and 81 can be avoided to minimize the possibility of the promoters failing completely. Following gRNA design, editing constructs can be created following steps along the lines of those described in Example 1 to generate edited plants, which can be further subjected to molecular and phenotypic characterization to select for the desired levels of relative expression and regulation of finiteness. Example 7. Evaluation of additional phenotypic traits of plants containing edited Dt1 alleles.

[0156] Further phenotypic characteristics of soybean plants containing the edited Dt1 allele were evaluated. All trait effects described in this example are reported as the delta of the mean relative to the indeterminate wild-type plants (unmodified soybean plants of maturity group 3.5) of a given entry.

[0157] Fields contained four rows of 10-ft long furrows with 20-inch spacing between furrows. Each area of ​​the field had 3-ft alleys between the furrows. A total of 288 seeds were sown per field. Each trial entry was replicated in 12 fields per experiment, and randomized mapping was performed with each trial entry replicate set containing enough fields containing wild-type comparison plants. Trait indices described below were collected for each field in the cultivated fields. The resulting trait values ​​reported are the delta of the mean, i.e., the difference between the trial mean and the comparison mean, for all 12 replicates. For this experimental design, a P value <0.2 is considered statistically significant. Scoring of growth phases was performed at the field level (containing approximately 15 consecutive plants) in a given 20-inch inner section of a uniform, undamaged field. A field was considered to have reached a particular developmental phase if 50% or more of the plants in the measured field plot were in that developmental phase. For large-scale quantitative comparisons, phase determinations are reported in terms of the number of days from planting to the desired phase.

[0158] Plant development and morphological traits, including date of full maturity, number of branches per plant, plant height, and plant lodging (standing position) were evaluated at the R8 growth phase. Full maturity was determined based on the number of days from planting date to the date when 95% of the pods on the plant reached mature pod color. Plant height was recorded as a direct measurement of the length of the linear main stem. Plant lodging was evaluated as described in Example 4 above. The results are shown in Table 9 below. [Table 9-1] [Table 9-2]

[0159] As shown in Table 7 of Example 4 above, plants with extreme indeterminate alleles such as AL398 (which exhibit increased GmTFL1b gene expression levels) exhibited significantly delayed maturation as well as delayed termination. However, as shown in Table 9 above, delayed termination in plants containing the AL398 allele was accompanied by moderately higher branch counts, increased plant height, and increased lodging. All plants with semi-determinate alleles matured within one day of indeterminate wild-type plants, except for plants with the AL437 allele, which matured more than one day earlier than the indeterminate wild-type plants. Plants containing semi-determinate alleles exhibited significantly reduced plant height compared to indeterminate wild-type plants, ranging from 2 inches to 30 inches. Plants containing semi-determinate alleles were more upright and exhibited lodging ratings that were 1 to 4.7 points (10% to 47%) lower than wild-type indeterminate plants. This is due in part to reduced plant height. Despite the reduction in plant height and lodging, most semi-determinate alleles allowed plants to produce a large number of branches per plant, which was not statistically different from indeterminate wild-type plants. Only plants carrying the AL437 allele had a significantly reduced number of branches.

[0160] Secondary yield components including number of nodes per plant, number of pods per plant, and number of pods per node were also evaluated. Secondary yield components were determined at the field level (containing approximately 15 consecutive plants) in a given 20-inch inner section of a uniform, undamaged field. In the R8 phase, all plants in a given section of each field were cut and wrapped to ensure that no pods were lost. The number of pods and nodes on each plant were systematically counted and recorded. Plant numbers were recorded and pods for a given field were pooled for primary yield component analysis. The number of pods per node was calculated from the counts. The results are shown in Table 10 below. [Table 10]

[0161] Plants carrying semi-finite alleles with significant early termination in arable fields also showed significantly improved standing in arable fields, without loss of branch number and without change in maturity beyond 1 day. These characteristics suggest that semi-finite alleles may improve standing without reducing productivity. However, early termination of the shoot tip may also result in loss of yield by reducing the number of productive nodes on the plant to an extent that cannot be offset by the improvements in node efficiency achieved by early assertion of apical dominance and complete dedication of resources to reproductive development. Plants carrying semi-finite alleles show some reduction in plant height depending on the allele, but these reductions are generally not associated with significant changes in productive nodes per plant. Consistent with node retention, most semi-finite alleles confer a number of pods per plant that is equal to or significantly higher than the number of pods per plant counted in indeterminate wild-type plants. Most semi-determinate plants tested have pod numbers per plant ranging from the same to 23 more pods than indeterminate wild-type plants. The increased pod number in semi-determinate plants is associated with improved node efficiency, as indicated by a significantly increased number of pods per node. The exceptions are the AL430a and AL411b alleles, which have significantly negative pod trait indices due to the early termination of Dt1 and the extreme reduction in plant height due to the near loss of function, as previously shown in Table 8.

[0162] Major yield components were also evaluated, including seeds per plant, 1000 seed weight, and grain yield estimates. Major yield components were determined at the field level by threshing pods collected from all plants in a given field and normalizing by the number of plants measured. Threshing returns the total seed weight and number of seeds. The 1000 seed weight per field is calculated by dividing the total seed weight by the number of seeds and multiplying by 1000. The grain yield estimate is a function of the estimated seeds per unit area multiplied by the single seed weight and a conversion factor of 45.375, which returns an estimate for relative comparisons only of 60 lb bushels per acre. The results are shown in Table 11 below. [Table 11-1] [Table 11-2]

[0163] The secondary yield components observed for most of the semi-determinant alleles described herein were comparable or significantly positive compared to indeterminate wild-type plants. These traits indicate that soybean standing can be significantly improved by editing without compromising yield. Alleles with positive secondary yield components also often showed increased seeds per plant in arable fields. These experiments showed some reduction in seed weight for most semi-determinant alleles. However, despite the reduction in seed weight, all plants carrying semi-determinant alleles with positive seed number still showed neutral to positive grain yield estimates. Alleles AL411b and AL430a, previously noted to have significantly negative pod trait indices due to extreme reduction in plant height, also showed reduced seed number per plant and grain yield estimates. This indicates a useful limit on the magnitude of the trait relative to the reduction in stem tip terminalization. These observations substantially support the possibility of using alleles conferring appropriate magnitudes of semi-determinance to enhance standing as well as yield potential in some cases.

[0164] The following above-ground traits were assessed: canopy coverage assessment, days from planting to the R1 phase (beginning of flowering), and days from planting to terminal pods (TP). Canopy coverage indicates the percentage of the field area that is occupied by plants. The assessment is based on remote RGB imaging data acquired by an unmanned aerial vehicle, which distinguishes between green and non-green pixels in the field when viewed from directly above the field. For example, a canopy coverage of 40% indicates that 60% of the planting space in the field is likely to be vacant. The terminal stage was determined by observing the date of terminal flower or terminal pod formation. The duration of the R1 and terminal pod phases was determined by continuous observation of the experimental fields until all fields reached the defined developmental phase. Flowering was determined by recording the date of flowering at any node on the main stem of the plant. Terminal pods were determined by recording the date when the terminal node of the main stem had one pod greater than 3 / 16 inch (0.5 cm) in length. The results are shown in Table 12 below. [Table 12]

[0165] The primary trait affected by edits to the Dt1 locus is termination date. The spectrum of changes in terminal traits resulting from edits to the Dt1 locus results in proportional downstream changes in plant height and standing habit. Semi-determinate alleles significantly reduce the number of days from planting to terminal pod set, ranging from 3.5 to 12 days earlier than indeterminate wild-type plants, and are associated with minimal changes in flowering and maturation. Plants carrying the AL437 allele began flowering within 1 day of indeterminate wild-type plants. Dt1 edits produced statistically significant changes of varying magnitude in termination, depending on the semi-determinate allele. Most measured aboveground traits of semi-determinate edited plants were not significantly different from indeterminate wild-type plants in the R1 growth phase.

[0166] Belowground traits of root dry weight, shoot dry weight, and shoot-to-root ratio, which are related to biomass, were also determined in the R1 phase. These traits were assessed at the field level (containing approximately 15 consecutive plants) in a given 20-inch inner section of a uniform, undamaged field. In the R1 phase, all plants in a given section of each field were completely removed from the ground, counted, separated into shoot and root sections, and packaged for transfer to the oven. After the plant samples were completely dried, weights were recorded for each field. Root and shoot biomass were reported as an average per plant. Shoot-to-root ratio was calculated by dividing the average shoot dry weight by the average root dry weight for each entry. The results are shown in Table 13 below. [Table 13]

[0167] Belowground biomass measurements and proportions for most plants carrying semi-determinate alleles were not significantly different from indeterminate wild-type plants in the R1 growth phase. One of the semi-determinate alleles, AL437, was an exception, being significantly negative for all three biomass indices, indicating a smaller overall plant size and disproportionately smaller shoots. This is evident from the significantly smaller canopy cover for this allele, as shown in Table 12.

[0168] Overall, these trait results demonstrate the utility of semi-determined editing to obtain unique alleles that can be the basis for more upstanding soybean varieties with stable or improved yield potential. Furthermore, the results show that editing of the Dt1 locus can create not only semi-determined alleles, but also near-determined, determinant, and extreme determinant alleles.

[0169] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the spirit and scope of the present disclosure as described in the specification and the appended claims. Moreover, it should be understood that all examples in the present disclosure are provided as non-limiting examples.

Claims

1. A modified plant, plant seed, plant part, or plant cell comprising a modification that reduces the expression or activity of TFL1 or a homolog thereof compared to the expression or activity of TFL1 or a homolog thereof in an otherwise identical plant, plant seed, plant part, or plant cell lacking the modification.

2. 2. The modified plant, plant seed, plant part, or plant cell of claim 1, wherein the modification is present in at least one allele of an endogenous TFL1 gene or a homolog thereof.

3. 3. The modified plant, plant seed, plant part, or plant cell of claim 2, wherein the TFL1 gene or a homolog thereof encodes a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:

2.

4. 4. The modified plant, plant seed, plant part, or plant cell of claim 2 or 3, wherein the modification is in a non-coding region of the TFL1 gene or a homolog thereof.

5. 5. The modified plant, plant seed, plant part, or plant cell of claim 4, wherein the non-coding region is selected from the group consisting of a promoter, an intron, a 5'-untranslated region, a 3'-untranslated region, and any combination thereof.

6. 6. The modified plant, plant seed, plant part, or plant cell of claim 5, wherein the modification is in the promoter of the TFL1 gene or a homolog thereof.

7. 7. The modified plant, plant seed, plant part or plant cell of any one of claims 1 to 6, wherein the plant is a legume, cotton plant or canola plant, or the plant seed, plant part or plant cell is a plant seed, plant part or plant cell of a legume, cotton plant, canola plant, corn plant, sorghum plant, rice plant, wheat plant, barley plant, tomato plant or pepper plant.

8. 8. The modified plant, plant seed, plant part, or plant cell of claim 7, wherein the plant is a legume or the plant seed, plant part, or plant cell is a plant seed, plant part, or plant cell of a legume.

9. 9. The modified plant, plant seed, plant part, or plant cell of claim 8, wherein the legume plant is a soybean plant, a kidney bean plant, a pea plant, a chickpea plant, an alfalfa plant, a peanut plant, a carob plant, a lentil plant, or a liquorice plant.

10. 10. The modified plant, plant seed, plant part, or plant cell of claim 9, wherein the legume is a soybean plant.

11. The modified plant, plant seed, plant part, or plant cell of claim 10, wherein the TFL1 gene is a TFL1b gene.

12. 12. The modified plant, plant seed, plant part, or plant cell of any one of claims 1 to 11, wherein the plant, plant seed, plant part, or plant cell is heterozygous for the modification.

13. 12. The modified plant, plant seed, plant part, or plant cell of any one of claims 1 to 11, wherein the plant, plant seed, plant part, or plant cell is homozygous for the modification.

14. 12. The modified plant, plant seed, plant part, or plant cell of any one of claims 1 to 11, wherein the plant, plant seed, plant part, or plant cell comprises a first modification in a first allele of the TFL1 gene and a second modification in a second allele of the TFL1 gene, wherein the first modification and the second modification are different from each other.

15. The modified plant, plant seed, plant part, or plant cell according to any one of claims 1 to 14, wherein the modification comprises a deletion, an insertion, a substitution, an inversion, or any combination thereof.

16. 16. The modified plant, plant seed, plant part, or plant cell of claim 15, wherein the modification is located at about 200 or more nucleotides from the 3' end of a sequence selected from the group consisting of SEQ ID NOs: 4, 67-77, 79, and 81.

17. 17. The modified plant, plant seed, plant part, or plant cell of claim 16, wherein the modification comprises a deletion.

18. The plant, the plant seed, the plant part, or the plant cell comprises a modification in at least one allele of a promoter of the TFL1b gene, the modification comprising: A deletion of 30 base pairs from nucleotide 1539 to nucleotide 1568 compared to the reference sequence SEQ ID NO:4; A deletion of 388 base pairs from nucleotide 1217 to nucleotide 1604 compared to the reference sequence SEQ ID NO:4; A deletion of 112 base pairs from nucleotide 1518 to nucleotide 1629 compared to the reference sequence SEQ ID NO:4; A deletion of 272 base pairs from nucleotide 951 to nucleotide 1222 compared to the reference sequence SEQ ID NO:4; A deletion of 44 base pairs from nucleotide 1364 to nucleotide 1407 compared to the reference sequence SEQ ID NO:4; A deletion of 150 base pairs from nucleotide 1367 to nucleotide 1516 compared to the reference sequence SEQ ID NO:4; A deletion of 1053 base pairs from nucleotide 754 to nucleotide 1806 compared to the reference sequence SEQ ID NO:4; A deletion of 104 base pairs from nucleotide 1216 to nucleotide 1319 compared to the reference sequence SEQ ID NO:4; A deletion of 807 base pairs from nucleotide 1159 to nucleotide 1965 compared to the reference sequence SEQ ID NO:4; A deletion of 455 base pairs from nucleotide 760 to nucleotide 1214 compared to the reference sequence SEQ ID NO:4; A deletion of 90 base pairs from nucleotide 939 to nucleotide 1028 compared to the reference sequence SEQ ID NO:4; a 37 base pair inversion in which the sequence from nucleotide 1029 to nucleotide 1065 of SEQ ID NO:4 has been deleted, inverted, and reinserted in the same position; A deletion of 599 base pairs from nucleotide 1066 to nucleotide 1664 compared to the reference sequence SEQ ID NO:4; A deletion of 601 base pairs from nucleotide 952 to nucleotide 1552 compared to the reference sequence SEQ ID NO:4; A deletion of 132 base pairs from nucleotide 1677 to nucleotide 1808 compared to the reference sequence SEQ ID NO:4; A deletion of 35 base pairs from nucleotide 1524 to nucleotide 1558 compared to the reference sequence SEQ ID NO:4; A deletion of 930 base pairs from nucleotide 476 to nucleotide 1405 compared to the reference sequence SEQ ID NO:4; A deletion of 195 base pairs from nucleotide 1365 to nucleotide 1559 compared to the reference sequence SEQ ID NO:4; A deletion of 633 base pairs from nucleotide 928 to nucleotide 1560 compared to the reference sequence SEQ ID NO:4; A deletion of 1221 base pairs from nucleotide 593 to nucleotide 1813 compared to the reference sequence SEQ ID NO:4; A deletion of 5 base pairs from nucleotide 1552 to nucleotide 1556 compared to the reference sequence SEQ ID NO:4; A deletion of 29 base pairs from nucleotide 1537 to nucleotide 1565 compared to the reference sequence SEQ ID NO:4; A deletion of 38 base pairs from nucleotide 1209 to nucleotide 1246 compared to the reference sequence SEQ ID NO:4; A deletion of 49 base pairs from nucleotide 1552 to nucleotide 1600 compared to the reference sequence SEQ ID NO:4; A deletion of 49 base pairs from nucleotide 1553 to nucleotide 1601 compared to the reference sequence SEQ ID NO:4; A deletion of 11 base pairs from nucleotide 1368 to nucleotide 1378 compared to the reference sequence SEQ ID NO:4; A deletion of 28 base pairs from nucleotide 1368 to nucleotide 1395 compared to the reference sequence SEQ ID NO:4; A deletion of 41 base pairs from nucleotide 1344 to nucleotide 1384 compared to the reference sequence SEQ ID NO:4; A deletion of 235 base pairs from nucleotide 1219 to nucleotide 1453 compared to the reference sequence SEQ ID NO:4; A deletion of 13 base pairs from nucleotide 1215 to nucleotide 1227 compared to the reference sequence SEQ ID NO:4; A deletion of 7 base pairs from nucleotide 1370 to nucleotide 1376 compared to the reference sequence SEQ ID NO:4; A deletion of 337 base pairs from nucleotide 1220 to nucleotide 1556 compared to the reference sequence SEQ ID NO:4; A deletion of 161 base pairs from nucleotide 1216 to nucleotide 1376 compared to the reference sequence SEQ ID NO:4; A deletion of 17 base pairs from nucleotide 1368 to nucleotide 1384 compared to the reference sequence SEQ ID NO:4; A deletion of 283 base pairs from nucleotide 1366 to nucleotide 1648 compared to the reference sequence SEQ ID NO:4; A deletion of 120 base pairs from nucleotide 1370 to nucleotide 1489 compared to the reference sequence SEQ ID NO:4; A deletion of 75 base pairs from nucleotide 1541 to nucleotide 1615 compared to the reference sequence SEQ ID NO:4; A deletion of 9 base pairs from nucleotide 1367 to nucleotide 1375 compared to the reference sequence SEQ ID NO:4; A deletion of 51 base pairs from nucleotide 1551 to nucleotide 1601 compared to the reference sequence SEQ ID NO:4; A deletion of 7 base pairs from nucleotide 1371 to nucleotide 1377 compared to the reference sequence SEQ ID NO:4; A deletion of 186 base pairs from nucleotide 1368 to nucleotide 1553 compared to the reference sequence SEQ ID NO:4; A deletion of 14 base pairs from nucleotide 1365 to nucleotide 1378 compared to the reference sequence SEQ ID NO:4; A deletion of 16 base pairs from nucleotide 1365 to nucleotide 1380 compared to the reference sequence SEQ ID NO:4; A deletion of 43 base pairs from nucleotide 1552 to nucleotide 1594 compared to the reference sequence SEQ ID NO:4; A deletion of 10 base pairs from nucleotide 1367 to nucleotide 1376 compared to the reference sequence SEQ ID NO:4; A deletion of 131 base pairs from nucleotide 1552 to nucleotide 1682 compared to the reference sequence SEQ ID NO:4; A deletion of 438 base pairs from nucleotide 954 to nucleotide 1391 compared to the reference sequence SEQ ID NO:4; A deletion of 26 base pairs from nucleotide 1535 to nucleotide 1560 compared to the reference sequence SEQ ID NO:4; A deletion of 111 base pairs from nucleotide 1534 to nucleotide 1644 compared to the reference sequence SEQ ID NO:4; A deletion of 663 base pairs from nucleotide 937 to nucleotide 1599 compared to the reference sequence SEQ ID NO:4; A deletion of 98 base pairs from nucleotide 1551 to nucleotide 1648 compared to the reference sequence SEQ ID NO:4; A deletion of 34 base pairs from nucleotide 1526 to nucleotide 1559 compared to the reference sequence SEQ ID NO:4; A deletion of 79 base pairs from nucleotide 1528 to nucleotide 1606 compared to the reference sequence SEQ ID NO:4; A deletion of 61 base pairs from nucleotide 1542 to nucleotide 1602 compared to the reference sequence SEQ ID NO:4; A deletion of 381 base pairs from nucleotide 1214 to nucleotide 1594 compared to the reference sequence SEQ ID NO:4; A deletion of 187 base pairs from nucleotide 1368 to nucleotide 1554 compared to the reference sequence SEQ ID NO:4; A deletion of 109 base pairs from nucleotide 1369 to nucleotide 1477 compared to the reference sequence SEQ ID NO:4; A deletion of 5 base pairs from nucleotide 1550 to nucleotide 1554 compared to the reference sequence SEQ ID NO:4; A deletion of 1267 base pairs from nucleotide 734 to nucleotide 2000 compared to the reference sequence SEQ ID NO:4; A deletion of 190 base pairs from nucleotide 1371 to nucleotide 1560 compared to the reference sequence SEQ ID NO:4; A deletion of 36 base pairs from nucleotide 1537 to nucleotide 1572 compared to the reference sequence SEQ ID NO:4; A deletion of 46 base pairs from nucleotide 1541 to nucleotide 1586 compared to the reference sequence SEQ ID NO:4; A deletion of 5 base pairs from nucleotide 1552 to nucleotide 1556 compared to the reference sequence SEQ ID NO:4; A deletion of 955 base pairs from nucleotide 669 to nucleotide 1623 compared to the reference sequence SEQ ID NO:4; A deletion of 38 base pairs from nucleotide 1521 to nucleotide 1558 compared to the reference sequence SEQ ID NO:4; A deletion of 109 base pairs from nucleotide 1369 to nucleotide 1477 compared to the reference sequence SEQ ID NO:4; A deletion of 15 base pairs from nucleotide 1540 to nucleotide 1554 compared to the reference sequence SEQ ID NO:4; A deletion of 458 base pairs from nucleotide 1217 to nucleotide 1674 compared to the reference sequence SEQ ID NO:4; A deletion of 81 base pairs from nucleotide 1546 to nucleotide 1626 compared to the reference sequence SEQ ID NO:4; A deletion of 89 base pairs from nucleotide 1547 to nucleotide 1635 compared to the reference sequence SEQ ID NO:4; and 17. The modified plant, plant seed, plant part, or plant cell according to any one of claims 11 to 16, selected from the group consisting of any combination thereof.

19. 17. The modified plant, plant seed, plant part, or plant cell of any one of claims 11 to 16, wherein the plant, plant seed, plant part, or plant cell comprises an alteration in the promoter of at least one allele of the TFL1b gene, the alteration being contained within the genomic region between nucleotides 1237 to 1570 of SEQ ID NO:4 of the reference sequence.

20. 20. The modified plant, plant seed, plant part, or plant cell of claim 19, wherein the modification comprises a deletion of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 consecutive nucleotides.

21. 21. The modified plant, plant seed, plant part, or plant cell of any one of claims 15 to 20, wherein the plant, plant seed, plant part, or plant cell comprises a chromosomal sequence of the TFL1b gene having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity to SEQ ID NO:4 in the region other than the deletion, insertion, substitution, or inversion.

22. The plant, the plant seed, the plant part or the plant cell is selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 341, 342, 343, 344, 345, 346, 350, 351, 352 19. The modified plant, plant seed, plant part, or plant cell of any one of claims 11 to 18, comprising a polynucleotide sequence selected from the group consisting of: 06, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128.

23. 23. The modified plant, plant seed, plant part, or plant cell of any one of claims 1 to 22, wherein the modification alters the expression level of the TFL1 protein compared to the expression level of the TFL1 protein in an otherwise identical plant, plant seed, plant part, or plant cell lacking the modification.

24. The modified plant, plant seed, plant part, or plant cell of claim 23, wherein the modification reduces the expression level of the TFL1 protein compared to the expression level of the TFL1 protein in an otherwise identical plant, plant seed, plant part, or plant cell lacking the modification.

25. 25. The modified plant of any one of claims 1 to 24, wherein the modification alters the definite phenotype of the plant compared to the definite phenotype of an otherwise identical plant lacking the modification.

26. 26. The modified plant of claim 25, wherein the modification increases the determinism of the plant compared to the otherwise identical determinism lacking the modification.

27. 27. The modified plant of any one of claims 1 to 26, wherein after planting, the modified plant reaches its terminal flowering date earlier than an otherwise identical plant lacking the modification.

28. 28. The modified plant of any one of claims 1 to 27, wherein the modified plant exhibits a reduced lodging rate compared to the lodging rate of an otherwise identical plant lacking the modification.

29. 29. The modified plant of any one of claims 1 to 28, wherein the modified plant exhibits substantially the same or increased yield compared to the yield of an otherwise identical plant lacking the modification.

30. 30. The modified plant of any one of claims 1 to 29, wherein the modified plant exhibits a reduced susceptibility to fungal disease compared to the susceptibility of the identical plant.

31. A polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128.

32. A guide RNA comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29.

33. 1. A method for producing a plant comprising a modified TFL1 gene, the method comprising: a) introducing an alteration into at least one target site in an endogenous TFL1 gene or a homologue thereof of a plant cell; b) identifying and selecting one or more plant cells of step (a) that contain said modification in said TFL1 gene or a homologue thereof; and c) regenerating at least one plant from at least one or more cells selected in step (b).

34. 34. The method of claim 33, wherein the target site is located in a non-coding region of the endogenous TFL1 gene or a homolog thereof.

35. 35. The method of claim 34, wherein the non-coding region is selected from the group consisting of a promoter, an intron, a 5'-untranslated region, a 3'-untranslated region, and any combination thereof.

36. 37. The method of claim 36, wherein the non-coding region is a promoter.

37. The method of claim 33, wherein the modification is facilitated by the presence of at least one site-specific genome modification enzyme in the plant cell.

38. The method of claim 37, wherein the site-specific genome modification enzyme is an RNA-guided nuclease, a zinc finger nuclease, a meganuclease, a TALE nuclease, a recombinase, a transposase, and any combination thereof.

39. The method of claim 38, wherein the site-specific genome modification enzyme is an RNA-guided nuclease, including Cas nuclease, Cpf1 nuclease, or a variant of any of these.

40. The method of claim 37, wherein the site-specific genome modification enzyme generates at least one strand break at the target site.

41. 34. The method of claim 33, wherein the modification is selected from the group consisting of a substitution, an insertion, an inversion, a deletion, a duplication, and combinations thereof.

42. 42. The method of claim 41 , wherein the modification is a deletion.

43. 43. The method of claim 42, wherein the deletion comprises a region of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, or at least 150 contiguous nucleotides.

44. A method for increasing the finiteness of a plant, comprising modifying at least one allele of an endogenous promoter of a TFL1 gene, such that production of TFL1 protein is reduced compared to production of TFL1 protein in an otherwise identical plant lacking said modification.

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